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Summer 8-13-2010
The Role of the Exocyst in Exocytosis and Cell
Migration
Jianglan Liu
University of Pennsylvania, [email protected]
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Liu, Jianglan, "The Role of the Exocyst in Exocytosis and Cell Migration" (2010). Publicly Accessible Penn Dissertations. 201.
http://repository.upenn.edu/edissertations/201
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For more information, please contact [email protected].
The Role of the Exocyst in Exocytosis and Cell Migration
Abstract
The exocyst, an evolutionarily conserved octameric protein complex, plays a crucial role in the tethering of
post-Golgi secretory vesicles to the plasma membrane for exocytosis and cell migration. How the exocyst is
targeted to sites of exocytosis and how this complex regulates cell migration are poorly understood. I have
carried out experiments to characterize Exo70, a component of the exocyst complex. Firstly, I found that
Exo70 directly interacts with phosphatidylinositol 4,5-bisphosphate through positively charged residues at its
C-terminus, and this interaction is critical for the plasma membrane targeting of Exo70. Using the ts045
vesicular stomatitis virus glycoprotein trafficking assay, I found that the Exo70-lipid interaction is critical for
the docking and fusion of post-Golgi secretory vesicles with the plasma membrane. Secondly, I demonstrate
that the exocyst plays a pivotal role in tumor cell invasion. Invadopodia are actin-rich membrane protrusions
formed by tumor cells that degrade the extracellular matrix for invasion. Depletion of the exocyst component
Exo70 or Sec8 led to failure in invadopodia formation in MDA-MB-231 cells expressing constitutively active
c-Src, whereas the overexpression of Exo70 promoted invadopodia formation. Disrupting the exocyst
function by RNA interference of EXO70 or SEC8, or by expression of a dominant negative fragment of Exo70
blocked the secretion of matrix metalloproteinases. I further found that the exocyst interacts with the Arp2/3
complex in cells with high invasion potential; blocking the exocyst-Arp2/3 interaction inhibited Arp2/
3-mediated actin polymerization and invadopodia formation. Finally, the exocyst also functions in directly
regulating Arp2/3-mediated actin polymerization. Using pyrene actin assay and total internal reflection
fluorescence microscopy, I found that Exo70 synergizes with WAVE2 to promote Arp2/3-mediated actin
polymerization and branching. In addition, I have examined how the Exo70-Arp2/3 interaction is regulated in
the cell downstream of growth factor signaling. Overall, these studies provide mechanistic insights to the
function of the exocyst in exocytosis and cell migration.
Degree Type
Dissertation
Degree Name
Doctor of Philosophy (PhD)
Graduate Group
Biology
First Advisor
Wei Guo
Keywords
Exocytosis, Exocyst, Invadopodia, The Arp2/3 complex, cell migration, actin polymerization
Subject Categories
Cell Biology
This dissertation is available at ScholarlyCommons: http://repository.upenn.edu/edissertations/201
THE ROLE OF THE EXOCYST
IN EXOCYTOSIS AND CELL MIGRATION
JIANGLAN LIU
A DISSERTATION
in
BIOLOGY
Presented to the Faculties of the University of Pennsylvania
in
Partial Fulfillment of the Requirements for the
Degree of Doctor of Philosophy
2010
Supervisor of Dissertation
_________________________________
Wei Guo, Associate Professor of Biology
Graduate Group Chairperson
_________________________________
Paul Sniegowski, Associate Professor of Biology
Dissertation Committee
Tatyana Svitkina, Associate Professor of Biology
Michael Lampson, Assistant Professor of Biology
Kimberly Gallagher, Assistant Professor of Biology
Margaret Chou, Associate Professor of Pathology and Laboratory Medicine
Mickey Marks, Associate Professor of Pathology and Laboratory Medicine
ACKNOWLEDGEMENTS
First of all, I would like to give my deepest gratitude to my mentor Dr. Wei
Guo. I regard Wei as my scientific father as he guided me into the wonderful
scientific field and directed me all the way till now. I am so impressed with his
enthusiasms and curiosity on science, his persistence on research and his extensive
knowledge. And I really appreciate that he gives me inspiring advices and warm
encouragements in all the time of research. I would benefit from his mentoring
throughout my entire scientific career.
Secondly, I really appreciate all the members in my thesis committee: Prof.
Tatyana Svitkina, Prof. Margaret M. Chou, Prof. Mickey Marks, Prof. Kimberly
Gallagher and Prof. Michael A.Lampson. They have committed much time and
efforts to follow my research and have always been a great source of constructive
suggestions and encouragements.
I enjoy the wonderful time that I have spent with all the previous and current
members of Guo lab. They make the lab such a pleasant place for everyday work.
They are not only my scientific partners, but also my incredible friends. In particular,
I would like to thank Dr. Bing He who has offered me so much help and valuable
advices. I appreciate Dr. Xiaofeng Zuo for his contribution to the first part of this
study. I am grateful to Dr Xiaoyu Zhang and Jian Zhang for their nice help since I
ii
entered the lab. I also want to thank Dr. Kelly Orlando for her help on my scientific
English writing. Finally, I want to thank all other lab folks for their constant
discussions and encouragements.
I would also like to extend thanks to the people who have been instrumental in
completion of this work. I am grateful to Prof. Susette C. Mueller, Dr. Vira V. Artym
and Mr. Peter Johnson for their collaboration on the invadopodia research; Prof. Yale
Goldman and Dr. Yujie Sun for their collaboration on the TIRFM experiments; Dr.
Jian Jing for giving me advices for zymography experiments; Prof. Roberto
Dominguez, Dr. Grzegorz Rebowski and Dr. Boczkowska Malgorzata for their help
on the pyrene actin assay.
Finally, I would like to give my special thanks to my family. I want to thank my
parents with my heart for their love, support and encouragement throughout my life.
Most of all, I would give my thanks to my dear husband, Peng Yue. He is my labmate,
my soulmate and my lifemate. His solid help for my research and his endless love for
my life make me strong enough to go this far. I want to give my last but most special
thanks to my little angel Shannon Xianning Yue. You make mommy’s life complete.
Love you forever.
iii
ABSTRACT
THE ROLE OF THE EXOCYST
IN EXOCYTOSIS AND CELL MIGRATION
Jianglan Liu
Supervisor: Dr. Wei Guo
The exocyst, an evolutionarily conserved octameric protein complex, plays a
crucial role in the tethering of post-Golgi secretory vesicles to the plasma membrane
for exocytosis and cell migration. How the exocyst is targeted to sites of exocytosis
and how this complex regulates cell migration are poorly understood. I have carried
out experiments to characterize Exo70, a component of the exocyst complex. Firstly, I
found that Exo70 directly interacts with phosphatidylinositol 4,5-bisphosphate
through positively charged residues at its C-terminus, and this interaction is critical
for the plasma membrane targeting of Exo70. Using the ts045 vesicular stomatitis
virus glycoprotein trafficking assay, I found that the Exo70-lipid interaction is critical
for the docking and fusion of post-Golgi secretory vesicles with the plasma
membrane. Secondly, I demonstrate that the exocyst plays a pivotal role in tumor cell
invasion. Invadopodia are actin-rich membrane protrusions formed by tumor cells that
degrade the extracellular matrix for invasion. Depletion of the exocyst component
Exo70 or Sec8 led to failure in invadopodia formation in MDA-MB-231 cells
iv
expressing constitutively active c-Src, whereas the overexpression of Exo70
promoted invadopodia formation. Disrupting the exocyst function by RNA
interference of EXO70 or SEC8, or by expression of a dominant negative fragment of
Exo70 blocked the secretion of matrix metalloproteinases. I further found that the
exocyst interacts with the Arp2/3 complex in cells with high invasion potential;
blocking
the
exocyst-Arp2/3
interaction
inhibited
Arp2/3-mediated
actin
polymerization and invadopodia formation. Finally, the exocyst also functions in
directly regulating Arp2/3-mediated actin polymerization. Using pyrene actin assay
and total internal reflection fluorescence microscopy, I found that Exo70 synergizes
with WAVE2 to promote Arp2/3-mediated actin polymerization and branching. In
addition, I have examined how the Exo70-Arp2/3 interaction is regulated in the cell
downstream of growth factor signaling. Overall, these studies provide mechanistic
insights to the function of the exocyst in exocytosis and cell migration.
v
Table of Contents
Chapter 1. Introduction································································································· 1
1.1 Overview··············································································································· 1
1.2 The exocyst complex···························································································· 3
1.3 Phosphoinositides in cell regulation and membrane dynamics·························· 56
1.4 Cell invasion and invadopodia structures··························································· 60
1.5 Thesis overview·································································································· 69
Chapter 2. Phosphatidylinositol 4,5-bisphosphate mediates the targeting of the
exocyst to the plasma membrane for exocytosis in mammalian cells························ 70
2.1. Introduction········································································································ 71
2.2. Results················································································································ 73
2.3. Discussion········································································································ 106
2.4. Materials and Methods····················································································· 110
Chapter 3. The role of the exocyst in matrix metalloproteinase secretion and actin
dynamics during tumor cell invadopodia formation················································· 116
3.1. Introduction······································································································ 117
3.2. Results·············································································································· 120
3.3. Discussion········································································································ 152
3.4. Materials and Methods····················································································· 156
vi
Chapter 4: The role of Exo70 in Arp2/3-mediated actin assembly during cell
migration··················································································································· 163
4.1. Introduction······································································································ 164
4.2. Results·············································································································· 170
4.3. Discussion and future perspectives·································································· 184
4.4. Materials and Methods····················································································· 194
Chapter 5: Discussion and future perspectives························································· 199
5.1. Impact of my research in the field··································································· 199
5.2. The molecular mechanism of exocyst targeting to the plasma membrane······ 200
5.3. A crosstalk between actin cytoskeleton and exocytosis machineries during cell
migration········································································································· 201
5.4. The functions of the exocyst in many cellular processes································· 202
5.5. Future perspectives·························································································· 202
References················································································································· 203
vii
List of Tables
Table 1. Comparison of the affinities of Exo70 and exo70-1 for phospholipids. ········ 80
Table 2. Exo70 C-terminus mutants. ············································································ 85
viii
List of Figures
Figure 1.1 An outline of membrane trafficking events in the cell.······························· 2
Figure 1.2 Four major steps in vesicular trafficking. ··················································· 4
Figure 1.3 Protein-protein interactions among the exocyst subunits. ·························· 9
Figure 1.4 Structural studies of the exocyst subunits. ··············································· 11
Figure 1.5 Subcellular localization of the exocyst in various cell types. ··················· 16
Figure 1.6 The exocyst is the downstream effector of a variety of Ras family of small
GTPases. ···················································································································· 44
Figure 2.1 Association of Exo70 with the plasma membrane (PM) in HeLa cells. ··· 75
Figure 2.2 The interaction between Exo70 and phospholipids in vitro. ···················· 79
Figure 2.3 Mutations at Exo70 C-terminus abolish the association between Exo70 and
the PM. ······················································································································· 84
Figure 2.4 Mutations at the C-terminus of Exo70 affect its interaction with
phospholipids. ············································································································ 87
Figure 2.5 Mutations in the exo70-1 mutant did not affect its interaction with TC10
(Q75L) or Sec8. ········································································································· 89
Figure 2.6 Exo70 is required for the PM localization of Sec8. ································· 92
Figure 2.7 Exocytosis of VSV-G ts045 is blocked in EXO70 siRNA knockdown
cells. ··························································································································· 95
Figure 2.8 VSV-G ts045 exocytosis defect in EXO70 siRNA knockdown cells
expressing the exo70-1 mutant. ··············································································· 102
ix
Figure 3.1 Knockdown of the exocyst inhibits invadopodial activity. ···················· 122
Figure 3.2 Overexpression of Exo70 stimulates invadopodial activity. ·················· 127
Figure 3.3 Localization of Exo70 at the focal degradation sites. ···························· 131
Figure 3.4 The exocyst is required for the secretion of MMPs in MDA-MB-231
(Y527F c-Src) cells. ································································································· 135
Figure 3.5 Zymography analyses of MMP-2 and MMP-9 secreted from
MDA-MB-231 (Y527F c-Src) cells overexpressing Exo70∆C (amino acids 1–408) or
full-length Exo70. ···································································································· 138
Figure 3.6 The interaction between Exo70 and the Arp2/3 complex is required for
invadopodia formation. ···························································································· 141
Figure 3.7 Exo70 is involved in Arp2/3-mediated actin polymerization in the
cell. ··························································································································· 147
Figure 4.1 rExo70 enhances actin polymerization mediated by the Arp2/3 complex
and WAVE2. ············································································································ 172
Figure
4.2
Elongation
and
branching
of
actin
filaments
visualized
by
TIRFM. ···················································································································· 175
Figure 4.3 GST-WAVE2 pulled down GFP/myc-tagged rExo70 from HeLa
cells. ························································································································· 179
Figure 4.4 Examine the in vitro interaction of GST-tagged rExo70 and His(6)-tagged
Arpc1 wt or Arpc1 (21E) mutant. ············································································ 182
x
Abbreviations
AMPAR, a-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid receptor.
aPKC, atypical protein kinase C.
ARE, apical recycling endosomes.
Arf, ADP ribosylation factor.
BFA, Brefeldin A.
COG, conserved oligomeric Golgi.
ECM, extracellular matrix
EGF, epidermal growth factor
ER, endoplasmic reticulum.
EST, expressed sequence tag
FRAP, fluorescence recovery after photobleaching.
GARP, Golgi-associated retrograde protein.
GEF, guanine nucleotide exchange factor.
GLUT4, glucose transporter 4
HDM, high-density microsomal membranes
HSS, high speed supernatant.
LDLR, LDL receptor.
LDM, low-density microsomal membranes
LUV, large unilamellar vesicle.
xi
MAGUK, membrane-associated guanylate kinase.
MDCK, Madin-Darby canine kidney.
MMP, matrix metalloprotease
NGF, nerve growth factor
NMDAR, N-methyl D-aspartate receptor.
NPF, nucleation-promoting factor.
NRK, normal rat kidney
PBS, phosphate-buffered saline.
PC, phosphotidylcholine.
PH, pleckstrin homology domain.
PI(4,5)P2, phosphatidylinositol 4,5-bisphosphate.
PKD, protein kinase D
PM, plasma membrane.
PS, phosphatidylserine.
PSD, postsynaptic density.
PI, phosphatidylinositol.
RBD, Ral binding domain.
RNAi, RNA interference.
shRNA, short hairpin RNA.
SNARE, soluble JV-ethylmaleimide-sensitive fusion attachment protein receptor.
xii
TIRFM, total internal reflection fluorescence microscopy.
TGN, trans-Golgi network.
tSNARE, target membrane-associated SNARE.
VSV-G, vesicular stomatitis virus G.
xiii
Jianglan Liu
THE ROLE OF THE EXOCYST IN EXOCYTOSIS AND CELL MIGRATION
Chapter 1
Chapter 1. Introduction
1.1. Overview
Membrane trafficking, or the transport of membrane-bound materials between
cellular endomembrane compartments and the plasma membrane, is essential for
delivery of proteins and other macromolecules to various sites inside and outside of
the cell (Figure 1.1). The membrane trafficking system involves distinct organelles,
including the endoplasmic reticulum (ER), Golgi apparatus, endosomal compartments,
and plasma membrane. In the biosynthetic pathway, secretory cargos are synthesized
and modified in the ER and then transported to the Golgi apparatus for further
modifications. Upon arrival at the trans-Golgi network (TGN), the cargos are sorted
and packaged into post-Golgi vesicles that take different routes towards the plasma
membrane or endosomal compartments, respectively. In the endocytic pathway,
membrane and proteins are internalized at the plasma membrane and are either
delivered through early and late endosomes to the lysosomes for degradation or
recycled back to the plasma membrane via recycling endosomes.
Between different membrane compartments, proteins and lipids are mostly
transported by small membrane-enclosed vesicles. The basic scheme of membrane
trafficking consists of several major steps: budding of vesicles from the donor
1
Jianglan Liu
THE ROLE OF THE EXOCYST IN EXOCYTOSIS AND CELL MIGRATION
Chapter 1
Figure 1.1
Figure 1.1 An outline of membrane trafficking events in the cell. In this diagram,
the exocytic and endocytic routes are illustrated with solid and dashed arrows,
respectively. In the biosynthetic-secretory pathway, cargo leaves the TGN or
recycling endosomes in vesicular carriers to the plasma membrane. In the endocytic
pathway, proteins are internalized and transported to early endosomes, and then either
travel through late endosomes to the lysosome to be degraded or return to the plasma
membrane through the recycling endosomes. Early endosomes may serve as sorting
stations for the next stages of cargo transport. Figure modified from Orlando and Guo,
2009.
2
Jianglan Liu
THE ROLE OF THE EXOCYST IN EXOCYTOSIS AND CELL MIGRATION
Chapter 1
compartment, transport of vesicles along cytoskeleton, tethering of the vesicles to
specific domains and fusion of vesicles with the target compartment. Coat proteins
and cytoskeleton elements play major roles during the budding and transport steps,
respectively. The tethering step, defined as the initial recognition and attachment of
secretory vesicles with the target membrane, is mediated by tethering proteins. In the
subsequent fusion step, the SNARE (soluble NSF attachment protein receptors)
proteins drive the final fusion of the two membranes (Cai et al., 2007, Figure 1.2).
Exocytosis is a fundamental membrane trafficking event in eukaryotic cells that
mediates the incorporation of membrane proteins and lipids into specific plasma
membrane domains as well as the secretion of vesicle contents to the exterior of the
cell. It is essential for a number of biological processes including cell growth,
morphogenesis and cell migration. Exocytosis is achieved by the fusion of secretory
vesicles with the plasma membrane, which is catalyzed by the assembly of the
SNARE complex. Before membrane fusion, the tethering of secretory vesicles to the
plasma membrane is thought to be mediated by the exocyst, an evolutionarily
conserved octameric protein complex.
1.2. The exocyst complex
Up to date, a number of proteins and protein complexes have been identified
3
Jianglan Liu
THE ROLE OF THE EXOCYST IN EXOCYTOSIS AND CELL MIGRATION
Chapter 1
Figure 1.2
Figure 1.2 Four major steps in vesicular trafficking. The diagram reveals the four
essential steps in vesicular transport. (1) Budding: coat proteins are recruited onto the
donor membrane to promote the formation of a vesicle. Cargo and SNAREs are
incorporated into the budding vesicle through interaction with coat subunits. (2)
Transport: the vesicle moves toward the acceptor compartment through a cytoskeletal
track. (3) Tethering: tethering factors work in conjunction with upstream regulators to
tether the vesicle to their acceptor membrane. (4) Fusion: the vesicle-associated
SNARE and the SNARE on the acceptor membrane assemble into a four-helix
bundled SNARE complex, which drives membrane fusion and the delivery of cargo.
Figure adapted from Cai et al., 2007.
4
Jianglan Liu
THE ROLE OF THE EXOCYST IN EXOCYTOSIS AND CELL MIGRATION
Chapter 1
and characterized that function at the tethering steps at various stages of membrane
trafficking (Pfeffer, 1999; Guo et al., 2000; Waters and Hughson, 2000; Whyte and
Munro, 2002). In particular, the tethering of post-Golgi secretory vesicles to the
plasma membrane is mediated by the evolutionarily conserved exocyst complex,
which is composed of Sec3, Sec5, Sec6, Sec8, Sec10, Sec15, Exo70 and Exo84.
1.2.1. The identification and molecular organization of the exocyst complex
Most exocyst components were originally identified in late 1970’s in a genetic
screen for temperature-sensitive secretory (sec) mutants of the yeast Saccharomyces
cerevisiae (Novick and Schekman, 1979; Novick et al., 1980). The screen identified
23 complementation groups of sec mutants defective in secretion, which could be
divided into two basic categories, 13 genes important for ER-to-Golgi and/or
intra-Golgi membrane trafficking and 10 genes required for Golgi-to-plasma
membrane (PM) transport (Novick et al., 1980 & 1981). These 10 genes are known as
the ‘‘late-acting’’ or “late” secretory genes and six out of them were later shown to
encode exocyst components: Sec3, Sec5, Sec6, Sec8, Sec10 and Sec15. Two other
exocyst components, Exo70 and Exo84, were biochemically identified from the
purified yeast complex or the homologous mammalian complex.
The observation that Sec15 and Sec8 associated with a 19.5 svedberg (19.5S)
5
Jianglan Liu
THE ROLE OF THE EXOCYST IN EXOCYTOSIS AND CELL MIGRATION
Chapter 1
particle in a linear density gradient (Bowser and Novick, 1991; Bowser et al., 1992)
leads to the hypothesis that some late SEC genes might be present in a high molecular
weight protein complex. Subsequently, Novick’s group purified this protein complex
from yeast lysates (TerBush et al., 1996; TerBush et al., 2001). This complex is
composed of the products of six late SEC genes Sec3, Sec5, Sec6, Sec8, Sec10 and
Sec15, and a novel gene product Exo70 (TerBush and Novick, 1995; TerBush et al.,
1996). All these components are present as single copy in the complex (TerBush et
al., 1996). The last yeast exocyst component, Exo84, was identified based on
sequence similarity to the mammalian homologue (Guo et al., 1999b). Genetic
analysis indicates that yeast Exo84, like the other exocyst subunits, is essential for
exocytosis (Zhang et al., 2005).
The identification of mammalian exocyst complex was largely promoted by the
identification of yeast exocyst complex as well as the timely realization of the
conservation of the secretory pathway from yeast to mammals. The mammalian Sec6
and Sec8 were identified by screening human genome expressed sequence tag (EST)
databases with the yeast SEC genes and then cloned from a rat brain cDNA library
(Ting et al., 1995). Similar to the yeast exocyst, mammalian Sec6 and Sec8
co-migrated on a continuous glycerol gradient at 17s, indicating that they are
components of a 600- to 700-kDa protein complex. Successively, the rat brain rSec6/8
6
Jianglan Liu
THE ROLE OF THE EXOCYST IN EXOCYTOSIS AND CELL MIGRATION
Chapter 1
(exocyst) complex was purified from rat brain lysates using antibodies against rSec6
and rSec8 by sequential column chromatography (Hsu et al., 1996). The purified
mammalian exocyst complex contained eight proteins, including rSec6 and rSec8,
with a stoichiometry of one copy of each protein within the complex. rSec5, rSec15,
rExo70 and rExo84 were subsequently identified by peptide sequencing using mass
spectrometry and cloned from cDNA database (Hsu et al., 1996; Kee et al., 1997).
The remaining two mammalian exocyst genes, Sec10 and Sec3, were identified
through search in EST database and GenBank based on sequence homology to yeast
genes, and then cloned from cDNA libraries (Guo et al., 1997; Matern et al., 2001).
Understanding the function of the exocyst at the molecular level will require
elucidation of the molecular organization of the complex. To explore the molecular
organization of the exocyst complex, the interactions between individual exocyst
components have been extensively studied using a variety of methods, including
yeast two-hybrid assay, protein binding assays with in vitro translated proteins and
purified recombinant proteins (For review, see Munson and Novick, 2006). The
interaction map is shown in Figure 1.3, which suggests that most exocyst subunits can
interact with multiple exocyst subunits in the complex. A number of interactions,
including Sec3-Sec5, Sec5-Sec6, Sec5-Exo84, Sec6-Sec8, Sec6-Secl0, Sec6-Exo70,
Sec8-Exo70, Sec10-Secl5, and Sec10-Exo70, have been found in both yeast and
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mammalian exocyst suggesting conserved interacting interfaces between different
components in the complex.
Whether individual exocyst component is required for exocyst assembly has
been examined by in vitro reconstitution assay. Mammalian exocyst components
were expressed and purified from E. coli, then mixed in a 1:1 molar ratio to
reconstitute the exocyst complex (Wang et al., 2004). Interestingly, the absence of
any single exocyst subunit did not affect the assembly of the remaining seven
subunits, which suggests that multiple interactions between the exocyst subunits are
sufficient for any of the seven subunits to associate with each other to form a complex
(Wang et al., 2004). On the other hand, studies in budding yeast indicate that the
integrity of the exocyst is largely perturbed in several exocyst mutants, including
sec3-2, sec5-24, sec6-4, sec 10-2 and sec15-1, at the restrictive temperature (TerBush
and Novick, 1995). These observations suggest that although no individual exocyst
component is essential for complex assembly in vitro, the function of at least several
exocyst components is critical for the assembly of the exocyst in vivo.
1.2.2. The structure of the exocyst complex
The recent crystal structure data have provided valuable insights into the
structure and molecular organization of the exocyst complex. So far, partial crystal
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Figure 1.3
Figure 1.3 Protein-protein interactions among the exocyst subunits. Interactions
among individual exocyst components has been studied using (1) yeast two-hybrid
assay (red), (2) protein binding assay with recombinant proteins purified from E. coli
(green) or (3) protein binding assay with in vitro translated proteins (blue). The
diagram was generated based on Munson and Novick, 2006. The interaction map
suggests that most exocyst subunit can interact with multiple exocyst subunits in the
complex.
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structures of five exocyst components have been solved. These include the near
full-length yeast and mouse Exo70 (Dong et al, 2005; Hamburger et al, 2006; Moore
et al, 2007); the C-terminal domains of Drosophila Sec15 (Wu et al, 2005), yeast
Exo84 (Dong et al., 2005) and yeast Sec6 (Sivaram et al., 2006); and the N-terminal
domain of yeast Sec3 (Yamashita et al., 2010; Baek et al., 2010). Despite the little
sequence homology among the exocyst components, most of their structures shared
some common features: they all exhibit rod-like structures consisting of two or more
consecutively packed helical bundles, with each bundle composed of three to five
α-helices connected by loops (Figure 1.4). Predictions of the secondary structure
suggest that the remaining unsolved domains and exocyst components may also be
composed of similar helical folds. The structural similarity among exocyst
components suggests that they evolved from an ancient common ancestor. In addition,
several subunits of the exocyst have been found to share distant sequence similarity
with subunits of two other tethering complexes that function at the Golgi apparatus,
the Golgi-associated retrograde protein (GARP) complex and the conserved
oligomeric Golgi (COG) complex (Whyte et al., 2001). The crystal structure of the
COG subunit COG2 reveals a six-helix bundle with a general resemblance to that of
the exocyst components (Cavanaugh et al., 2007). These findings suggest that the
exocyst, the COG and the GARP complexes, which are all constructed from helical
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Figure 1.4
Figure 1.4 Structural studies of the exocyst subunits. The diagram shows crystal
structures of near full length yeast Exo70 (Dong et al, 2005; Hamburger et al, 2006),
and the C-terminal domains of Drosophila Sec15 (Wu et al, 2005), yeast Exo84
(Dong et al, 2005), and yeast Sec6 (Sivaram et al, 2006), and N-terminus of yeast
Sec3 (Yamashita et al., 2010; Baek et al., 2010). Except for Sec3N, these exocyst
subunits all display rod like structures composed of two or more consecutively
packed helical bundles, each consists of three to five a-helices linked by loops. The
core domain of Sec3N consists of a semi-open seven-stranded β-barrel. Figure
adapted from Munson and Novick, 2006 and Yamashita et al., 2010.
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bundles, are structurally related and might have diverged from a common ancestor to
mediate vesicle tethering at various stages of membrane trafficking.
The structure of individual exocyst components also provides a clue to the
assembly of the exocyst complex. For example, Exo70 has an extended rod-shaped
structure. Initial mapping of the binding sites of Sec8 and Sec10 on the Exo70
structure indicates that they have an extended interface with Exo70 along the length
of the structure, suggesting that Sec8 and Sec10 themselves also have extended
rod-like structures (Dong et al., 2005). Therefore, the assembly of the exocyst
complex probably involves the packing of at least several rod-shaped subunits in an
elongated side-to-side fashion. This is consistent with the previously described
‘Y’-shaped structure of the mammalian exocyst complex after fixation (Hsu et al.,
1998, see later).
It is worth noting that although the distribution of electronic charges is not
conserved in yeast and mammalian Exo70 molecules, both of them share a C-terminal
electropositive domain. Indeed, the C-terminal domain is the most highly conserved
portion of Exo70. In this region, homologous residues form a surface patch of basic
residues at the extreme C-terminal tip of the rod, suggesting a conserved function for
this last domain. In many cases the association of proteins with the PM can be
mediated by interactions with the negatively charged phospholipids in membrane via
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clusters of basic residues (for reviews, see McLaughlin et al., 2002; Balla, 2005).
Therefore, the C-terminal basic patch of Exo70 suggests a possible interaction with or
proximity to the membrane. It would be intriguing to examine whether Exo70 directly
interacts with the membrane.
Recently, the crystal structure of the N-terminal domain of yeast Sec3 (Sec3N)
was resolved (Yamashita et al., 2010; Baek et al., 2010). Different from the above
four rod-like structures, Sec3N consists of a semi-open seven-stranded β-barrel. The
barrel is capped at one end by a C-terminal α-helix, whereas at the other end of the
barrel, three inter-strand variable loops form the canonical phosphoinositide-binding
pocket (Figure 1.4). The structure of Sec3N represents a typical pleckstrin homology
(PH) domain, which belongs to a structural superfamily including the
phosphotyrosine-binding, Ena/VASP homology, and Ran-binding domains (Lemmon,
2004). These domains lack sequence similarity, but all share this core structure. The
structure of Sec3N suggests that it may directly associate with the plasma membrane
through its PH domain.
While the yeast Sec3N (amino acids 1-320) is composed of β-barrel, the region
following the PH domain is predicted to be predominantly helical in structure.
Residues 320–470 are predicted with high probability to form a coiled-coil structure;
fold recognition programs further suggest that the region starting from residue 610 to
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the C terminus of Sec3 is composed of a series of long helices separated by short
loops (Baek et al., 2010). This is consistent with the signature pattern of the structure
of other exocyst subunits -- the packed helical bundles.
The overall organization of the purified mammalian exocyst complex was
revealed by Quick-Freeze/Deep-Etch Electron Microscopy (Hsu et al., 1999).
Without fixation with glutaraldehyde, the complex shows variable conformations
usually as a set of four or six arms 4-6 nm in width and 10-30 nm in length which
radiate outward from a central point. The arms appear to attach to the body through a
flexible hinge region, allowing the arms to extend from the body at varying angles.
Following glutaraldehyde fixation, the exocyst complex displays a much more
uniform and packed Y-shaped or T-shaped structure, with a 30 nm x 13 nm stem and
two 6 nm x l5 nm arms branched away from one end of the central body (Hsu et al,
1998). The difference in the shape of fixed and unfixed complex suggest that the
exocyst might undergo conformational changes when it functions in vesicle tethering.
Or it may represent assembled and disassembled complex, respectively. The structure
of fixed complex may more closely resemble the native assembled exocyst structure,
in which the rod-like subunits pack against one another along their length; whereas
the structure represented in the unfixed preparation is in the process of disassembly
into individual subunits.
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To date, the description of the exact subunit organization and structure of the
holo-exocyst complex is still incomplete. The elucidation of the structure of other
individual subunits and, ultimately, the intact complex at higher resolution will
clearly improve our understanding of the function of the exocyst and the molecular
mechanism of vesicle tethering.
1.2.3. Subcellular localization of the exocyst
1.2.3.1. In budding yeast
In budding yeast, the exocyst exhibits a cell cycle-dependent localization
pattern that represents sites of active exocytosis and cell surface expansion (Finger et
al., 1998; Mondesert et al., 1997; TerBush and Novick, 1995) (Figure 1.5). As cells
enter the cycle, exocyst components are found in a patch at the pre-bud site. During
bud emergence, the exocyst is concentrated at the tip of the bud, corresponding to the
apical growth of the daughter cell. When the daughter cell has grown to a certain size
and their growth pattern has switched from apical to isotropic, the exocyst proteins
are correspondingly redistributed to the entire surface of the growing bud. At the time
of cytokinesis, exocyst components are redistributed to the mother-daughter junction,
where active exocytosis takes place for the completion of cytokinesis and separation
of the two cells (TerBush et al, 1995; Finger et al, 1998). This pattern of localization
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Figure 1.5
Figure 1.5 Subcellular localization of the exocyst in various cell types. The
diagram reveals the localization of the exocyst in budding yeast, developing neurons,
polarized epithelial cells, and migrating cells. The plasma membrane domains where
the exocyst is localized are marked by red. In budding yeast, the exocyst is
concentrated at the bud tip in small-budded cells, and relocates to the
mother-daughter junction during cytokinesis. In developing neurons, the exocyst is
enriched in axonal branch points, the growth cone of the extending neurites, and
discrete puncta along axons where mature synapses will form. In polarized epithelial
cells, the exocyst is enriched at the apical portion of the lateral membrane. In
migrating cells, the exocyst is recruited to the leading edge of the plasma membrane.
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mirrors the pattern of deposition of newly synthesized materials at the cell surface.
Consistent with the cortical localization, biochemical analyses reveal that about
20-25% of the exocyst proteins are associated with the plasma membrane in budding
yeast (Bowser and Novick, 1991; Bowser et al, 1992).
In yeast, while all the exocyst components are localized to the growing end of
the daughter cells, they seem to have different targeting mechanisms. Sec3 is
localized to the bud tip independent of actin cables, along which the vesicles are
transported (Finger et al., 1998; Boyd et al., 2004). A portion of Exo70 is transported
to sites of exocytosis on vesicles, but approximately half also localizes independently
of vesicle traffic (Boyd et al., 2004). By contrast, the remaining exocyst components
are associated with exocytic vesicles and delivered to sites of exocytosis through
actin cables (Boyd et al., 2004; Zajac et al., 2005; Zhang et al., 2005). These results
led to the hypothesis that Sec3 and Exo70 associate with the plasma membrane and
interact with the rest of the exocyst components on the arriving vesicles, which
promotes the assembly of the exocyst complex to tether the secretory vesicles to the
plasma membrane. Consistently, recent structural studies have revealed that Exo70
and Sec3 may directly interact with the plasma membrane through basic residues or
PH domain, respectively, which supports the model that Exo70 and Sec3 are potential
landmarks in the exocyst complex (Dong et al., 2005; Hamburger et al., 2006; Moore
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et al., 2007; Yamashita et al., 2010; Baek et al., 2010).
Septins are additional regulators of the localization of the exocyst in yeast.
Septins are a family of GTPases conserved from yeast to mammals that play central
roles in cell division and cell polarization (For reviews, see Spiliotis and Nelson,
2006; Barral and Kinoshita, 2008). Septins act as membrane diffusion barriers
between different membrane domains and as molecular scaffolds for the localization
of many signaling proteins, bud site selection proteins, and chitin synthases. In
budding yeast, septins polymerize to form ring-like structure at the bud neck
throughout the entire budding process (Longtine and Bi, 2003). The intact septin ring
is essential for the spatial restriction of the exocyst in the bud during isotropic growth
(Barral et al, 2000) as well as the recruitment of the exocyst to the bud neck during
cytokinesis (Dobbelaere and Barral, 2004). However, how septin undergoes
highly-regulated functional transitions from spatial restriction during isotropic growth
to protein recruitment during cytokinesis is unknown. Interestingly, the mammalian
exocyst was shown to coimmunoprecipitate with septins and microtubules from rat
brain extracts (Hsu et al., 1998; Vega and Hsu, 2001; Vega and Hsu, 2003). It
remains under investigation whether there is a direct interaction between exocyst and
septin, and whether septin mediates the spatial regulation of the exocyst through this
interaction.
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1.2.3.2. In mammalian cells
The subcellular localization of the exocyst in mammalian cells is much more
complex than that in yeast cells (Figure 1.5).
a) Epithelial cells
The establishment of epithelial polarity involves the initial formation of
cell–cell adhesion and the subsequent specialization of structurally and functionally
distinct apical and basal-lateral plasma membrane domains. The subcellular
distribution of the exocyst complex dramatically changes during development of
epithelial polarity. In single polarized Madin-Darby canine kidney (MDCK) cells in
contact with the substratum (contact-naive cells), the exocyst components are
diffusely distributed in the cytosol. Upon initiation of E-cadherin-mediated cell
adhesion, they are rapidly recruited to the plasma membrane at sites of cell–cell
contact (Grindstaf et al., 1998; Yeaman et al., 2001; Yeaman et al, 2004). In fully
polarized cells, the exocyst is sorted out to the apex of the lateral membrane and
co-localized with components of tight junction and nectin complexes, but only
partially overlapped with E-cadherin, which also localized to the rest of the forming
lateral membrane (Grindstaf et al., 1998; Yeaman et al, 2004). Consistent with this
spatiotemporal
redistribution,
the
exocyst
co-fractionates
with
membranes
specifically enriched in apical junctional proteins (Yeaman et al, 2004). The plasma
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membrane localization of the exocyst in polarized epithelial cells is dependent upon
the integrity of the cell-cell adhesion (Grindstaff et al, 1998; Yeaman et al, 2004). It
was shown that E-cadherin and nectin-2α mediates the recruitment of the exocyst to
the site of cell-cell adhesion. The exocyst components were recruited to the cell-cell
contacts when E-cadherin and nectin-2α were co-expressed in fibroblasts (Yeaman et
al, 2004). Biochemical studies also indicate that exocyst is associated with apical
junctional, proteins, including E-cadherin and nectin-2α, in polarized epithelial cells
(Yeaman et al, 2004).
In addition to the plasma membrane localization, the exocyst components were
also shown to be localized in membrane-bound structures at the perinuclear region of
epithelial cells (Yeaman et al., 2001; Prigent et al., 2003; Folsch et al., 2003; Oztan et
al., 2007). However, which intracellular compartment at the prenuclear region does
the exocyst associate with is still under investigation. Some studies found that the
exocyst complex is present on both TGN and plasma membrane in normal rat kidney
(NRK) cells that formed either fibroblast-(NRK-49F) or epithelial-like (NRK-52E)
intercellular junctions (Yeaman et al., 2001). In contrast, other studies showed that
the exocyst was associated with the recycling endosomal compartments in MDCK
cells. (Prigent et al., 2003; Folsch et al., 2003; Oztan et al., 2007).
The controversy in exocyst localization may reflect the growth conditions of
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the cells, the degree of cellular polarization, fixation methods, differential recognition
of junctional versus intracellular populations of the exocyst subunits by different
antibodies (Yeaman et al., 2001, 2004), and the possibility that the exocyst complex
may exist in different conformational states (extended or closed) depending on its
localization in the cell (Munson and Novick, 2006).
b) Neurons
The localization of the exocyst in neurons is largely dependent on the
differentiation state of the neuronal cells. In developing neurons that have not formed
synapses or in cultured neurons that are in the process of differentiation, the exocyst
is found in the cell body, as well as in axonal branching points, the growth cone of the
extending neurites, and discrete puncta along axons where mature synapses will form
(Hsu et al., 1996; Kee et al., 1997; Hazuka et al., 1999; Vega and Hsu, 2001; Brown
et al., 2001; Murthy et al., 2003). In contrast, in mature neurons, the exocyst
components are largely absent from mature synapses, where synaptic vesicle cycling
takes place (Hazuka et al, 1999). The localization pattern suggests that exocyst is
involved in synaptogenesis and neurite outgrowth, but not required for the cycling of
synaptic vesicles and neurotransmitter release at the presynaptic membranes. On the
other hand, exocyst components have been found to be associated with the
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postsynaptic membrane of mature synapse, and have been implicated in the delivery
of glutamate receptors to the postsynaptic membrane for synaptic transmission (Sans
et al, 2003; Gerges et al, 2006).
c) Cytokinesis
During cell division, the exocyst is initially localized to centrosome/spindle
poles in interphase, then with mitotic spindles in mitosis, and later enriched at the
central spindles and midbody during cytokinesis. The localization of the exocyst at
the abscission site could also be detected in cells ectopically expressing exocyst
components (Chen et al., 2006). The spatial regulation of the exocyst is coupled to
the dynamic redistribution of its interacting Ral GTPases (Chen et al., 2006; Cascone
et al., 2008). During cytokinesis, the recruitment of the exocyst to the midbody was
shown to be mediated by centriolin, a coil-coiled protein that is localized to the
midbody and required for abscission. Centriolin directly interacts with the exocyst
subunit Sec15 and depleting centriolin disrupts the midbody localization of the
exocyst (Gromley et al., 2005).
d) Migrating cells
Cell migration requires the coordination of polarized exocytosis and actin
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cytoskeleton remodeling to support dynamic reorganization of the plasma membrane
at the leading edge. The initial step of cell migration is the formation of cell
protrusions in the direction of cell movement. Overexpression of Exo70 induces
numerous actin-based protrusive structures resembling filopodia (Wang et al., 2004;
Xu et al., 2005; Zuo et al., 2006). And Exo70 is localized at the tips of filopodia,
where it is colocalized with the actin staining. In migrating cells, the exocyst subunits
are enriched at the leading edges of plasma membranes free of cell-cell contacts,
where they are colocalized with the Arp2/3 complex (Rosse et al., 2006; Zuo et al.,
2006). This localization pattern of the exocyst at the leading edge reflects its role in
mediating vesicular traffic to this region as well as its direct interaction with the
Arp2/3 complex (Zuo et al., 2006).
The recruitment of the exocyst to the leading edge may involve different
molecular mechanisms. It was shown that RalB expression is required for promoting
both exocyst assembly and localization to the leading edge of moving cells (Rosse et
al., 2006). Recently, it was reported that the polarized delivery of the exocyst to the
leading edge of migrating epithelial cells is dependent on aPKCs (Rosse et al., 2009).
Interestingly, it was shown that aPKC localization at the leading edge is also
dependent on the exocyst. Potential molecular links of how these regulators
coordinate to mediate the exocyst localization are still under investigation. It is
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interesting to note that the absence of ongoing membrane traffic did not affect the
leading edge localization of the exocyst subunit Exo70, as revealed by the persistence
of its leading edge localization in cells treated with Brefeldin A (BFA), which inhibits
vesicular transport within the Golgi apparatus (Rosse et al., 2006). This is consistent
with the finding in budding yeast that the polarized localization of Exo70 (and Sec3)
is independent of actin cytoskeleton and on-going membrane traffic. It remains to be
determined whether the leading edge localization of other exocyst subunits is
sensitive to BFA treatment.
In migrating prostate tumor cells, the exocyst was shown to be co-localized
with cell–substrate paxillin-containing focal complexes that are newly formed within
protrusive cellular extensions (pseudopods) at the leading edge (Spiczka et al., 2008).
Biochemical studies also showed that the exocyst is associated with these
paxillin-containing complexes within pseudopods. Localization of the exocyst within
the pseudopods is dependent on Ral GTPases, which control the association between
Sec5 and paxillin.
e) Primary cilium
The exocyst components have also been detected at the basal body, a centriole
derived organelle, of the primary cilium in polarized epithelial cells (Rogers et al.,
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2004; unpublished data from Guo lab), reminiscent of the colocalization of the
exocyst with centrosomes in interphase cells (Chen et al, 2006).
1.2.4. Cellular functions of the exocyst
1.2.4.1. Functions of the exocyst in exocytosis
1. Budding yeast
Most of the exocyst subunits were originally discovered as SEC gene products
in budding yeast due to their essential function in secretion (Novick et al., 1980).
Temperature-sensitive mutants of the exocyst components are defective in the
secretion of invertase, which results in intracellular accumulation of the enzyme.
Furthermore, electron microscopic studies revealed the accumulation of 80-nm
Golgi-derived secretory vesicles in the mutant cells, suggesting that the exocyst
functions at the post-Golgi stage of exocytosis. To further define which step the
exocyst functions during post-Golgi trafficking, the vesicle accumulation patterns are
compared between the exocyst and myo2 mutants by electron microscopic analysis.
In budding yeast, during the apical and isotropic growth of the daughter cell,
post-Golgi secretory vesicles are delivered to the bud through actin cables by Myo2, a
yeast type V myosin. In myo2 mutants, secretory vesicles accumulate almost
exclusively in the mother cell. In contrast, in the exocyst mutants, the initial
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accumulation of secretory vesicles occurs within the bud of the cell, suggesting that
the transport of vesicles from donor compartments to the plasma membrane is not
affected. To further confirm this, the phenotype of the double mutant for Myo2 and
exocyst components was examined, which accumulates vesicles in the mother cell, as
does the myo2 single mutant (Govindan et al, 1995; Walch-Solimena et al, 1997).
Thus, the exocyst appears to function at a stage subsequent to the delivery of vesicles
to the vicinity of the plasma membrane. On the other hand, yeast genetic studies
showed that overexpression of Sso1, a yeast homolog of the mammalian t-SNARE
protein syntaxin, can suppress mutations in the exocyst mutants (Aalto et al., 1993).
In addition, in exocyst mutants, assembly of the SNARE complex is blocked (Grote
et al, 2000). Thus, the exocyst function is required for the assembly of the SNARE
complex and the fusion of the vesicles with the plasma membrane. Therefore, the
exocyst functions downstream of the vesicle transport machineries, but upstream of
the SNARE complex. It has been accepted in the field that the exocyst functions as a
molecular tether which mediates the initial contact of the secretory vesicles with the
plasma membrane prior to vesicle docking and fusion. This is also consistent with the
localization of the exocyst at the site of plasma membrane where active exocytosis
and membrane expansion occur.
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2. Mammalian cells
In animal cells, the role of the exocyst in exocytosis and cell surface expansion
has been demonstrated in a variety of cell types. Comparing to yeast cells, the
functions of the exocyst in mammalian cells are much more complex. While the yeast
exocyst mainly functions at the plasma membrane, the mammalian exocyst seems to
functions at multiple stages during exocytosis. Moreover, the mammalian exocyst is
not required for all types of exocytic events in animal cells. The following are several
examples of the functions of mammalian exocyst:
a) Epithelial cells
In fully polarized MDCK epithelial cells, addition of function-blocking Sec8
antibodies to streptolysin-O–permeabilized cells perturbed the membrane targeting of
the basolateral membrane protein LDL receptor (LDLR), but not the apical membrane
protein p75NTR. This is consistent with the localization of the exocyst to the apex of
the lateral membrane. Therefore, it has been proposed that the exocyst specifies the
delivery of vesicles to the basolateral domain, but not the apical domain in polarized
epithelial cells (Grindstaff et al., 1998). However, this hypothesis has been
challenged by the recent findings that the exocyst is involved in the targeting of
vesicles to both the apical and basolateral domains of the cell (Oztan et al., 2007;
Cresawn et al, 2007). Introducing Sec8 antibodies to partially permeabilized
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epithelial cells revealed exocyst requirements for several endocytic pathways
including
basolateral
recycling,
apical
recycling,
and
basolateral-to-apical
transcytosis by (Oztan et al., 2007). It was also found that expressing a
dominant-negative C-terminal fragment of Sec15 affected the exocytosis of newly
synthesized apical membrane domain marker endolyn, which is delivered en route
through the Rab11-positive apical recycling endosomes to the plasma membrane
(Cresawn et al, 2007). Although it is not clear whether the block of secretion is
imposed at the plasma membrane or the donor membrane (see below), these results
clearly indicate that the exocyst function is required for the targeting of a subset of
apical-targeted cargos.
In epithelial cells, the exocyst components were found to be localized to both
the plasma membrane and perinuclear membrane compartments, presumably TGN
and/or recycling endosomes (Yeaman et al., 2001; Prigent et al., 2003; Folsch et al.,
2003; Oztan et al., 2007). Blocking distinct pools of the exocyst by specific
antibodies resulted in defects in exocytosis at different stages. In non-polarized NRK
cells, selective perturbation of the TGN pool of the exocyst disrupted the transit of the
vesicular stomatitis virus G (VSVG) proteins to the plasma membrane, which
resulted in cargo accumulation in a perinuclear region. In contrast, selectively
perturbing the plasma membrane pool of the exocyst did not affect the transport of
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VSVG from TGN to the plasma membrane, but disrupted the plasma membrane
incorporation of the proteins, which resulted in cargo accumulation near the plasma
membrane (Yeaman et al., 2001). In polarized MDCK cells, vesicle budding was
reconstituted from apical recycling endosomes (ARE) in mechanically perforated
cells. Addition of antibodies against Sec8 resulted in a significant inhibition of cargo
release from the reconstituted ARE (Oztan et al., 2007), suggesting that the exocyst
may also play some role in steps that precede vesicle targeting, possibly including
cargo exit or vesicle transport. These results suggest that different pools of the
exocyst function in different vesicular trafficking stages: while the plasma membrane
pool functions in vesicle tethering, the perinuclear pool might function at an earlier
stage at the donor compartment.
b) Adipocytes
Insulin stimulates glucose transport in adipocytes and muscle by inducing the
redistribution of glucose transporter 4 (GLUT4) from intracellular locations to the
plasma membrane. GLUT4 trafficking is a highly regulated multi-step process that
includes translocation, targeting, docking and fusion of GLUT4-containing vesicles
with the plasma membrane. The first evidence to reveal the role of the exocyst in
GLUT4 transport is its interaction with the GTPase TC10 (Inoue et al., 2003). TC10
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interacts with Exo70 and recruits Exo70 as well as Sec6 and Sec8 to the plasma
membrane in response to insulin signaling, where the multiprotein complex is
assembled (Inoue et al., 2003; Ewart et al., 2005). Knockdowns of exocyst
components by RNA interference blocked insulin-stimulated glucose uptake (Inoue et
al., 2006). Overexpression of the N-terminus of Exo70 (Exo70-N), which acts as a
dominant negative mutant, inhibited the fusion of the Glut4 vesicles with the plasma
membrane, without affecting the transport of the vesicles to the cell periphery (Inoue
et al., 2003). In addition to TC10, another G protein RalA was also shown to function
in glucose transport downstream of insulin signaling through its interaction with the
exocyst (Chen et al., 2007). How these regulations are coordinated to mediate the
function of the exocyst still needs clarification.
The function of the exocyst in GLUT4 vesicle exocytosis requires its
association with lipid rafts. Upon insulin stimulation, the exocyst is recruited to the
lipid rafts and then assembled there, which sets up targeting sites for GLUT4 vesicles.
The anchoring of the exocyst at the lipid rafts is likely mediated by the interaction
between Sec8 and the PDZ domain-containing protein SAP97, a MAGUKs family
protein expressed in lipid rafts. Depleting SAP97, which prevented the lipid raft
recruitment of the exocyst, or disrupting the exocyst function, blocked the
translocation of Glut4 vesicles to the lipid raft and prevented the membrane
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incorporation of GLUT4 (Inoue et al, 2006). On the other hand, Exo70 was shown to
interact with Snapin, a ubiquitous protein known to associate with SNAP23, a major
t-SNARE regulating GLUT4 vesicle trafficking (Rea et al., 1998). Snapin was
predominantly targeted to the plasma membrane and depletion of Snapin in
adipocytes using RNA interference inhibits insulin-stimulated glucose uptake. Thus,
it is proposed that Exo70 associates with SNARE machinery through the interaction
with Snapin, which promotes the anchoring of Exo70 at exocytosis sites of GLUT4
vesicles (Bao et al., 2008). Collectively, these studies provide insights into the
mechanism of the targeting of the exocyst to GLUT4 vesicle exocytosis sites during
glucose uptake.
All the above studies used newly differentiated 3T3-L1 adipose cells to study
GLUT4 transport. Recently, a group investigated the effects of Exo70 on tethering
and fusion of GLUT4 vesicles in primary isolated rat adipose cells (Lizunov et al.,
2009). They utilized total internal reflection (TIRF) microscopy to visualize the
trafficking of GLUT4 vesicles, in which the tethering and fusion steps can be
observed separately. They found that overexpression of Exo70 in the presence of
insulin did not further promote the fusion rate and exposure of GLUT4. In sharp
contrast to the previous studies, the Exo70-N mutant induced tethering of GLUT4
vesicles independent of insulin, however it did not lead to fusion and exposure of
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GLUT4 at the plasma membrane. Upon insulin stimulation, the stationary
pre-tethered GLUT4 vesicles in Exo70-N mutant cells underwent fusion without
relocation. Taken together, their data suggest that fusion of GLUT4 vesicles is the
rate-limiting step regulated by insulin downstream of Exo70-mediated tethering.
The secretion of insulin itself is also dependent on the exocyst. In pancreatic β
cells, the exocyst is implicated in the secretion of insulin-containing dense core
vesicles. Overexpressing truncated, dominant negative exocyst mutants inhibited the
docking of the insulin vesicles to the plasma membrane and substantially reduced
insulin secretion in response to glucose stimulation. However, the final fusion step
was not affected by these mutants (Tsuboi et al., 2005).
c) Neurons
In developing neurons, the exocyst is essential for neurite extension and
synaptogenesis, processes that require active plasma membrane addition and
remodeling. Overexpressing a dominant negative fragment of Sec10 blocked neurite
outgrowth in cultured neuronal cells treated with nerve growth factor (NGF) (Vega
and Hsu, 2001). In contrast, the exocyst was revealed to be dispensable for
neurotransmitter release from synaptic vesicles at mature synapse (Hazuka et al.,
1999; Brown et al., 2001; Murthy et al., 2003). In sec5 mutants in Drosophila,
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synaptic transmission continued to be robust despite the decline of maternal Sec5
protein (Murthy et al., 2003). The function of the exocyst in neurite outgrowth and
synaptogenesis but not in synaptic vesicle exocytosis well corresponds to the
localization of the exocyst in developing neurons.
The molecular mechanisms underlying the function of the exocyst in neurons
are still under investigation. RalA was reported to regulate exocyst function in neurite
branching (Lalli and Hall, 2005). PAR-3 and atypical protein kinase C (aPKC) were
shown to associate with the exocyst in a RalA-dependent manner, which is essential
for early stages of neuronal polarization (Lalli, 2009). Moreover, TC10-Exo70
interaction was shown to be essential for membrane expansion and axonal
specification in developing neurons (Dupraz et al., 2009).
In neurons, the exocyst is also involved in the dendritic delivery of the NMDA
receptor
(NMDAR)
and
AMPA
receptors
(AMPARs)
to
postsynaptic
membrane/postsynaptic density (PSD), which is essential for synaptic transmission
and synaptic plasticity. The role of the exocyst in the delivery of NMDAR is thought
to involve interactions between Sec8, which contains a PDZ-binding domain at its
C-terminus, and the PDZ-containing proteins -- postsynaptic density protein-95 and
synapse-associated protein 102 (Riefler et al, 2003; Sans et al, 2003). Overexpressing
a Sec8 mutant defective in PDZ binding inhibited the surface exposure of the
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NMDAR (Sans et al, 2003). During AMPARs delivery, the exocyst function is
implicated in at least two stages. While overexpression of a dominant negative
C-terminal fragment of Sec8 abolished the transport of AMPARs toward PSD, a
dominant negative N-terminal fragment of Exo70 blocked the insertion of the
receptors to the plasma membrane (Gerges et al, 2006). This observation is consistent
with the notion that the exocyst functions in multiple vesicular traffic stages in
exocytosis.
1.2.4.2. Functions of the exocyst in endosomal recycling
In addition to functioning in biosynthetic secretory pathway, the exocyst has
also been implicated in the endocytic recycling pathway. Recycling endosomes
mediate the transport of the internalized plasma membrane receptors back to the cell
surface and are major sources of cargos destined to the plasma membrane in many
types of cells. The role of the exocyst in endocytic recycling is consistent with the
localization of the mammalian exocyst to the recycling endosomes. Loss of exocyst
function blocks the recycling of internalized cargos and results in their accumulation
in recycling endosomes. For example, depletion of Sec5 or dominant inhibition of
Sec10 affects the function and the morphology of the recycling pathway (Prigent et al,
2003). In polarized epithelial cells, disrupting the exocyst function by treating
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permeabilized cells with specific antibodies blocked multiple endocytic recycling
pathways, including basolateral recycling, apical recycling, and basolateral-to-apical
transcytosis (Oztan et al., 2007). Overexpression of Sec15 also blocked the recycling
of transferrin receptors and resulted in its accumulation in the recycling endosomes
(Zhang et al, 2004).
The exocyst has also been shown to be involved in endocytic recycling in other
organisms. Disrupting the functions of the Drosophila exocyst components Sec5,
Sec6, and Sec15 in epithelial cells leads to E-Cadherin accumulation in an enlarged
Rab11 recycling endosomal compartment and inhibits E-Cadherin delivery to the
membrane (Langevin et al., 2005). In Exo84 mutants at advanced stages of epithelial
degeneration in the Drosophila embryo, apical and adherens junction proteins
accumulate in an expanded recycling endosome compartment in the Drosophila
embryo (Blankenship et al., 2007). Drosophila oocytes homozygous for a sec5
hypomorphic mutant were defective in endocytic recycling of the yolk receptor and
blocked yolk uptake (Murthy and Schwarz, 2004; Sommer et al, 2005).
Recycling endosomes was also found to act as intermediates in biosynthetic
secretory pathway after the cargos exit the Golgi apparatus (Hoekstra et al, 2004;
Ang et al, 2004). Therefore, the roles of the exocyst in exocytosis and endocytic
recycling pathways are likely to be interrelated, and the exocyst could function
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analogously to mediate the exocytosis of newly synthesized cargos, or cargos
internalized from the plasma membrane for recycling.
1.2.4.3. Other functions of the exocyst
a) Cell polarity
Membrane traffic is a major contributing factor to cell polarization. Thus, the
exocyst, as a key component functioning in exocytosis, is highly involved in cell
polarity establishment.
In budding yeast, cell polarity is established through the localization and
activation of Cdc42 at the intrinsic bud site (for review, see Park and Bi, 2007).
Cdc42 mediates directional exocytosis and polarized cell growth through its
regulations of the components of exocytic pathways, such as the exocyst. In several
cdc42 mutants, the exocyst components fail to localize to the bud tip or
mother-daughter junction (Zhang et al, 2001). In turn, polarized exocytosis is also
required for the robust asymmetric localization of Cdc42, which is thought to be
associated with post-Golgi secretory vesicles and delivered to the bud tip through
exocytic/recycling endocytic pathways (Ayscough et al., 1997; Wedlich-Soldner et
al., 2003; 2004; Irazoqui et al., 2005; Zajac et al., 2005). Mutations in the exocyst
component Sec5 not only lead to mislocalization of Cdc42, but also of Bem1, a
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polarity establishment component which interacts with Cdc42 and its guanine
nucleotide exchange factor (GEF) Cdc24 (Zajac et al., 2005). Thus, the exocyst may
contribute to yeast cell polarity by reinforcing the localization of polarity
determinants, such as Cdc42, to the site of polarization, which in turn regulates
polarized exocytosis by controlling the polarity of actin cytoskeleton and the exocyst.
In addition, the exocyst component Sec15 was found to directly interact with Bem1
and recruit Bem1 as well as Cdc42 and Cdc24 to the bud tip (Zajac et al., 2005;
France et al., 2006). The interaction between Sec15 and Bem1 may also contribute to
this positive feedback loop. Collectively, these observations suggest that the exocyst
and the polarity-establishment complex coordinate to establish and maintain cell
polarity.
In multicellular organisms, cell–extracellular matrix (ECM) and cell–cell
interactions are important for polarity establishment. E-cadherin is one of the most
important junction proteins for the establishment and maintenance of the
apical-basolateral asymmetry in epithelial cells (Gumbiner et al. 1988; Uemura et al.
1996). The polarization of the epithelial cells might also apply a cyclical regulatory
mechanism analogous to the polarization of the yeast cells: E-cadherin engagement is
necessary for localization of the exocyst. Members of the exocyst complex are
recruited to the apex of the lateral membrane through the association with junctional
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complexes containing E-cadherin during the establishment of cell polarity in MDCK
cells (Grindstaff et al. 1998; Yeaman et al. 2004). In turn, E-cadherin itself is
delivered as a cargo via secretory vesicles to sites of polarity. Proper localization of
the exocyst in epithelial cells is necessary for the specific delivery of polarity
determinants including E-cadherin to the proper plasma membrane domains
(Grindstaff et al. 1998; Langevin et al. 2005); disruption of exocyst function led to
intracellular accumulation of E-cadherin (Langevin et al. 2005; Blankenship et al.
2007). In addition to this cyclical regulatory network, several exocyst components
were shown to affect epithelial polarity on their own. Overexpression of GFP-tagged
Exo70 significantly impaired the formation of tight epithelial monolayers, in which
the E-cadherin staining is much more diffuse compared with the crisp pattern in
parental cells (Matern et al., 2001). On the other hand, MDCK cells overexpressing
Sec10 were significantly taller than control cells when grown as a monolayer. In
addition, these cells formed cysts more efficiently and rapidly, and generated more
branching tubules from cysts upon HGF stimulation (Lipschutz et al., 2000).
b) Cytokinesis
Due to its role in targeted membrane addition through polarized exocytosis, the
exocyst function is essential for cytokinesis. In the budding yeast S. cerevisiae, the
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exocyst mediates vesicle fusion at the mother-daughter junction during cytokinesis.
Perturbing the exocyst function in anaphase cells resulted in accumulation of
secretory vesicles around the bud neck and impairs actomyosin-ring contraction and
cell cleavage (Novick et al, 1980; Salminen and Novick, 1989; Dobbelaere and
Barral, 2004; VerPlank and Li, 2005). In the fission yeast S. pombe, exocyst
components localize to the actomyosin ring (Wang et al., 2002). Mutants of the
exocyst component Sec8 accumulated 100nm secretory vesicles near the division
septum and cannot complete extracellular separation of the two daughter cells.
In mammalian cells, disruption of the exocyst function leads to cytokinesis
regression and abscission failure (Fielding et al, 2005; Gromley et al, 2005; Chen et
al, 2006). Reduction of exocyst levels by RNAi blocked the delivery of the recycling
endosome-derived vesicles to the cleavage furrow, a process important for membrane
addition during furrow ingression and abscission (Fielding et al, 2005). Disruption of
the exocyst also led to accumulation of exocytic vesicles at the midbody during
abscission (Gromley et at, 2005). Overall, these results suggest that the exocyst is
required for both furrow regression and abscission by mediating membrane
incorporation at cleavage furrow or midbody during cytokinesis of the animal cells,
which is well consistent with its localization at both sites.
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c) Cell migration and cell invasion
The exocyst has also been shown to be involved in cell motility. The initial step
of cell migration is the formation of cell protrusions in the direction of cell movement.
In mammalian cells, Sec5 was shown to be required for RalA-induced filopodia
formation (Sugihara et al., 2002). Overexpression of Exo70 promoted numerous
actin-based protrusive structures resembling filopodia (Wang et al., 2004; Zuo et al.,
2006), whereas inhibition of Exo70 by RNA interference (RNAi) or antibody
microinjection blocked the formation of actin-based membrane protrusions induced
by activated Cdc42 or Racl (Wang et al., 2004; Zuo et al., 2006; Pommereit and
Wouters, 2007). Depletion of Exo70 by RNAi affected various aspects of cell motility:
Exo70 knockdown prevented the recruitment of Arp2/3 complex to the leading edge
and impaired the formation of lamellipodia. As a result, the Exo70 RNAi treatment
affected cell migration in the wound healing assay and reduced the directional
persistence of individual migrating cells (Zuo et al., 2006). Similarly, depletion of
other exocyst components, including Sec5, Sec8, Sec 10 or Exo84, also affected cell
motility by reducing the velocity of cell migration (Rosse et al., 2006; Rosse et al.,
2009).
How does the exocyst contribute to cell migration is still under investigation.
First, the exocyst may contribute to cell migration via its vesicle tethering function in
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exocytosis. In prostate tumor cells, interference with exocyst activity impaired the
efficient exocytosis of newly synthesized α5-integrin to the plasma membrane and
inhibited tumor cell motility and matrix invasiveness (Spiczka and Yeaman, 2008).
On the other hand, it is also likely that the exocyst contributes to cell migration
through some novel mechanisms distinct from its principle role in vesicle tethering.
One such mechanism is to coordinate cytoskeleton remodeling with vesicle
trafficking. It was shown that Sec5-mediated filopodia can still form in the absence of
ongoing membrane trafficking processes (Sugihara et al., 2002). The exocyst has
been shown to associate with the Arp2/3 complex through direct interaction between
their subunits Exo70 and Arpcl, respectively (Zuo et al., 2006). Based on these
observations, it would be intriguing to examine whether the exocyst contributes to
cell migration by directly regulating actin dynamics.
d) Ciliogenesis
The exocyst has also been implicated in primary cilia formation. Sec10
overexpression resulted in increased ciliogenesis (Zuo et al., 2009). On the other hand,
deletion of Sec10 by short hairpin RNA (shRNA) blocked primary ciliogenesis (Zuo
et al., 2009). Similarly, depleting other exocyst components were also shown to
disrupt ciliogenesis (unpublished data from Guo lab).
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1.2.5. Regulation of the exocyst by the Ras family of small GTPases
Exocytosis is highly regulated in eukaryotic cells. Vesicle tethering, as the
initial contact of secretory vesicles with the specific domains of the plasma
membrane, is thought to confer specificity on the subsequent SNARE-mediated
fusion. Therefore, spatial and temporal regulation of the exocyst is crucial to
determining where and when exocytosis takes place. Consistent with this notion, the
exocyst has been found to be under the control of a number of small GTPases in a
variety of cells (for review, see Novick and Guo, 2002; He and Guo, 2009) (Figure
1.6).
1.2.5.1. Rab and Arf GTPases
Rab GTPases, constituting one of the most abundant families of Ras-like small
GTPases, are master regulators of membrane trafficking. There are at least 60 Rab
proteins and they are associated with distinct membrane compartments along both
exocytic and endocytic pathways and regulate vesicular trafficking at distinct stages
through their interactions with specific tethering proteins and cytoskeleton elements (for
reviews, see Pfeffer, 2001; Behnia and Munro, 2005; Grosshans et al., 2006).
The first GTPase found to interact with the exocyst is Sec4, the founding member
of the Rab family (Salminen and Novick, 1987). Sec4 regulates exocytic trafficking from
the TGN to the plasma membrane in budding yeast and was found to be associated with
secretory vesicles and the plasma membrane (Salminen and Novick, 1987; Goud et al.,
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1988). Similar to the exocyst, Sec4 also localizes to active growth sites during the yeast
cell cycle (Novick and Brennwald, 1993). The exocyst subunit Sec15 was found to
directly and specifically interact with the GTP-bound form of Sec4 through its effector
domain (Guo et al., 1999). Furthermore, loss of Sec4p function leaves the exocyst in a
partially assembled state (Guo et al., 1999). On the other hand, overproduction of Sec4p
partially suppressed the secretion defects in sec15 temperature-sensitive mutant
(Salminen and Novick, 1989). These observations support the hypothesis that the exocyst
is a downstream effector of Sec4: Sec4 interacts with the exocyst subunit Sec15 and
triggers further interactions between Sec15 and other exocyst components to promote
exocyst assembly, and eventually leads to targeting of secretory vesicles with specific
plasma membrane domains.
In addition to Sec4, Sec15 was also shown to interact with Sec2, the guanine
nucleotide exchange factor (GEF) of Sec4 (Medkova et al., 2006). Sec2 directly
interacts with the GTP-bound form of Ypt32, a Rab GTPase regulating vesicle
budding from the TGN (Jedd et al. 1997), and this interaction recruited Sec2 to
secretory vesicles. Sec15 and Ypt32 bind to Sec2 in a competitive manner, which
forms a negative feedback loop. A hypothesis has been proposed on the regulation of
the Rab signaling cascade: Ypt32 interacts with Sec2, and this interaction mediates
the recruitment of Sec2 to the post-Golgi secretory vesicles. Once on the vesicle,
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Figure 1.6
Figure 1.6 The exocyst is the downstream effector of a variety of Ras family of
small GTPases. Summary of the interactions between the exocyst and Ras-like small
GTPases. In budding yeast, the Rab GTPase Sec4 interacts with the Sec15 on the
secretory vesicles; Rhol and Cdc42 GTPases interact with Sec3; Rho3 and Cdc42
interact with Exo70. In mammalian cells, the Rab GTPase Rab11 interacts with
Sec15 at the recycling endosomes. The Rho GTPase TC10 interacts with Exo70. The
Ral GTPases, including RalA and RalB, interact with Sec5 and Exo84. Arf6 interacts
with Sec10.
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Sec2 activates Sec4, which in turn recruits Sec4 and its effector Sec15 to the
secretory vesicles, thereby releasing Ypt32 from Sec2 (Medkova et al., 2006).
Both in mammalian cells and Drosophila, Sec15 has been found to be a
downstream effector of Rab11 (Zhang et al., 2004; Wu et al., 2005; Langevin et al.,
2005; Beronja et al., 2005). Rab11 is a Rab GTPase that regulates vesicular transport
from recycling endosomes to the plasma membrane (Ullrich et al., 1996; Ren et al.,
1998). Sec15 directly interacts with the GTP-bound form of Rab11 through a single
alpha helix in its most C-terminal helical bundle (Zhang et al., 2004; Wu et al., 2005).
Consistent with the physical interaction, Sec15 and other exocyst components were
shown to be localized to Rab11-positive recycling endosomes in both mammalian and
Drosophila cells (Oztan et al., 2007; Langevin et al., 2005). The exocyst plays an
important role in endocytic recycling pathways, which likely involves its interaction
with Rab11. Disrupting the interaction between endogenous Sec15 and Rab11 by
overexpressing a dominant-negative Sec15 C-term fragment abolished transcytosis
from the basolateral apical domains in polarized MDCK cells (Oztan et al., 2007).
However, at which step does Rab11 regulate the exocyst function is still under
investigation: recruitment of the exocyst to the recycling endosomes or the assembly
of the exocyst or cargo release from recycling endosomes?
The homologues of Ypt32, Sec2 and Sec4 in mammalian cells are Rab11,
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Rabin8 and Rab8, respectively (Walch-Solimena et al., 1997; Hattula et al., 2002).
Recently, it has been shown that the “Rab cascade” is conserved from yeast to
mammals: Rab11, in its GTP-bound form, interacts with Rabin8 and kinetically
stimulates the guanine nucleotide-exchange activity of Rabin8 toward Rab8 (Knödler
et al., 2010). Furthermore, it has been suggested that these Rab GTPases coordinate
with each other in the regulation of vesicular trafficking during primary ciliogenesis
(Yoshimura et al., 2007; Nachury et al., 2007; Knödler et al., 2010). Since the
exocyst is the downstream effector of Rab11 and has also been implicated in
ciliogenesis (Zuo et al., 2009; unpublished data from Guo lab), it will be interesting
to elucidate the molecular connection between the Rab proteins and the exocyst
complex during primary ciliogenesis.
It is worthy noting that several other tethering factors have also been shown to
interact with specific Rab GTPases that regulate the corresponding membrane
trafficking stages. The regulation of specific tethering proteins by distinct sets of Rab
GTPases may be a common mechanism that assures membrane traffic fidelity.
The Arf (ADP Ribosylation Factor) proteins comprise a conserved family of
the Ras superfamily of small GTPases which act to regulate membrane traffic and
organelle structure (For review, see Gillingham and Munro, 2007). The major cellular
functions of Arf6 include uptake and recycling of plasma membrane proteins,
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organization of the actin cytoskeleton and membrane dynamics, which are essential
for cell spreading, cell motility and phagocytosis (For review, see D’Souza-Schorey
& Chavrier 2006). It has been shown that the exocyst subunit Sec10 acts as a
downstream
effector
of
Arf6
(Prigent
et
al,
2003).
Expression
of
a
constitutively-active Arf6 promotes the redistribution of Sec10 from the recycling
endosomes to the plasma membrane during cell spreading, whereas expression of a
dominant negative N-terminal Sec10 fragment interferes with Arf6-induced
membrane spreading (Prigent et al, 2003). Collectively, these results suggest that
Arf6 recruits the exocyst from the recycling endosomes to the plasma membrane to
promote membrane recycling toward specialized plasma membrane regions.
1.2.5.2. Rho GTPases
The Rho GTP-binding proteins are the key regulators of a wide range of
cellular processes including cytoskeleton organization, membrane trafficking, cell
polarization, cell growth and gene transcription (For review, see Bustelo et al., 2007).
In budding yeast, the Rho GTPase Cdc42 is a central regulator for the establishment
of yeast cell polarity during bud formation (For review, see Park and Bi, 2007). In
several cdc42 mutants, the exocyst components were depolarized (Zhang et al, 2001).
Biochemical analyses have revealed that Cdc42 in its GTP-bound state directly
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interacts with the amino terminus of Sec3 and regulates the polarized localization of
the exocyst. Disruption of this interaction leads to secretion and polarity defects
(Zhang et al. 2001; Zhang et al. 2008). In addition to Cdc42, the polarity of the
exocyst in budding yeast is also regulated by another Rho GTPase, Rho1 (Guo et al,
2001). Disrupting Rhol function led to mislocalization of the exocyst components
(Guo et al, 2001). Sec3 directly interacts with the GTP-bound Rhol through its amino
terminus, and functional Rho1 is required both to establish and to maintain the
polarized localization of Sec3 (Guo et al, 2001). Rho1 and Cdc42 compete with each
other for their binding to Sec3 in vitro (Zhang et al. 2001). In contrast to Cdc42
which functions in the establishment of cell polarity, the function of Rhol has been
implicated in the maintenance of polarized cell growth (For review, see Park and Bi,
2007). Therefore, Cdc42 and Rho1 may regulate Sec3 at different cell cycle stages or
physiological conditions. However, Sec3 is not the only mediator of the effect of Rho
GTPases on the exocyst, because some members of the complex are correctly
targeted independently of the interaction between Rho and Sec3, and no discernable
defects in exocytosis or cell growth were found with the deletion of the N-terminal
Rho-binding domain of Sec3 (Guo et al, 2001). These results reveal that additional
pathway(s) may also contribute to the polarized localization of the exocyst besides
the Rho-Sec3 interaction.
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While Sec3 is the downstream effector of both Rho1 and Cdc42, Exo70 has
been shown to be the downstream effector of another Rho GTPase, Rho3, which was
reported to regulate both exocytosis and actin cytoskeleton (Adamo et al, 1999;
Robinson et al, 1999; Dong et al, 2005). However, mutations in Exo70 that disrupt its
interaction with Rho3 did not affect polarized localization of exocyst components
(Adamo et al, 1999; Roumanie et al, 2005). The functional implication of
Rho3-Exo70 interaction still remains unclear. It was speculated that in addition to
Rho3, other factor(s) interact with Exo70 and regulate its activity. One such candidate
could be Cdc42, because the phenotypes of exo70 mutants and cdc42-6, a particular
mutant allele of Cdc42, are quite similar. Both of them are primarily defective in the
secretion of a subset of exocytic vesicles, which carry cargos such as the
endoglucanase Bgl2 that are needed for plasma membrane expansion and cell wall
remodeling. Furthermore, the secretion defect in exo70 and cdc42-6 mutants is most
pronounced at the early budding stage (He et al., 2007, Adamo et al., 2001). The
phenotypic similarity between cdc42-6 and exo70 mutants suggests that Exo70
functions downstream of Cdc42. However, a physical interaction between activated
recombinant Cdc42 and Exo70 was not detected (He et al., 2007a). Interestingly, a
recent study found that C-terminal prenylation of Cdc42 as well as Rho3 promotes
their interactions with Exo70 (Wu et al., 2010). Gain-of-function mutants in Exo70
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were also identified that potently suppress mutants in Rho3 and Cdc42 deficient in
exocytic function (Wu et al., 2010). Furthermore, several novel loss-of-function
mutants in Exo70 were isolated which displayed phenotypes consistent with the
notion that Exo70 acts as an effector for both Rho3 and Cdc42 function in polarized
exocytosis (Wu et al., 2010).
Rho proteins are also involved in polarized exocytosis in mammalian cells
(Kroschewski et al., 1999; Musch et al., 2001; Rogers et al., 2003). It has been shown
that Exo70 is a downstream effector of TC10, a Rho GTPase sharing sequence
similarity with Cdc42 (Inoue et al., 2003). Exo70 interacts with GTP-bound TC10
through its N-terminus. Expression of the active form of TC10 in adipocytes recruited
Exo70 from the cytoplasm to the plasma membrane. Overexpression of full-length
Exo70 led to increase in insulin-mediated glucose uptake. On the other hand,
overexpression of a dominant-negative N-terminal portion of Exo70 (Exo70-N)
blocked glucose uptake. Exo70-N did not affect the translocation of GLUT4. Instead,
it probably inhibited the targeting of GLUT4-containing vesicles to the plasma
membrane. This study reveals a molecular connection between the exocyst and Rho
protein in mammalian cells. The TC10-Exo70 interaction has also been found in
neuronal cells in response to NGF stimulation. Both Exo70 and TC10 antagonized the
activation of N-WASP by Cdc42, suggesting that the TC10-Exo70 interaction is
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involved in the regulation of actin cytoskeleton during formation of membrane
protrusions (Pommereit and Wouters, 2007). In addition, the TC10–Exo70 complex is
also implicated in membrane expansion and axonal specification in developing
neurons (Dupraz et al., 2009). In hippocampal pyramidal neurons in culture,
membrane expansion at the axonal growth cone is regulated by IGF-1 through a
cascade involving TC10 and the exocyst complex. TC10 and Exo70 are essential for
the polarized externalization of IGF-1 receptor, which is necessary for axon
specification (Dupraz et al., 2009).
1.2.5.3. Ral GTPases
Ral GTPases also belong to the Ras superfamily of small GTPases, which are
found only in animal cells and play an important role in signaling pathways. Ral
proteins have been implicated in the regulation of a diverse array of cellular processes
such as membrane traffic, actin-cytoskeleton dynamics, cell migration, cell
proliferation and oncogenesis (For review, see Feig, 2003). Ral has two isoforms:
RalA and RalB, which play distinct roles in the cell. Using yeast two-hybrid screens
and pull-down assays, the exocyst components Sec5 and Exo84 have been identified
as downstream effectors of RalA (Brymora et al., 2001; Moskalenko et al., 2002;
Polzin et al., 2002; Sugihara et al., 2002). Sec5 and Exo84 also bind to RalB but with
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a substantially lower affinity compared to RalA (Shipitsin and Feig, 2004).
Biochemical and structural studies indicate that Sec5 and Exo84 share overlapping
binding sites on the RalA effector-domain and compete for the binding to RalA
(Fukai et al., 2003; Mott et al., 2003; Jin et al., 2005). Depletion of RalA by siRNA
results in destabilization or disassembly of the exocyst complex, suggesting that Ral
might regulate exocytosis by promoting the proper assembly of the exocyst complex
(Moskalenko et al., 2002).
The functional implications of the Ral-exocyst interaction in exocytosis have
been extensively studied in various cell types. In polarized MDCK cells, inhibition of
the interaction between RalA and Sec5 using the Ral-binding domain (RBD)
prevented protein targeting to the basolateral domain (Moskalenko et al., 2002).
Expression of constitutively-activated RalA enhanced protein delivery to the
basolateral membrane, whereas expression of the RalA mutant which can not bind to
the exocyst failed to promote enhanced secretion (Shipitsin and Feig, 2004). Thus,
exocyst binding to active RalA is required for the Ral GTPases to regulate cellular
secretion. In neuroendocrine PC12 cells, both expression of activated Ral and
disruption of RalA-exocyst interaction using Sec5 RBD were found to block the
secretion of secretory granules (Moskalenko et al., 2002). In another study,
expression of wild type RalA, but not the RalA mutant deficient in Sec5 binding,
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enhanced GTP-dependent exocytosis. On the other hand, disruption of endogenous
RalA-exocyst interaction by adding Sec5 RBD inhibited GTP-dependent exocytosis
(Wang et al., 2004). In adipocytes, RalA is activated in response to insulin
stimulation and regulates the delivery of Glut4 vesicles to the plasma membrane
(Chen et al., 2007). Knockdown of RalA by RNAi or disruption of RalA-Sec5
interaction by overexpressing Sec5 RBD inhibited Glut4 transport (Chen et al., 2007).
Collectively, these findings strongly suggest that RalA binding to the exocyst is
required for RalA to regulate exocytosis. Since RalA has been found to be associated
with a variety of cellular compartments - the plasma membrane (Ngsee et al., 1991),
secretory vesicles (Bielinski et al., 1993; Mark et al., 1996; Chen et al., 2007) or
recycling endosomes (Shipitsin and Feig, 2004; Chen et al., 2006), it will be
interesting to test where and how RalA regulates the exocyst.
Since many cellular processes require functional exocytosis, the RalA-exocyst
interaction has been reported to function in many exocytosis-based cellular processes.
The exocyst has been implicated in epidermal growth factor (EGF)-induced
lamellipodia protrusions through RalA activation. Disruption of the Ral-exocyst
interaction by overexpressing the dominant negative Sec5 RBD inhibited
lamellipodia formation in COS cells in response to EGF stimulation (Yoshizaki et al.,
2006). In differentiating neurons, RalA is needed for cell body spreading and neurite
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branching, which seems to be mediated by the exocyst. Overexpression of wild type
RalA, but not a RalA mutant specifically defective in exocyst binding, stimulated
neurite branching (Lalli and Hall, 2005). In addition, exocyst is also involved in
neuronal polarity through interaction with RalA. Expression of a constitutively
activated RalA mutant that loses the ability to interact with the exocyst complex
resulted in non-polarized neurons (Lalli, 2009). During cytokinesis, the exocyst is
colocalized with RalA at the midbody. Knockdown of RalA or Sec8 led to
incomplete abscission and cytokinesis failure (Chen et al, 2006). During invasive cell
migration, the exocyst is colocalized with focal complex proteins within the
pseudopods through the interaction between Sec5 and paxillin which is dependent on
Ral GTPases (Spiczka and Yeaman, 2008). Overexpression of Ral-uncoupled Sec5
mutants or RNAi-mediated depletion of Ral or RalB inhibited the exocyst interaction
with paxillin which inhibited tumor cell motility (Spiczka and Yeaman, 2008).
Recently, the Ral-exocyst interaction has been implicated in membrane nanotube
formation, which is a new type of cell-cell communication dependent on the
formation of thin membranous nanotubes between adjacent cells (Hase et al., 2009).
Further investigations are needed to elucidate how RalA regulates exocyst function
during these processes.
In addition, the exocyst has also been implicated in several cellular processes
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that seem to be exocytosis-independent, such as RalA-induced filopodia formation
(Sugihara et al., 2002), RalA-induced human cell transformation (Lim et al., 2005),
and the anti-apoptotic function of RalA during the formation of Drosophila sensory
organ (Balakireva et al, 2006). How the exocyst functions in these processes remains
puzzled.
Even though RalA and RalB are 80% identical, the interaction of the exocyst
and RalB is implicated in distinct cellular processes compared to that of the exocyst
and RalA. The RalB-exocyst interaction was reported to function in the activation of
the atypical IkappaB kinase TBK1, a key mediator of the host defense upon virus
infection (Chien et al, 2006). RalB activation promotes the interaction between Sec5
and TBK1, which leads to the activation of TBK1 to initiate host defense pathway in
response to virus exposure. Intriguingly, during oncogenic transformation, the same
mechanism has been applied in cancer cells which antagonizes the apoptotic
programs and leads to tumor cell survival (Chien et al, 2006). The role of
RalB-exocyst interaction during cytokinesis is still under debate. One study showed
that in contrast to RalA, which is required for the recruitment of the exocyst to the
cytokinetic furrow in early cytokinesis, RalB is required for the targeting of the
exocyst to the midbidy during the late abscission step (Cascone et al., 2008).
However, another study showed that RalA on its own is sufficient for the
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relocalization of the exocyst proteins through the whole process of cell division
(Chen et al., 2006). The RalB-exocyst interaction has also been implicated in cell
migration (Rosse et al, 2006). In contrast to the study from Spiczka and Yeaman as
described above, in which RalA and RalB were both found to contribute to cell
motility (Spiczka and Yeaman, 2008), this study showed that RalB on its own drives
cell migration. They found that RalB activation and the RalB-Sec5 interaction were
enhanced under the conditions that promoted cell migration. RalB expression is
required for promoting both exocyst assembly and recruitment of the exocyst to the
leading edge. In addition, Knockdown of RalB or exocyst components largely
inhibited the velocity of cell migration. In contrast, RalA is neither activated by cell
motility, nor needed for the leading edge targeting of the exocyst during cell
migration (Rosse et al, 2006).
The apparent discrepancies of the roles of RalA and RalB reported by different
groups have not been solved yet. Different cell types or growth conditions may
partially account for these differences.
1.3. Phosphoinositides in cell regulation and membrane dynamics
1.3.1. Composition and spatial distribution of phosphoinositides
The biological membrane is mostly made of phospholipids, which have a polar
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group
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and
two
hydrocarbon
tails.
Among
the
Chapter 1
phospholipids,
phosphatidylinositol (PI) is a minor but important component of the inner leaflet
(Hokin 1985; Berridge et al., 1999). The polar head group of PtdIns is inositol, which
can be phosphorylated on specific carbons of the inositol ring. Reversible
phosphorylation of the inositol ring of PtdIns at positions 3, 4 and 5 results in the
generation of seven phosphoinositide species including phosphatidylinositol
phosphate -- PI(3)P, PI(4)P, PI(5)P, phosphatidylinositol bisphosphate -- PI(3,4)P2,
PI(3,5)P2, PI(4,5)P2, and phosphatidylinositol trisphosphate PI(3,4,5)P3.
Each of the seven phosphoinositides has a unique subcellular distribution:
PI(4,5)P2 and PI(3,4,5)P3 are mostly found at the plasma membrane, PI(4)P is
associated with the Golgi, PI(3)P and PI(3,5)P2 are associated with various types of
endosomes, and PI(3,5)P2 and PI(5)P are associated with multi-vesicular bodies (De
Matteis and Godi 2004; Behnia and Munro 2005; Di Paolo and De Camilli 2006).
The differential intracellular distribution of each of these phospholipids not only
contributes to defining the identities of these membrane compartments, but also
makes these lipids optimal signaling molecules in a diverse array of cellular processes
including cytoskeletal dynamics and vesicular trafficking.
The spatial restriction and steady-state levels of specific phosphoinositides are
achieved by a combination of localized synthesis by specific kinases and rapid
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turnover by phosphotases that prevent the lipid spreading between compartments. The
localizations of phosphoinositide kinases and phosphatases are tightly controlled. All
phosphoinositide kinases are themselves peripheral membrane proteins and, in many
cases, their localizations are regulated by organelle-specific GTPases. For example,
the endosomal PI-3-OH kinase (PI(3)K) Vps34 that makes PI(3)P is recruited by the
endosomal GTPase Rab5 (Murray et al., 2002), whereas the PI(5)K that makes PI
(4,5)P2 is recruited to the plasma membrane by Arf6 (Krauss et al., 2003).
1.3.2. Molecular mechanisms for phosphoinositides in protein targeting
Phosphoinositides are involved in a number of signaling events through the
binding of their head groups to cytosolic proteins or cytosolic domains of membrane
proteins. Thus, they can regulate the function of integral membrane proteins, or
recruit cytoskeletal and signaling components to the plasma membrane. Typically,
proteins bind to phosphoinositides through electrostatic interactions with the negative
charges of the phosphate(s) on the inositol ring. In some cases, adjacent hydrophobic
amino acids enhance the interaction through a partial embedding into the bilayer
(Lemmon, 2003; Balla, 2005). Protein surfaces that interact with phosphoinositides
are either composed of folded modules, such as the pleckstrin homology domain
(Lemmon, 2003; Balla, 2005; Hurley and Meyer, 2001), or of clusters of basic
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residues within unstructured regions, such as those found in many actin regulatory
proteins (for example, profilin) (Yin and Janmey, 2003). There is no clear distinction
between these two types. Furthermore, unstructured peptides can undergo folding
upon
binding
to
phosphoinositide
head
groups.
The
number
of
phosphoinositide-binding modules is rapidly expanding and their diverse properties
greatly amplify the signaling potential of phosphoinositides.
1.3.3. Cellular functions of PI(4, 5)P2
PI(4,5)P2, which is enriched in the plasma membrane, is involved in almost all
of the cellular events that occur at the cell surface. In addition, it plays a major part in
the transduction of extracellular signals, either through its metabolites or the
fluctuations of its own levels.
PI(4,5)P2 has an important role in endocytosis owing to its functions as a
co-receptor for the recruitment and regulation of endocytic proteins to the plasma
membrane (Wenk and Camilli, 2004; Owen et al., 2004). PI(4,5)P2 interacts with all
the known endocytic clathrin adaptors such as AP-2 and AP180/CALM as well as
many other endocytic factors including dynamin, which controls the fission reaction
(Gaidarov and Keen, 1999; Wenk and De Camilli, 2004; Owen et al., 2004).
PI(4,5)P2 is also implicated in exocytosis because it plays a critical role at a
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pre-fusion stage, also known as the vesicle priming step. It acts both in cis on plasma
membrane proteins and in trans on vesicle proteins (Martin, 1998; Bai et al., 2004).
Importantly, in-trans interactions probably cooperate with SNARE pairing in
marking the plasma membrane as the appropriate partner for the fusion of these
organelles.
PI(4,5)P2 along with small GTPases, participates in the activation of a variety
of actin regulatory proteins at the plasma membrane, thus regulates cell motility,
cytokinesis and a wide variety of other processes. A well-characterized mechanism
regulating actin assembly that involves PI(4,5)P2 is the Arp2/3-mediated nucleation
of branched actin networks. PI(4,5)P2, in concert with the small GTPase Cdc42, binds
N-WASP and releases its autoinhibition which allows its binding to and activation of
the Arp2/3 complex (Pohatgi et al., 2000; Pollard and Borisy 2003).
1.4. Cell invasion and invadopodia structures
When cultured on a flat extracellular matrix substratum, invasive tumor or
transformed cells extend actin-rich protrusions emanating from their ventral surfaces
into the matrix and displaying focalized proteolytic activity towards the substrate.
1.4.1. The identification and general features of invadopodia
Invadopodia were first discovered by Wen-Tien Chen in fibroblast cells
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transformed by the v-src oncogene, which encodes a constitutively active
non-receptor tyrosine kinase (v-Src) (Chen, 1989). Src-transformed fibroblasts grown
on fibronectin formed ventral protrusions with degradative properties. Furthermore,
vSrc was found to co-localize with the sites of fibronectin degradation (Chen et al.,
1985). In addition to Src-transformed fibroblast cells, invadopodia have also been
found in cell lines or primary tumor cells from malignant melanoma (such as
A375MM), breast/mammary carcinoma cells (such as MDA-MB-231) and other
malignant tumor cells (Baldassarre et al., 2003; Artym et al., 2006; Chuang et al.,
2004).
There are several criteria to recognize invadopodial structures under the light
microscope: 1) roundish actin-rich protrusions at the ventral surface of the cell, 2)
sites with the colocalization of cortactin and phosphotyrosine (Bowden et al., 2006),
3) not confined to the cell periphery, and 4) are associated with the degradation of the
extracellular matrix (Artym et al., 2006). There are other features that can be used to
identify invadopodia at least in some cell lines, such as their localization proximal to
the Golgi apparatus (Baldassarre et al., 2003) and their extended half-life of up to 2 h
or more (Yamaguchi et al., 2005; Baldassarre et al., 2003) as compared to other
protrusive adhesions.
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1.4.2. Structure of invadopodia
The description of the ultrastructural features of invadopodia still remains
incomplete. Through transmission electron microscopy, Chen made an initial
observation on the invadopodial structures in transformed fibroblasts and found that
they are thin protrusions extending from the plasma membrane into the underlying
ECM (Chen, 1989). Later, a detailed ultrastructural analysis was carried out in the
melanoma cell line A375MM using a correlative confocal light electron microscopy
technique, through which individual areas of ECM degradation with matching
invadopodia were first identified under the light microscope, analyzed using the
electron microscope and reconstituted in three dimensions (Baldassarre et al., 2003).
In this study, invadopodia were shown to be originated from profound invaginations
of the ventral plasma membrane surface, which averaged 8 µm in width and 2 µm in
depth. From these invaginations, many surface protrusions emanated and sometimes
penetrated into the matrix, which exhibited lengths of about 500 nm and diameters
ranging from hundreds of nanometers to a few micrometers. These protrusions were
consistent with the “invading” structures originally described by Chen. However,
they seemed to be part of more complex superstructures. More recently, the
connections between invadopodial protrusions and the cell body were revealed by
electron microscopy tomography experiments (Baldassarre et al., 2006). They found
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that the Golgi complex is always polarized in a direction towards the invadopodial
area, suggesting a tight relationship between proteolytic activity and membrane/
protein transport.
1.4.3. Molecular components of invadopodia
1.4.3.1. Initial events at the cell-ECM interface
It has been accepted that the initiation of invadopodia formation is largely
triggered by the engagement of cell surface integrins through ECM components
(Nakahara et al., 1996& 1997; Mueller et al., 1999). α3β1 and α5β1 integrins were
the first two pairs shown to be involved in the formation of invadopodia, albeit the
specific integrin combination might be cell-type dependent. Integrin activation leads
to the activation of the Rho family of GTPases to regulate actin cytoskeleton, thus
promoting membrane-protrusive and proteolytic activity, leading to invadopodia
formation and cell invasion. In addition to their role in signaling, integrins also
spatially and temporally regulate metalloprotease activity, so to focalize the
degradation process. For example, integrins can coordinate with the membrane-type
matrix metalloprotease MT1-MMP to regulate membrane proteolytic activity through
the activation of matrix prometalloproteinase-2 (MMP2) (Deryugina et al., 2001). In
some cases, invadopodia formation can also be triggered by activation of the EGF
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receptor followed by activation of the signaling cascade leading to activation of the
actin polymerization machinery (Yamaguchi et al., 2005).
1.4.3.2. Signaling molecules in invadopodia
Many molecular effectors have been characterized in the integrin
engagement-initiated signaling cascade in invadopodia, including protein kinases,
small GTPases and effector proteins.
a) Protein kinases
Invadopodia were initially identified as specialized membrane protrusions
enriched in Src non-receptor tyrosine kinase (Src) (Chen 1989). Cells failed to initiate
invadopodia formation and matrix degradation when treated with tyrosine
phosphorylation
inhibitors
(Bowden
et
al.,
2006).
On
the
other
hand,
constitutively-active Src was found to be able to stimulate invadopodia formation in
fibroblasts and breast carcinoma cells (Chen et al., 1985; Artym et al., 2006).
Therefore, Src activity is essential for invadopodia formation and matrix degradation.
Recently, several classes of serine/threonine kinases have been implicated in
invadopodia formation. For example, protein kinase D (PKD), a family of
serine/threonine protein kinases involved in a variety of fundamental functions
including vesicular trafficking, cell shape, cell motility and adhesion (Rykx et al.,
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2003; Wang, 2006), was the first serine/threonine kinase to be implicated in
invadopodia activity (Bowden et al., 1999). ERK1/2, part of the mitogen-activated
protein kinase (MAPK) pathways, has also been connected to invadopodia biogenesis
(Tague et al., 2004; Ayala et al., 2008). However, how these kinases contribute to
invadopodia formation remains to be clarified.
b) Small GTPases
Cdc42, Rac and Rho GTPases have all been shown to be involved in
invadopodia formation albeit in various cell types. While depletion of Cdc42 by
RNAi or overexpression of a constitutively inactive Cdc42 mutant inhibited
invadopodia formation in the metastatic MTLn3 adenocarcinoma cell line
(Yamaguchi et al., 2005), overexpression of a constitutively active mutant of Cdc42,
but not Rac or Rho, promoted dot-like degradation in RPMI17951 melanoma cells
(Nakahara et al., 2003). In addition, transfected Cdc42 but not RhoA or Rac, was
detected at invadopodia in A375MM melanoma cells through immunofluorescence
microscopy. However, In SNB19 and U87MG glioma cells, depletion of Rac1 by
siRNA disrupted lamellipodia and invadopodia formation (Chuang et al., 2004). In
another study, endogenous active form of RhoA was shown to be localized in
invasive protrusions of Src transformed NIH 3T3 fibroblasts (Berdeaux et al., 2004).
Further investigations are required to obtain a clearer understanding of the functions
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of Rac1, Cdc42 and RhoA during invadopodia formation.
ARF6 has been found to be localized at invadopodia in MDA-MB-231 breast
cancer cells (Hashimoto et al., 2004) and in LOX melanoma cells (Tague et al., 2004).
Disruption of Arf6 by RNAi or expression of GTP-binding deficient mutants blocked
ECM degradation and matrigel invasion. Furthermore, ARF6 activity at invadopodia
was shown to be dependent on ERK activation (Tague et al., 2004).
c) Effector proteins
The multi-domain actin-binding protein cortactin is one of the key effector
proteins during invadopodia formation. Cortactin was first found to be associated
with invadopodia in a complex with paxillin and PKD (Bowden et al., 1999), and
later shown to be essential for invadopodia formation (Artym et al., 2006). Live cell
imaging of c-src-expressing carcinoma cells showed that aggregation of cortactin to
the invadopodial initiation site is an early step during invadopodia formation, which
is followed by MT1-MMP targeting to these sites and the subsequent degradation of
underlying matrix substrates (Artym et al., 2006). It was proposed that cortactin may
couple actin assembly to the secretory machinery through the regulation of MMP
secretion (Clark et al., 2007). A recent study showed that multiple phosphorylation
events by Src, ERK1/2 and PAK(s) converge onto cortactin and are crucial for the
regulation of invadopodia function (Ayala et al., 2008).
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Dynamin 2 was recently found at invadopodia-forming sites and colocalized
with other invadopodia markers as F-actin, cortactin and phosphotyrosine. It was also
shown to be required for invadopodia function (Baldassarre et al., 2003 McNiven et
al., 2004). The novel Src substrate Tks5, a scaffold protein containing five SH3 and
one PX domains, was recently identified at invadopodia of Src-transformed
fibroblasts and several cancer cell lines and shown to be required for invadopodia
formation and function (Abram et al., 2003, Seals et al., 2005).
In addition, many components of the actin-based membrane remodeling
machinery have been reported to be involved in invadopodia biogenesis, which
include the Arp2/3 complex, N-WASP (Wiskott-Aldrich syndrome protein) (Mizutani
et al., 2002; Yamaguchi et al., 2005), and the actin depolymerizing factor cofilin
(Yamaguchi et al., 2005).
1.4.3.3. Degradation of the ECM during invadopodia formation
The proteolytic activity of invadopodia is mainly due to members of the matrix
metalloprotease (MMP) family. MMPs are synthesized as inactive proenzymes and
become activated through proteolytic removal of a prodomain. MMPs are either
secreted from the cell (such as MMP-2 and MMP-9) or anchored to the plasma
membrane as integral proteins (membrane-type MMPs). MMP-2 was the first MMP
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found to be enriched at invadopodia which led to the recognition of invadopodia as
protease-rich membrane protrusions (Monsky et al., 1993). MT1-MMP has been
considered as a master regulator of the degradative activity of invadopodia in a
variety of cell types (Nakahara et al., 1997; Clark et al., 2007). MMP-9 was also
found at invadopodial structures such as those of the metastatic breast cancer cells
(Bourguignon et al., 1998) and leukemia cells (Redondo-Muñoz et al., 2006).
Metalloproteases are crucial for invadopodia formation and cell invasion.
However, how they are specifically targeted to invadopodia is still unclear. The
secretory pathway might play an important role, as persistent ECM degradation
requires an active transport of protease-delivering carriers to specific sites of ECM
degradation. But it still remains elusive how this transport is organized.
1.4.4. Invadopodia vs podosomes
Another similar structure, termed podosome, is formed by a number of normal
cell types including osteoclasts, smooth muscle cells and macrophages (Linder, 2007).
Invadopodia and podosomes share many structural and functional characteristics,
including a dependence on actin assembly and Src kinase signaling (Gimona and
Buccione, 2007; Buccione et al., 2004). However, they differ in size, turn-over rate,
cell types and molecular components that are involved (Linder, 2007). It is still
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unclear whether podosomes and invadopodia are distinct structures or whether they
are two similar structures of the cellular response to contact with the substratum.
1.5. Thesis overview
The exocyst, as a vesicle tethering machinery, regulates vesicle trafficking at
the vicinity of the plasma membrane. Although genetic, cell biological and
biochemical studies in various systems have shown important roles of the exocyst in a
multitude of developmental processes, many uncertainties still exist in the field. I am
asking two fundamental questions in my thesis work: how does the exocyst mediate
vesicle tethering to the plasma membrane? What is the role of the exocyst in cell
migration and tumor cell invasion?
My thesis has three major parts that describe the studies on exocyst functions in
various aspects. Chapter 2 presents a molecular mechanism of how the exocyst
associates with the plasma membrane. Chapter 3 describes the role of the exocyst in
tumor cell invadopodia formation by mediating the secretion of matrix
metalloproteinases at focal degrading sites and regulating Arp2/3-mediated actin
dynamics. Chapter 4 further discusses the molecular mechanism of how the exocyst
regulates actin assembly and contributes to cell migration and cell invasion.
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Chapter 2
Chapter 2. Phosphatidylinositol 4,5-bisphosphate mediates the targeting of
the exocyst to the plasma membrane for exocytosis in mammalian cells
Abstract
The exocyst is an evolutionarily conserved octameric protein complex that
mediates the tethering of post-Golgi secretory vesicles at the plasma membrane for
exocytosis. To elucidate the mechanism of vesicle tethering, it is important to
understand how the exocyst physically associates with the plasma membrane (PM).
In this study, I report that the mammalian exocyst subunit Exo70 associates with the
PM through its direct interaction with phosphatidylinositol 4,5-bisphosphate
(PI(4,5)P2). Furthermore, I have identified key conserved residues at the C-terminus
of Exo70 that are crucial for the interaction of Exo70 with PI(4,5)P2. Disrupting
Exo70-PI(4,5)P2 interaction abolished the membrane association of Exo70. I have
also found that wild-type Exo70 but not the PI(4,5)P2-binding–deficient Exo70
mutant is capable of recruiting other exocyst components to the PM. Using the ts045
vesicular stomatitis virus glycoprotein trafficking assay, I demonstrate that
Exo70-PI(4,5)P2 interaction is critical for the docking and fusion of post-Golgi
secretory vesicles, but not for their transport to the PM.
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2.1. Introduction
Exocytosis is essential in a variety of cellular functions, ranging from the
release of hormones to the incorporation of membrane proteins for cell growth and
morphogenesis. The late stage of exocytosis is a multistep process including
directional transport, tethering, docking, and fusion of post-Golgi secretory vesicles
with the plasma membrane (PM). The tethering step, defined as the initial contact of
secretory vesicles with the PM prior to SNARE-mediated docking and fusion (Pfeffer,
1999; Guo et al., 2000; Waters and Hughson, 2000; Whyte and Munro, 2002), is
mediated by the exocyst, an evolutionarily conserved octameric complex composed
of Sec3, Sec5, Sec6, Sec8, Sec10, Sec15, Exo70, and Exo84 (for review, see Guo et
al., 2000; Hsu et al., 2004; Munson and Novick, 2006; Wang and Hsu, 2006). In
budding yeast, the exocyst components localize to the growing end of the daughter
cell ("bud tip"), where active exocytosis and membrane addition take place (TerBush
and Novick, 1995; Finger et al., 1998, Guo et al., 1999). This localization pattern
contrasts that of the membrane fusion machine, the t-SNAREs, which are evenly
distributed along both the mother and daughter cell membrane (Brennwald et al.,
1994). In mammalian cells, the exocyst components were found in the cytosol,
recycling endosomes and trans-Golgi network (Yeaman et al., 2001; Fölsch et al.,
2003; Ang et al., 2004; Langevin et al., 2005). However, they are recruited to the PM
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during a number of cellular processes. For example, in epithelial cells, the exocyst is
recruited to the adherens junction region upon cell–cell contact, where it mediates
protein and membrane addition at the basolateral domain (Grindstaff et al., 1998;
Yeaman et al., 2001); in developing neurons, the exocyst is localized to the growing
neurites, where it mediates membrane expansion (Hazuka et al., 1999; Vega and Hsu,
2001); during cell migration, the exocyst is recruited to the leading edges of the PM
(Rosse et al., 2006; Zuo et al., 2006).
The mechanism by which the exocyst mediates vesicle tethering to the PM is
unclear. One key question yet to be resolved is how the exocyst itself associates with
the PM. Using fluorescence recovery after photobleaching (FRAP) analyses and
immunoelectron microscopy, Boyd et al. (2004) have shown that Exo70 is stably
localized to the yeast bud tip membrane and remains polarized even when the actin
cables are disrupted, suggesting that Exo70 is a candidate in this complex involved in
membrane targeting of the exocyst. In MDCK cells, extragenically expressed
GFP-tagged Exo70 is localized to the PM near cell–cell contacts, suggesting that
Exo70 may mediate PM association independent of the rest exocyst components in
these cells (Matern et al., 2001). Recent structural studies have revealed that Exo70
contains a number of conserved basic residues that cluster on a surface patch at the
C-terminal end of the tertiary structure that may directly bind to the PM (Dong et al.,
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2005; Hamburger et al., 2006; Moore et al., 2007). In fact, the C-terminal sequence of
Exo70 is the most evolutionarily conserved region of this protein.
Here, I report that mammalian Exo70 directly interacts with PI(4,5)P2 in the
PM through the positively charged residues at its C-terminus. I have also identified
key residues in Exo70 that are crucial for this interaction. Finally, using the ts045
vesicular stomatitis virus glycoprotein (VSV-G) trafficking assay, I found that the
Exo70-lipid interaction is critical for PM stages of exocytosis, but not for the
trafficking steps through ER and Golgi. My study revealed a molecular mechanism
by which the exocyst directly interacts with the PM that is critical for vesicle
tethering and exocytosis.
2.2. Results
Association of Exo70 with the Plasma Membrane in HeLa Cells
I have tested the localization of GFP-tagged rat Exo70 in HeLa cells. As shown
in Figure 2.1A, GFP-Exo70 was enriched at the PM as revealed by serial optical
sectioning from different axes (Figure 2.1A). In addition, cells expressing
GFP-Exo70 exhibited filopodia-like structures as previously reported (Wang et al.,
2004; Xu et al., 2005; Zuo et al., 2006). In order to map the region of Exo70 that is
required for its association with the PM, I examined the localization of a series of
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GFP-tagged Exo70 truncates in HeLa cells. As shown in Figure 2.1B, Exo70 with its
C-terminus deleted (amino acids 1-408; Exo70-∆C) was cytosolic, whereas the
C-terminal domain Exo70 (amino acids 403-653; Exo70-CT) was enriched at the PM
(Figure 2.1B), suggesting a critical role of Exo70 C-terminus in membrane
association. The crystal structure of yeast Exo70 revealed that this protein is a long
rod composed mainly of α-helices that fold into four domains (named domains A, B,
C, and D; Dong et al., 2005 ; Hamburger et al., 2006). Domain D (the C-terminal
114 amino acids) is the most evolutionarily conserved domain in Exo70 that contains
a number of basic residues that cluster into an electro-positive surface patch at the
C-terminal tip of the rod. Because in many cases the association of proteins with the
PM can be mediated by interactions with the negatively charged phospholipids in
membrane via clusters of basic residues (for reviews, see McLaughlin et al., 2002;
Balla, 2005), it is likely that mammalian Exo70 directly associates with the PM
through those positively charged residues at its C-terminus. I sought to examine
whether domain D is required for the PM localization of Exo70 by generating
truncations of Exo70, in which the last 95 amino acids (aa 559-653) or the last 45
amino acids (aa 609-653) were deleted. The resulting Exo70 fragments, Exo70 (aa
1-558) and Exo70 (aa 1-608), were both cytosolic (Figure 2.1B), indicating that
domain D of mammalian Exo70, especially the last 45 amino acids, is necessary for
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Figure 2.1 Association of Exo70 with the plasma membrane (PM) in HeLa cells.
(A) GFP-Exo70 was detected at the PM. HeLa cells were transfected with
GFP-tagged Exo70, fixed, and observed using a confocal microscope. Serial optical
sections in the x-z and y-z planes were taken. (B) Various truncates of Exo70 were
tested for their localization at the PM. HeLa cells were transfected with GFP-tagged
Exo70 and Exo70 truncates as diagramed. The cells were fixed, stained, and observed
under microscope. Full-length and the C-terminal fragments of Exo70 containing
domain D (aa 558-653) are associated with the PM. +, membrane association.
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the association of Exo70 with the PM. I have also examined the localization of Exo70
(aa 403-558) and Exo70 (aa 403-608), which are C-terminal fragments of Exo70 with
the last 95 amino acids (aa 559-653) or the last 45 amino acids (aa 609-653) deleted,
and found that both of the fragments were cytosolic (Figure 2.1B). I next tested
whether the C-terminal fragments of Exo70 containing aa 403-653 or aa 540-653
(domain D) were able to associate with the PM. As shown in Figure 2.1B, both
fragments were localized to the PM, suggesting that domain D of Exo70 is sufficient
for its PM localization. A portion of these fragments was also found in the nucleus,
probably resulting from nonspecific retention of the GFP fusion. Collectively, these
data suggest that the C-terminus of Exo70 is both necessary and sufficient for the PM
localization of Exo70.
Exo70 Directly Interacts with Phospholipids through Its C-Terminus
Based on sequence analysis, domain D of Exo70 contains a number of
positively charged residues that are well conserved in yeast. Moreover, based on the
crystal structure, the basic residues cluster onto a surface patch at the C-terminal tip
in the folded Exo70 protein. Therefore, it is likely that Exo70 directly interacts with
negative charged phospholipids through this basic patch. To test this hypothesis, I
examined the binding between recombinant GST-Exo70 and various phospholipids by
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cosedimentation assay using the methods as previously described (Papayannopoulos
et al., 2005; Hokanson and Ostap, 2006). PI(4,5)P2 is the major phosphatidylinositol
species which comprises 1–5% of the total lipids in the PM (for review, see
McLaughlin et al., 2002). Binding assays were carried out with large unilamellar
vesicle (LUVs) composed of the neutral PC and PI(4,5)P2 at 5% or the acidic
phospholipid PS in molar percentages of 20, 40, and 60%. Because the recombinant
Exo70 protein could only access the outer leaflet of the reconstituted LUV vesicles,
the effective PI(4,5)P2 and PS in the binding reaction were only half of the total. As
shown in Figure 2.2A, Exo70 bound to LUVs containing 5% PI(4,5)P2, but not to
LUVs containing 100% neutral PC. While Exo70 also bound to PS, the binding was
weak unless the molar ratio of PS in the LUVs was raised to 60%. As a control, GST
did not bind to LUVs with any lipid composition.
I have also measured the affinity of Exo70 for various lipids (Figure 2.2B). The
bound Exo70 was quantified and plotted against the lipid concentration with the
equation: B = BmaxX/[Kd + X], where Kd is the dissociation constant and X and B
represent the concentrations of the free Exo70 and the bound Exo70, respectively.
The Kd was calculated by nonlinear regression. As shown in Table 1, Exo70 bound to
LUVs composed of 5% PI(4,5)P2 with a Kd of 13.9 ± 3.0 µM and bound to LUVs
composed of 60% PS with a Kd of 46.3 ± 9.7 µM. The Kd value for PI(4,5)P2 would
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Figure 2.2 The interaction between Exo70 and phospholipids in vitro. (A)
GST-Exo70 purified from bacteria (0.15 µM) was incubated with liposomes (200 µM)
containing 100% PC, 5% PI(4,5)P2, 20% PS, 40% PS, and 60% PS. After
centrifugation at 150,000 x g for 30 min, the proteins in supernatant (S) and pellet (P)
were subjected to SDS-PAGE and visualized by SYPRORed staining. Exo70 bound
to vesicles containing 5% PI(4,5)P2 or 60% PS. It also bound weakly to vesicles
containing 40% PS. GST did not bind to liposomes with any lipid composition in the
cosedimentation assay. (B and C) The interaction of Exo70 with phospholipids.
Exo70, 0.15 µM, was incubated with increasing concentrations of LUVs composed of
(B) 100% PC, 5% PI(4,5)P2, 20% PS, 40% PS, and 60% PS or (C) PI(3)P, PI(4)P,
PI(3,5)P2, PI(4,5)P2, and PI(3,4,5)P3. The percentage of bound Exo70 was plotted
with the increasing liposome concentration with a single rectangular hyperbola
equation (B = BmaxX/[Kd + X]) using SigmaPlot. Each point is the average of three
measurements. Error bars, SD.
Table 1. Comparison of the affinities of Exo70 and exo70-1 for phospholipids.
LUV Composition
100% PC
20% PS
60% PS
5% PIP2
Kd (µM) for Exo70
>> 400
>> 400
46.30±9.7
13.92±3.0
Kd (µM) for exo70-1
>> 400
>> 400
146.23±14.6
>> 400
* The Kd was obtained by rectangular hyperbolae equation using the SigmaPlot software. For
some of the bindings, the Kd was >> 400 because the binding never reached saturation.
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be much smaller if expressed in terms of pure PI(4,5)P2, because the molar ratio of
PI(4,5)P2 is only 5% of the total lipids in the reconstituted LUVs. Overall, these
results suggest that Exo70 may associate with the PM through its direct interaction
with PI(4,5)P2.
To determine the specificity of Exo70-PI(4,5)P2 interaction, I have tested the
binding of Exo70 to four additional phosphoinositides: PI(3)P, PI(4)P, PI(3,5)P2, and
PI(3,4,5)P3. In mammalian cells, PI(4)P and PI(4,5)P2 are the two most abundant
phosphoinositides enriched in the Golgi and PM, respectively (Di Paolo and De
Camilli, 2006). PI(4,5)P2 constitutes more than 95% of the bis-phosphoinositides in
mammals (Bonangelino et al., 2002). PI(3)P and PI(3,5)P2 are mainly localized to the
endosomal compartments (for reviews, see Behnia and Munro, 2005; Di Paolo and
De Camilli, 2006). PI(3,4,5)P3 is present in negligible amount in mammalian cells at
the "resting" state, but is up-regulated at specific PM domains in response to certain
stimuli (Insall and Weiner, 2001). Recently, PI(3,4,5)P3 was found to be localized to
the basolateral domain of MDCK cells (Gassama-Diagne et al., 2006). As shown in
Figure 2.2C, Exo70 hardly binds to PI(3)P; the affinity of Exo70 for PI(4,5)P2 (Kd =
13.92 ± 3.0 µM) is ~4–5-fold higher than PI(3,5)P2 (Kd = 58.34 ± 9.2 µM) and more
than 10-fold higher than PI(4)P (Kd = 173.12 ± 15.5 µM). The affinity of Exo70 for
PI(3,4,5)P3 (Kd = 15.55 ± 4.6 µM) is similar to that of PI(4,5)P2.
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Mutations in Domain D Disrupt the Plasma Membrane Association of Exo70
The direct binding of Exo70 with PI(4,5)P2 suggests a critical role for the
domain D basic residues in the PM targeting of Exo70. To determine the responsible
residues that are essential for Exo70 to bind to the PM, the conserved basic residues
in domain D of Exo70 (Figure 2.3A) were targeted for mutagenesis and the resulting
mutants were tested for their cellular localization (summarized in Table 2). Among
the 10 mutants I tested, 6 failed to associate with the PM. The other four mutations
had no effect on Exo70 localization, suggesting that not all of the basic residues are
required for PI(4,5)P2 binding. I focused on one of the mutants, named exo70-1, in
which residues K632 and K635 have been mutated to alanine. As shown in Figure
2.3B, GFP-tagged wild-type Exo70 (GFP-Exo70) was enriched at the PM, whereas
GFP-tagged exo70-1 was distributed diffusely throughout the intracellular regions.
These results indicate that K632 and K635 are necessary for the PM localization of
Exo70.
To directly test whether K632 and K635 are required for the physical
association of Exo70 with the PM, I performed subcellular membrane fractionation
assay using HeLa cells transfected with GFP-tagged wild-type Exo70 or exo70-1.
Cell lysates were fractionated, and the amount of Exo70 or exo70-1 in the PM,
cytosol, and other fractions was examined. As shown in Figure 2.3C, wild-type
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Exo70 was present in the PM fraction, whereas exo70-1 was mostly found in the
cytosol. The amount of Exo70 and exo70-1 in the HDM (mostly ER membrane) and
LDM (mostly Golgi and endosomal compartments) fractions was almost the same.
During my studies, I have noticed that GFP-tagging of Exo70 may promote its
translocation from cytosol to the PM. It is likely that GFP-tagging induces
conformational changes on Exo70, exposing the lipid-binding site on Exo70; and
mutations on the C-terminus of Exo70 may disrupt its membrane-association (see
below). That may explain the observed clear PM versus cytosol distribution of
GFP-Exo70 versus GFP-exo70-1 in Figure 2.3. The membrane fractionation data,
together with the fluorescence localization results, indicate that K632 and K635 are
critical residues in Exo70 that are involved in the physical association of Exo70 with
the PM.
Next, I tested the interaction of the mutant exo70-1 protein with phospholipids
in the cosedimentation assay. As shown in Figure 2.4 and Table 1, the interaction
between exo70-1 and 5% PI(4,5)P2 was almost abolished, suggesting that K632 and
K635 are critical residues for the binding of Exo70 to PI(4,5)P2. Interestingly, the
binding of exo70-1 for PS was only partially affected, suggesting that these two
residues may confer certain degree of specificity for PI(4,5)P2.
It was previously shown that Exo70 directly interacts with the constitutively
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Figure 2.3 Mutations at Exo70 C-terminus abolish the association between
Exo70 and the PM. (A) Sequence alignment between the C-termini (domain D) of
yeast Exo70 and rat Exo70. The mutated residues in the exo70-1 mutant were marked
in grey. (B) The localization of the exo70 mutant, exo70-1, in HeLa cells. HeLa cells
were transfected with GFP-tagged wild-type Exo70 as a control (left) and exo70-1
(right). The exo70-1 mutant failed to associate with the PM. (C) Membrane
fractionation was performed to examine the localization of GFP-Exo70 and
GFP-exo70-1 in HeLa cells. Equal amounts of proteins from each fraction of the cells
expressing Exo70 and exo70-1 were loaded on SDS-PAGE. The total proteins in each
lane were detected by SYPRORed staining. Exo70 was detected by Western blot.
LDM, low-density microsomal membranes, mostly the Golgi fraction; HDM,
high-density microsomal membranes, mostly the ER fraction.
Table 2. Exo70 C-terminus mutants.
Mutants
Mutation sites
exo70-1
K632A, K635A
exo70-2
∆634-636aa
exo70-3
K571A, E572A
D564A, K565A
exo70-4
K571A, E572A
exo70-5
∆571-573aa
K628A, K632A
exo70-6
R573A, K575A
exo70-7
K628A, K632A
exo70-8
R620A
exo70-9
K635A
exo70-10
R573A, K575A
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Localization
Cytoplasm
Cytoplasm
Cytoplasm
Cytoplasm
Cytoplasm
Cytoplasm
Plasma membrane
Plasma membrane
Plasma membrane
Plasma membrane
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activated form of TC10 (Q67L; Inoue et al., 2003). I then examined whether
mutations in exo70-1 affect this interaction. Cell lysates transfected with GFP-tagged
Exo70 or exo70-1 were incubated with GST-TC10 (Q67L) conjugated to the
glutathione-Sepharose for binding reaction. Exo70 or the exo70 mutant bound to the
Sepharose was detected by Western blot using an anti-GFP antibody. The amount of
exo70-1 bound to the TC10 beads was similar to that of the wild-type Exo70 (Figure
2.5A). As a control, neither the wild-type Exo70 nor exo70-1 bound to the GST beads.
The result indicates that the two key basic residues mutated in exo70-1 are not
required for its interaction with TC10. Therefore, the loss of PM association of the
exo70 mutant is unlikely due to impaired interaction with TC10. The binding results
strongly suggest that Exo70 associates with the PM through its direct interaction with
PI(4,5)P2. In addition to TC10, I have also examined the binding of exo70-1 with
another exocyst component Sec8. I found that mutations on exo70-1 did not affect its
binding with Sec8 in the cell (Figure 2.5B). This result is consistent with the previous
reports in mammalian and yeast cells that the interaction of Exo70 with the other
exocyst subunits for complex assembly is mediated by the N-terminal domains of
Exo70 (Inoue et al., 2003; Dong et al., 2005).
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Figure 2.4 Mutations at the C-terminus of Exo70 affect its interaction with
phospholipids. (A) GST-tagged Exo70 and exo70-1 (0.15 µM) were used in the lipid
cosedimentation assay. The binding of exo70-1 to 5% PI(4,5)P2 was abolished,
whereas its interaction with 60% PS was reduced. (B) Binding curves of Exo70 and
exo70-1 to LUVs containing various phospholipids.
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Figure 2.5 Mutations in the exo70-1 mutant did not affect its interaction with
TC10 (Q75L) or Sec8. (A) Mutations in exo70-1 did not affect its interaction with
TC10 (Q75L). Glutathione Sepharose conjugated with GST or GST-TC10 (Q75L)
was incubated with either GFP-tagged wild type Exo70 or the exo70 mutant. The
input and the bound Exo70 or exo70 mutant were analyzed by western blot using
anti-GFP monoclonal antibody. The lower panel was a Commassie blue stained gel
showing the amounts of GST-TC10 and GST (as a control) used in the binding assay.
(B) The exo70-1 mutant binds Sec8. Lysates from HeLa cells expressing GST-tagged
Exo70 or exo70-1 were incubated with Glutathione Sepharose. The binding of Sec8
to Exo70 or exo70-1 were detected by western blotting using an anti-Sec8
monoclonal antibody. GST-tagged Exo70 or exo70-1 bound to the beads was detected
using an anti-GST monoclonal antibody. Cell lysates expressing GST was used as a
negative control.
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Exo70 Recruits Sec8 to the Plasma Membrane
I next asked whether Exo70 is involved in recruiting other exocyst components
to the membrane. It has been shown that the exocyst subunits were mostly localized
in the cytoplasm in cultured HeLa cells (Zuo et al., 2006). I then examined whether
an increase of Exo70 at the PM would recruit Sec8 to the membrane. GFP-Exo70 and
GFP-exo70-1 were expressed in HeLa cells, and the localization of Sec8 in these cells
was detected by immunofluorescence staining using the anti-Sec8 mAb 2E12. As
shown in Figure 2.6, both GFP-Exo70 and Sec8 were detected at the PM. In contrast,
in cells expressing GFP-exo70-1 that is defective in binding PI(4,5)P2, Sec8 was
found in the cytoplasm and intracellular membrane structures. This result suggests
that the wild type, but not the mutant Exo70, is able to recruit other members of the
exocyst to the PM. Because exo70-1 maintains its ability to interact with Sec8, the
lack of PM association of Sec8 in cells expressing GFP-exo70-1 is unlikely resulted
from a defect in exocyst complex assembly.
Exo70 Is Essential for Exocytosis of VSV-G ts045 at the Plasma Membrane
Because the exocyst functions to tether post-Golgi secretory vesicles at the PM,
the physical interaction between Exo70 and phospholipids may play an important role
in exocytosis. Here I examined the effect of Exo70 depletion on the trafficking of a
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Figure 2.6 Exo70 is required for the PM localization of Sec8. GFP-Exo70 and
GFP-exo70-1 were transfected into HeLa cells. Sec8 in the transfected cells was
detected by the anti-Sec8 monoclonal antibody 2E12. Sec8 was detected at the PM in
cells expressing GFP-Exo70 (top panel), but not in cells expressing GFP-exo70-1
(bottom panel).
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temperature-sensitive VSV-G mutant (ts045) to the PM. At the restrictive temperature
(40°C), the VSV-G ts045 mutant is reversibly misfolded and retained in the ER. Upon
temperature shift to 32°C, the protein correctly folds and is transported through the
Golgi apparatus to the PM. I treated HeLa cells with EXO70 siRNA specific for
human Exo70 (hExo70), and the luciferase siRNA was used as a control. The
treatment reduced the level of Exo70 by more than 90% without affecting the level of
other exocyst subunits such as Sec8 (Figure 2.7A). Cells treated with EXO70 siRNA
or luciferase siRNA were transfected with GFP-tagged VSV-G mutant ts045
(VSV-G-GFP). Cells were then kept at 40°C overnight and shifted to 32°C for various
times before being examined by fluorescence microscopy. The translocation of
VSV-G-GFP inside the cells was shown by GFP fluorescence, whereas the
incorporation of VSV-G-GFP to the PM (at the 90-min point) was evaluated by
immunostaining using the 8G5 mAb against the extracellular domain of VSV-G
(Lefrancois and Lyles, 1982). As shown in Figure 2.7B, in luciferase siRNA-treated
cells, VSV-G-GFP was retained in the ER before the cells were shifted from 40 to
32°C (0 min). After the shift, the protein was transported from ER to the Golgi
apparatus around 30 min and then to the cell surface at 90 min. The incorporation of
VSV-G-GFP at the PM at 90 min was clearly detected by immunostaining of
nonpermeabilized cells with the 8G5 antibody (Figure 2.7B). In EXO70
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Figure 2.7 Exocytosis of VSV-G ts045 is blocked in EXO70 siRNA knockdown
cells. (A) HeLa cells were treated with EXO70 siRNA and Luciferase siRNA (as
control). Exo70 was knocked down as detected by Western blot. The amount of Sec8
in these cells was not affected. The anti-Exo70 monoclonal antibody (13F3) and
anti-Sec8 monoclonal antibody (2E12) were used in the Western blot analysis. (B)
Luciferase siRNA-treated HeLa cells were transfected with VSV-G-GFP, kept at 40°C
overnight, and shifted to 32°C for 0, 15, 30, 60, and 90 min in the presence of
cycloheximide (100 µg/ml). The cells were then fixed and stained as described in
Materials and Methods. VSV-G was transported from ER, to the Golgi, and to the PM.
(C) In EXO70 knockdown cells, VSV-G transport to the PM through the endoplasmic
reticulum and Golgi complex was normal. However, the incorporation of VSV-G
(stained by the 8G5 monoclonal antibody recognizing the extracellular domain of
VSV-G) was considerably delayed. Instead of being detected at the surface at 90 min
as in control cells, VSV-G was detectable after 180 min from temperature arrest. (D
and E) VSV-G association with various intracellular compartments was quantified in
cells expressing luciferase siRNA (D) or EXO70 siRNA (E). For the quantification,
boundaries of the whole cell, Golgi region and cell periphery region were outlined,
and VSV-G fluorescence in these areas was then quantified using ImageJ 1.73v
software after subtraction of background outside the cell. (F) Quantification of
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fluorescence intensity of surface VSV-G signal stained by the 8G5 monoclonal
antibody. Boundary of the cell surface was outlined, and average fluorescence
intensity of surface VSV-G signal was quantified using ImageJ 1.73v software and
then divided by the perimeter of the cell surface. Three independent experiments (20
cells each) were carried out. Error bars, SD. p < 0.01.
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siRNA-treated cells, the translocation of VSV-G-GFP from the ER to the Golgi
apparatus and from the Golgi to the PM followed time courses similar to that in
control siRNA-treated cells. However, at 90 min, the extracellular exposure of
VSV-G-GFP was hardly detectable in nonpermeabilized cells; instead, it could only
be detected after 180 min (Figure 2.7C), suggesting that the fusion of the exocytic
vesicles with the PM was substantially delayed. Quantification of the GFP signal in
different intracellular compartments indicates that the cellular distributions of
VSV-G-GFP at various time points after the temperature shift were similar in EXO70
siRNA and luciferase siRNA-treated cells (Figure 2.7, D and E): at 0 min, most of the
VSV-G-GFP was located at the ER (88% for luciferase siRNA treatment and 84% for
EXO70 siRNA treatment); at 30 min, a majority of VSV-G-GFP was located at the
Golgi apparatus (91% for luciferase siRNA and 92% for EXO70 siRNA treatment);
and at 90 min, VSV-G-GFP was located at the cell periphery (88% for luciferase
siRNA and 74% for EXO70 siRNA treatment). In contrast, quantification of the
surface VSV-G signal displayed a different pattern in EXO70 siRNA and luciferase
siRNA-treated cells. As shown in Figure 2.7F, at 0-, 15-, and 30-min points,
fluorescence intensity of exposed VSV-G was diminutive both in control and EXO70
siRNA-treated cells, which indicates that VSV-G-GFP was translocating in the
intracellular compartments; at 60 and 90 min, fluorescence intensity of surface
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VSV-G was much less in EXO70 siRNA-treated cells than in control siRNA-treated
cells; at 180 min, fluorescence intensity of exposed VSV-G in EXO70 siRNA-treated
cells began to appear at the cell surface. These results suggest that the incorporation
of the exocytic VSV-G vesicles with the PM was delayed in cells treated with EXO70
siRNA. Collectively, these results demonstrated that Exo70 is probably not required
for the transport of VSV-G from the intracellular compartments to the cell periphery,
but is critical for the efficient tethering or subsequent docking/fusion of
VSV-G–containing exocytic vesicles with the PM.
The Interaction of Exo70 with PI(4,5)P2 Is Important for the Exocytosis of
VSV-G ts045
To examine the role of the Exo70-PI(4,5)P2 binding in exocytosis, it is
necessary to specifically disrupt the interaction between Exo70 and phospholipids in
vivo. I therefore tested the transport of VSV-G in EXO70 siRNA knockdown cells
transfected with the rat exo70 mutant, exo70-1, that is deficient in the PI(4,5)P2
binding. EXO70 siRNA-treated cells transfected wtih wild-type rat Exo70 (rExo70
WT) were used as control. While rat Exo70 is over 90% identical in protein sequence
to human Exo70, on the nucleotide level, it cannot be targeted by the EXO70 siRNA
oligos used in this study. The level of Exo70 knockdown as well as the expression of
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GST-rExo70 and GST-rexo70-1 is shown in Figure 2.8A.
In EXO70 siRNA-treated cells, only a negligible amount of VSV-G-GFP was
detected on the cell surface after 90 min of growth at the permissive temperature
(Figure 2.8B). Expression of wild-type rat Exo70 restored normal surface
incorporation of VSV-G (VSV-G-myc) in EXO70 siRNA knockdown cells (Figure
2.8C); therefore the rat Exo70 can serve as a rescue reagent for the RNAi experiment
in HeLa cells. In contrast, in EXO70 siRNA-treated cells expressing the exo70-1
mutant that is deficient in PI(4,5)P2 binding, the surface exposure of VSV-G-myc was
disrupted, whereas the translocation of VSV-G-myc from the intracellular
compartments to the cell periphery was not affected (Figure 2.8D). VSV-G-myc
rather than VSV-G-GFP was used here so that different pairs of proteins in the cells
could be stained (GST-rExo70 and myc vs. GST-rExo70 and 8G5). Quantification of
cells with surface VSV-G indicates that expressing wild-type rat Exo70 in EXO70
siRNA-treated cells restores VSV-G surface exposure in the majority of the cells
(from 18 to 61%), whereas expressing rat exo70-1 does not have an obvious effect
(Figure 2.8E). These results suggest that the interaction of Exo70 with membrane
lipids is essential for the exocytosis of VSV-G at the PM.
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Figure 2.8 VSV-G ts045 exocytosis defect in EXO70 siRNA knockdown cells
expressing the exo70-1 mutant. (A) EXO70 siRNA knockdown cells were
transfected with GST-tagged rat Exo70 (GST-rExo70) or rat exo70 mutant
(GST-rexo70-1). The amount of endogenous Exo70 in HeLa cells, and the amount of
extragenically expressed GST-tagged rat Exo70 were detected by anti-Exo70
monoclonal antibody (top panel). The total proteins in the cell lysates were detected
by SYPRORed staining (bottom panel). (B) VSV-G-GFP trafficking in HeLa cells
transfected with EXO70 siRNA. The cells were grown at 40°C overnight after
transfection. The cells were then shifted to 32°C for 0, 30, 60, and 90 min in the
presence of cycloheximide, fixed, and stained as described in Materials and Methods.
The 8G5 monoclonal antibody was used to detect the surface-incorporated VSV-G. (C
and D) VSV-G-myc trafficking was examined in EXO70 siRNA knockdown cells
expressing GST-rExo70 (C) or GST-rexo70-1 (D). VSV-G-myc and GST-tagged
wild-type rExo70 (C) or exo70-1 (D) were cotransfected into EXO70 knockdown
HeLa cells. VSV-G transport to the PM was rescued in the EXO70 siRNA knockdown
cells expressing wild-type rExo70, whereas VSV-G transport was not rescued in the
EXO70 siRNA knockdown cells expressing exo70-1. (E) Quantification of the
percentage of cells with surface VSV-G staining.
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2.3. Discussion
To understand the molecular basis of vesicle tethering at the PM, it is important
to clarify how the tethering complex itself, the exocyst, is targeted to the PM. Here I
identified a direct interaction between the exocyst component Exo70 and PI(4,5)P2;
and demonstrated that this interaction is essential for the recruitment of the exocyst to
the PM. Furthermore, disruption of this interaction impaired later stages of exocytosis
of post-Golgi secretory vesicles at the PM.
PI(4,5)P2 and PS are the major negatively charged lipids in the PM. The
observation that Exo70 binds to both 5% PI(4,5)P2 and 60% PS indicates that the
interaction of Exo70 with the phospholipids is electrostatic in nature. This type of
interaction has been found in a number of proteins, such as N-WASP and MARCKS
(McLaughlin and Murray, 2005). Comparing the charges of PI(4,5)P2 versus PS at the
physiological pH, LUVs composed of 5% PI(4,5)P2 have approximately the same
amount of effective charges as LUVs composed of 15–20% PS. However, the
interaction of Exo70 with LUVs composed of 20% PS is much weaker. Mutations on
exo70-1 that eliminate some of the positive charges in the Exo70 C-terminus nearly
abolished the ability of Exo70 to bind PI(4,5)P2; however, the ability of this mutant to
bind PS at high concentrations (≥60%) was only partially affected. These data suggest
that Exo70 has significant binding specificity for PI(4,5)P2 over PS. I have also
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examined the interaction of Exo70 with other phosphoinositides and observed its
selectivity
for
PI(4,5)P2
over
the
stereoisomeric
PI(3,5)P2
and
other
monophosphorylated phosphoinositides. I have also found that Exo70 binds
PI(3,4,5)P3 with an affinity that is comparable to that for PI(4,5)P2. PI(3,4,5)P3 has
recently been found to be localized to the basolateral domain in MDCK cells
(Gassama-Diagne et al., 2006), and the exocyst has been implicated in basolateral
vesicle targeting (Grindstaff et al., 1998). In other types of mammalian cells, although
the concentration of PI(3,4,5)P3 is low at the PM in resting cells, it can be rapidly
up-regulated in response to extracellular stimuli. It is therefore possible that Exo70
binds to PI(3,4,5)P3 under certain physiological circumstances or in certain cell types.
In yeast, our lab has found that the amount of the exocyst complex associated
with the PM was much lower in the temperature-sensitive mss4 mutant cells, in
which the PI(4,5)P2 level in the PM was reduced (data not shown), indicating that
PI(4,5)P2 mediates the membrane targeting of the exocyst. Moreover, the structural
analysis of Exo70 provided important insights into the potential mechanism of
membrane association of Exo70 (Dong et al., 2005; Hamburger et al., 2006). On the
basis of the crystal structure information of yeast Exo70, I have made mutations on
the rat Exo70 residues K632 and K635 (exo70-1), which are positively charged amino
acids well conserved in the yeast Exo70 sequence. My in vitro vesicle sedimentation
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experiments demonstrated that these mutations disrupted the PI(4,5)P2-binding.
Furthermore, the VSV-G trafficking analysis further revealed its functional
importance in exocytosis at the PM. Later, Moore et al. (2007) resolved the crystal
structure of mouse Exo70. Analysis of the structure indicates that the point mutations
on exo70-1 are localized on the loop between Helix 18 and 19 on the surface of the
mouse Exo70, which is almost identical to that of the yeast Exo70.
Taking advantage of the VSV-G trafficking assay, I was able to assess the role
of Exo70 in various stages of membrane traffic. More importantly, using the exo70-1
mutant in the EXO70 RNAi knockdown cells, I was able to specifically examine the
functional importance of Exo70-PI(4,5)P2 interaction in VSV-G exocytosis. The
exocyst has been found in various cellular compartments, including Golgi and
recycling endosomes, in addition to the PM (Yeaman et al., 2001; Fölsch et al., 2003;
Ang et al., 2004; Langevin et al., 2005). Here I found that the Exo70-PI(4,5)P2
interaction is not involved in the early stages of VSV-G trafficking through the
endoplasmic reticulum and Golgi. Instead, it is critical for the PM events such as
vesicle tethering and fusion. When the Exo70-PI(4,5)P2 interaction was perturbed, the
transport of VSV-G to the PM was barely changed. However, the incorporation of
VSV-G into the PM was significantly impaired, as revealed by the 8G5 antibody
specifically recognizing the extracellular domain of VSV-G. Similarly the exocyst has
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been implicated in tethering and fusion of Glut4-containing vesicles in 3T3-L1
adipocytes (Inoue et al., 2003; Ewart et al., 2005; Tsuboi et al., 2005). It is possible
that other exocyst components also interact with phospholipids (Moskalenko et al.,
2003). However, specific disruption of Exo70-PM interaction is sufficient to block
exocytosis in mammalian cells.
The interaction of the exocyst with the PM is an important step in vesicle
tethering. When and where this association takes place may regulate the kinetics and
location of exocytosis. In the budding yeast S. cerevisiae, the exocyst complex is
specifically concentrated at the growing tip of the daughter cell (the "bud tip"), which
is the site of active exocytosis and cell surface expansion. Moreover, Exo70 primarily
functions at the early stages of the yeast cell cycle, suggesting a temporal control of
Exo70 function (He et al., 2007a). In mammalian cells, growth factor signaling
involving small GTPases may mediate the subunits assembly, translocation of the
exocyst from intracellular compartments to, or activation of the exocyst at, the PM
(Sugihara et al., 2002; Moskalenko et al., 2002, 2003; Inoue et al., 2003; Takaya et
al., 2004; Zuo et al., 2006). Future work will be focused on the identification and
characterization of proteins that temporally and/or spatially regulate Exo70 and other
exocyst components using different eukaryotic systems.
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2.4. Materials and Methods
DNA Plasmid Construction
Wild-type rat Exo70 (rExo70) cDNA and various truncates of Exo70 were
cloned in-frame into pEGFP-C1 for expression as green fluorescent protein (GFP)
fusions or in pEBG for expression as glutathione S-transferase (GST) fusion in
mammalian cells. rExo70 was also cloned into pGEX-KG (a modified form of
pGEX-2T, from Amersham Biosciences, Piscataway, NJ) for expression as GST
fusion in bacteria. The Exo70 mutants were generated using the QuikChange
site-directed mutagenesis kit (Stratagene, La Jolla, CA). All the constructs were
confirmed by nucleotide sequencing.
Cell Culture and RNA Interference Experiments
HeLa cells were cultured at 37°C in DMEM supplemented with 10% fetal
bovine serum and 100 U/ml penicillin and 100 µg/ml streptomycin in a 5% CO2
incubator. For RNA interference (RNAi) experiments, cells were grown to 50%
confluence and transfected with small interfering RNA (siRNA) duplexes using
Oligofectamine (Invitrogen, Carlsbad, CA). The human Exo70 siRNA target
sequence is 5'-GGTTAAAGGTGACTGATTA-3'. The control Luciferase GL2 siRNA
target sequence is 5'-AACGTACGCGGAATACTTCGA-3'. The efficiency of Exo70
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knockdown was determined by Western blot.
Confocal Microscopy
Transfected
HeLa
cells
were
grown
on
coverslips,
washed
with
phosphate-buffered saline (PBS), fixed in 4% paraformaldehyde at room temperature
for 12 min, washed, permeabilized for 5 min with PBST (PBS-Tween), and blocked
for 10 min with 2% bovine serum albumin in PBST. The coverslips were incubated
sequentially with primary and secondary antibodies for fluorescence observation
using the Leica TCS SL laser-scanning confocal microscope (63x objective; Deerfield,
IL). Images were processed with Adobe Photoshop (Adobe Systems, San Jose, CA;
version 7.0).
Large Unilamellar Vesicle Sedimentation Assay
Large unilamellar vesicle (LUV) sedimentation assay was performed as
previously described (Hokanson and Ostap, 2006). Phospholipids were purchased
from Avanti Polar Lipids (Alabaster, AL). LUVs with a 100-nm diameter were
prepared by size extrusion. Various lipids were mixed at different molar ratios, dried
with nitrogen stream, and resuspended at a concentration of 2 mM in a buffer
containing 12 mM HEPES, pH 7.0, and 176 mM sucrose. The mixed lipids were
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subjected to five cycles of freeze-thaw and a 1-min bath sonication before being
passed through 100-nm filters using a mini-extruder. LUVs were dialyzed overnight
in the HNa100 buffer (10 mM HEPES, pH 7.0, 100 mM NaCl, 1 mM EGTA, and 1
mM dithiothreitol [DTT]). The percentages of phosphotidylserine (PS), PI(3)P, PI(4)P,
PI(3,5)P2, PI(4,5)P2, and PI(3,4,5)P3 indicated in the text are the molar percentages of
total PS and PIPs with the remainder being phosphatidylcholine (PC). Lipid
concentrations are given as total lipid. The binding of Exo70 to LUVs was
determined by sedimentation assays in 200 µl total volume using TLA-100 rotor
(Beckman Coulter, Fullerton, CA). Sucrose-loaded LUVs were precipitated at
150,000 x g for 30 min at 25°C. The supernatants and pellets were subjected to 10%
SDS-PAGE and stained with SYPRORed (Invitrogen) for quantification of free and
bound materials with the Image Quant software (Molecular Dynamics, Sunnyvale,
CA).
Membrane Fractionation
HeLa cells were plated in 10-cm dishes at 1.5 x 106 cells per dish. The next day
cells were transfected with DNA by FuGene6 reagent and incubated at 37°C
overnight. Homogenization and subcellular fractionation of the cells to isolate the PM
fraction, cytosol, the low-density microsomal fraction (LDM), and the high-density
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microsomal fraction (HDM) were performed basically as previously described
(Weber et al., 1988). All steps were performed at 4°C in the presence of a protease
inhibitor cocktail. The distribution of Exo70 and Sec8 were detected by monoclonal
antibodies (kind gifts of Dr. Shu-Chan Hsu, Rutgers University).
GST Pulldown Assay
HeLa cells were transfected with GFP-tagged Exo70 or exo70-1, and the cells
were lysed in a buffer containing 20 mM Tris-HCl, pH 7.5, 25 mM KCl, 1 mM
MgCl2, 0.5 mM EGTA, 1 mM DTT, 0.5% Triton X-100, and protease inhibitors. Cell
lysates were incubated overnight with glutathione-Sepharose conjugated with GST or
GST-TC10 (Q75L) at 4°C. After incubation, the beads were washed five times with
the lysis buffer, and the bound proteins were analyzed by Western blot using an
anti-GFP antibody. To detect the interaction of Exo70 and Sec8 in the cell, HeLa cells
were transfected with GST-tagged Exo70 or exo70-1, and cell lysates were incubated
overnight with glutathione-Sepharose beads at 4°C. After incubation, the beads were
washed, and the bound proteins were detected by Western blot using anti-GST or
anti-Sec8 monoclonal antibodies.
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VSV-G Trafficking Assay
HeLa cells were transfected with EXO70 siRNA. luciferase siRNA was used as
the negative control. After 24 h of the siRNA treatment, HeLa cells were transfected
with VSV-G-45ts-GFP mutant and immediately placed at 40°C. After overnight
growth, the cells were shifted to 32°C for 0, 15, 30, 60, and 90 min in the presence of
cycloheximide (100 µg/ml). The cells were then fixed for GFP observation or
immunofluorescence. The 8G5 mAb against the extracellular domain of VSV-G was
kindly provided by Dr. Douglas Lyles (Wake Forest University). No detergent was
used in the immunofluorescence procedure. Cells with surface VSV-Gs were
quantified, and statistical analyses were performed using Student's t test. In some
cases, HeLa cells were transfected with VSV-G-myc and GST-Exo70 or GST-exo70-1
after 24 h of the EXO70 siRNA treatment. The cells were divided into two sets based
on their treatments. For Set I, the cells were fixed, permeabilized, and stained with
anti-myc mAb (9E10) and anti-GST polyclonal antibody to test the intracellular
traffic of VSV-G and to detect the expression of Exo70 or exo70-1 at 0-, 30-, 60-, and
90-min points. For Set II, cells of the 90-min point group were first stained with the
8G5 antibody, then permeabilized, and stained with anti-GST polyclonal antibody.
Anti-mouse Alexa488 and anti-rabbit Alexa594 were used as secondary antibodies for
the above experiments. For the quantification of surface VSV-G signals at various
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points, boundary of the cell surface was outlined, and average fluorescence intensity
of surface VSV-G signal was quantified using ImageJ 1.73v software and then
divided by the perimeter of the cell surface. For the quantification of VSV-G in
different membrane compartments, boundaries of the whole cell, the Golgi, and the
cell periphery were outlined, and VSV-G fluorescence in these areas was then
quantified using ImageJ 1.73v software after subtraction of background outside the
cell using the following equations:
Golgi (%) = FluorescenceGolgi /Fluorescencetotal
Cytoplasm (%) = Fluorescencecytoplasm /Fluorescencetotal.
Cell periphery (%) = (Fluorescencetotal -Fluorescencecytoplasm -FluorescenceGolgi)/Fluorescencetotal.
* This work has been published in Molecular Biology of the Cell in November 2007.
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Chapter 3. The Role of the Exocyst in Matrix Metalloproteinase Secretion
and Actin Dynamics during Tumor Cell Invadopodia Formation
Abstract
Invadopodia are actin-rich membrane protrusions formed by tumor cells that
degrade the extracellular matrix for invasion. Invadopodia formation involves
membrane protrusions driven by Arp2/3-mediated actin polymerization and secretion
of matrix metalloproteinases (MMPs) at the focal degrading sites. The exocyst
mediates the tethering of post-Golgi secretory vesicles at the plasma membrane for
exocytosis and has recently been implicated in regulating actin dynamics during cell
migration. Here, I report that the exocyst plays a pivotal role in invadopodial activity.
With RNAi knockdown of the exocyst component Exo70 or Sec8, MDA-MB-231
cells expressing constitutively active c-Src failed to form invadopodia. On the other
hand, overexpression of Exo70 promoted invadopodia formation. Disrupting the
exocyst function by siEXO70 or siSEC8 treatment or by expression of a dominant
negative fragment of Exo70 inhibited the secretion of MMPs. I have also found that
the exocyst interacts with the Arp2/3 complex in cells with high invasion potential;
blocking
the
exocyst-Arp2/3
interaction
inhibited
Arp2/3-mediated
actin
polymerization and invadopodia formation. Together, my results suggest that the
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exocyst plays important roles in cell invasion by mediating the secretion of MMPs at
focal degrading sites and regulating Arp2/3-mediated actin dynamics.
3.1. Introduction
One of the most important features of cancer is the ability of the tumor cells to
break through tissue barriers and invade into surrounding tissues. The initial step of
tumor cell invasion is the formation of cell protrusions in the direction of cell
movement. Invadopodia are specialized membrane protrusions formed by invading
tumor cells that extend into the extracellular matrix (ECM). At the ultrastructural level,
invadopodia are filament-like extensions from the ventral surface of the cells adherent
to matrix that range from hundreds of nanometers to a few micrometers in diameter
and reach 500 nm in length (Buccione et al., 2004 ; Marx, 2006; Linder, 2007).
Time-lapse image analysis has shown that invadopodia are formed de novo at the cell
periphery and their lifetime varies from minutes to several hours (Yamaguchi et al.,
2005). Invadopodial protrusions are actin-based structures enriched in actin-associated
proteins, adhesion proteins, matrix proteases such as matrix metalloproteinases
(MMPs) and signaling proteins that regulate the actin cytoskeleton and membrane
remodeling. However, how multiple processes including signaling, protease secretion,
and actin assembly are coordinated to generate a functional ECM-degrading machine
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remains poorly understood.
Focal degradation of tissue barriers by MMPs plays a crucial role in tumor
invasion. The matrix-degrading capability of invadopodia is largely dependent on
MMPs, including MMP-2, MMP-9, and MT1-MMP (Gimona and Buccione, 2006;
Linder, 2007). MMP-2 was the first MMP found to localize at invadopodia of
Src-transformed fibroblasts (Monsky et al., 1993). MMP-9 was also found at
invadopodial structures such as those of the metastatic breast cancer cells
(Bourguignon et al., 1998) and leukemia cells (Redondo-Muñoz et al., 2006).
MT1-MMP is a transmembrane protease essential for invadopodial activity (Itoh and
Seiki, 2006). The secretion of a combination of MMPs is important for effective
invasion. However, the exact mechanism by which MMPs are targeted to and
exocytosed at invadopodia-forming sites is elusive.
In addition to MMP secretion, the formation of cell protrusions also involves
the assembly of branched actin network at the leading edge. The Arp2/3 complex is
the core machinery that nucleates actin for the generation of branched filamentous
actin networks. During invadopodia formation, the Arp2/3 complex has been shown
to play a critical role in the formation of degrading protrusions (Yamaguchi et al.,
2005). However, how actin assembly is controlled during invadapodia formation is
still not well understood.
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The exocyst is an evolutionarily conserved octameric protein complex that
mediates the tethering of secretory vesicles to the plasma membrane for exocytosis
(Guo et al., 2000; Hsu et al., 2004; Munson and Novick, 2006; He and Guo, 2009).
The exocyst complex is involved in a number of cellular processes that require
polarized exocytosis, including yeast budding, neurite extension, epithelia
polarization, and cytokinesis (for review, see Hsu et al., 2004). Here I hypothesize
that the exocyst is involved in invadopodial activities via regulating the secretion of
MMPs at the focal degradation sites. Recently, the exocyst has also been shown to be
involved in actin-based membrane protrusion and cell migration (Zuo et al., 2006;
Rosse et al., 2006). The exocyst component, Exo70, directly interacts with the Arp2/3
complex, and this interaction is important for the regulation of actin assembly at the
leading edge of migrating cells (Zuo et al., 2006). Therefore it is likely that the
exocyst also regulates actin dynamics during invadopodia formation through its
interaction with the Arp2/3 complex.
Here, I report that the exocyst plays a pivotal role in invadopodial activity. I
have found that blocking the exocyst function inhibits invadopodial formation. RNAi
knockdown of the exocyst component Exo70 or Sec8 in MDA-MB-231 cells
expressing mutant Y527F c-Src abolished the secretion of MMPs, whereas the
overexpression of Exo70 promoted MMP secretion. In addition, the exocyst-Arp2/3
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interaction is important for actin assembly during invadopodia formation. Together,
these findings suggest that the exocyst coordinates protease secretion and
cytoskeleton dynamics during tumor invasion.
3.2. Results
The exocyst is necessary for invadopodial activity
A matrix degradation assay was applied to study invadopodia formation. This
assay
involves
culturing
cells
on
coverslips
coated
with
thin,
fluorochrome-conjugated gelatin matrices. Local proteolytic activity was revealed by
the appearance of dark areas lacking fluorescence in the bright fluorescent matrix
(Artym et al., 2006). MDA-MB-231 parental cells were used in the experiments,
along with cells stably transfected with the constitutively active c-Src (Y527F c-Src),
which display increased invasive potential.
To investigate the role of the exocyst in invadopodia formation, I examined the
effect of siRNA-mediated knockdown of endogenous exocyst subunits on
invadopodia formation in c-Src–activated cells. Using different siRNA oligos that
target EXO70 (siEXO70(1) and siEXO70(2)) and SEC8 (siSEC8), I observed effective
knockdown of the target proteins by Western blot (82% for siEXO70(1), 67% for
siEXO70(2) and 69% for siSEC8 (Figure 3.1A, left panel). The knockdown cells
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formed much fewer invadopodia as indicated by fewer matrix degradation sites and a
smaller matrix degradation area. Correspondingly, the number of actin puncta
localized to sites of matrix degradation was also reduced in the knockdown cells
(Figure 3.1B). I have also tested whether the expression of rat Exo70 is able to rescue
the defect of invadopodia formation in siEXO70-treated MDA-MB-231 (Y527F c-Src)
cells. While rat Exo70 is more than 90% identical in amino acid sequence to human
Exo70, it is not targeted by the human EXO70 siRNA oligonucleotides used in this
study. The level of Exo70 knockdown and the expression of GFP-rExo70 are shown
in Figure 3.1A (right panel). I found that the defect of invadopodia formation in
siEXO70-treated cells was restored by the expression of rat Exo70 (Figure 3.1C).
Quantification of the results from four independent experiments was carried out in
siEXO70, siSEC8, control siRNA-treated cells, and siEXO70-treated cells expressing
GFP-rExo70. The degradation level was calculated by dividing the total area of the
degraded zones per cell by the area of the whole cell. Quantification of degradation
levels was included in Materials and Methods, and representative photographs were
shown in Figure 3.1D. The percentage of cells with different degradation levels was
calculated for each treatment. As shown in Figure 3.1E, most of the EXO70 or SEC8
siRNA-treated cells failed to form detectable invadopodia (77% for siEXO70(1), 68%
for siEXO70(2) and 72% for siSEC8), whereas most of the control siRNA-treated
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Figure 3.1 Knockdown of the exocyst inhibits invadopodial activity. (A)
Expression levels of the exocyst subunits Exo70 and Sec8 in MDA-MB-231 (Y527F
c-Src) cells transfected with siRNAs. Lysates were prepared from cells transfected
with siRNA oligos targeting Luciferase (Control siRNA), Exo70 (two different oligos:
siEXO70(1) and siEXO70(2)), or Sec8 (siSEC8). siRNA treatments led to significant
reduction in the amounts of Exo70 and Sec8 in the cells. The levels of Exo70 and
Sec8 in cell lysates were analyzed by Western blot (left panel). In the rescue
experiment, siEXO70(1) knockdown cells were transfected with GFP-tagged rat
Exo70 (GFP-rExo70). The amounts of endogenous Exo70 and GFP-rExo70 were
detected by Western blot (right panel). The amounts of actin were also examined as
loading controls. (B) MDA-MB-231 (Y527F c-Src) cells with RNAi knockdown
were cultured on Alexa 568–labeled gelatin film for 4 h. Cells were then fixed and
stained with phalloidin-Alexa 488 to label F-actin. Individual and merged images of
F-actin (green) and gelatin (red) were shown. RNAi knockdown of Exo70 or Sec8
decreased the amount of focal degradation (the black dots). (C) Gelatin degradation
assay was performed in siEXO70(1) knockdown cells expressing GFP-rExo70.
Individual and merged images of GFP-rExo70 fluorescence (green), F-actin (blue),
and gelatin (red) are shown. The expression of GFP-rExo70 in siEXO70(1)
knockdown cells rescued invadopodia formation in these cells. (D) Representative
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photographs showing the quantification of matrix degradation in the cell. a) The
original image of the labeling of Alexa 568-gelatin. b) The area of degraded matrix in
the field was measured using ImageJ 1.73v software. The image was first converted
to 8 bit grayscale files. Then automatic thresholding function of ImageJ was applied
to the image, followed by manually adjusting the maximum threshold value by
moving the bottom horizontal bar in the “Threshold” window from right to left until
the upper threshold line just touches the edge of the histogram while keeping the
minimum threshold value zero. This adjustment allows optimal matching to the
original fluorescence images taken from the microscope. The AlexaFluor568-gelatin
image was thresholded at 0-127 after the above adjustments. c) The boundary of the
black dots and the total degradation area were obtained by automatic outlining. There
are 41 degrading spots and the total degradation area is 3909.000 pixel. d) The area of
the whole cell (37274.7 pixel) was obtained by manually outlining the boundary of
the cell surface. Degradation level (10.487%) was then calculated as the total
degradation area divided by the total area of the cell. (E) Comparison of focal
degradation in cells with exocyst knockdown. Four independent measurements (50
cells each) for each siRNA treatment (left panel) and rescue experiment (right panel)
were carried out. Error bars, SD. *p < 0.01. Scale bar, 5 µm.
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cells formed invadopodia (81%; left panel). The expression of rat Exo70 in
siEXO70(1)-treated cells largely rescued the degradation defects in these cells. The
percentage of cells that formed invadopodia was restored from 26% to 68%) (Figure
3.1E, right panel).
I have also examined whether the exocyst is able to induce the formation of
invadopodia. As shown in Figure 3.2A, in the parental MDA-MB-231 cells,
expression of GFP-tagged Exo70 promoted invadopodia formation by increasing
both the numbers of invadopodia and areas of degradation in each transfected cell,
whereas expression of GFP alone did not cause any change in invadopodia formation
(data not shown). The percentage of cells with different degradation levels in
GFP-Exo70 and control vector-transfected cells was quantified. As shown in Figure
3.2B, the percentage of cells with focal matrix degradation was substantially
increased in Exo70-transfected cells (31, 35, and 23% for 0–1, 1–5, and >5%
degradation levels, respectively) compared with control cells (18, 10, and 5% for 0–1,
1–5, and >5% degradation levels, respectively). I also examined whether the exocyst
is able to induce invadopodia formation in Y527F c-Src–expressing cells. As shown
in Figure 3.2, C and D, expression of GFP-tagged Exo70 further promoted
invadopodia formation in transfected cells. Expression of GFP alone was used as a
negative control. Collectively, these results suggest that the exocyst plays an essential
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Figure 3.2 Overexpression of Exo70 stimulates invadopodial activity. (A)
MDA-MB-231 cells were transfected with GFP-tagged Exo70 (GFP-Exo70), plated
on fluorescent Alexa 568–labeled gelatin film for 4 h, and then processed for
microscopy. Individual and merged images of GFP-Exo70 fluorescence (green),
F-actin (blue), and gelatin (red) are shown. Overexpression of GFP-Exo70 stimulated
focal degradation in MDA-MB-231 cells (top panel). Cells transfected with GFP
alone were used as negative control (bottom panel). (B) Quantification of degradation
areas in cells overexpressing Exo70. Three independent measurements (~70 cells
each) for each treatment were carried out. Error bars, SD. *p < 0.01. Scale bar, 5 µm.
(C) MDA-MB-231 (Y527F c-Src) cells were transfected with GFP-tagged Exo70
(GFP-Exo70), plated on fluorescent Alexa 568 labeled-gelatin film for 4 hrs, and then
processed for microscopy. Individual and merged images of GFP-Exo70 fluorescence
(green), F-actin (blue) and gelatin (red) were shown. Expression of GFP alone did not
change the ability of c-Src-expressing cells to form invadopodia (bottom panel).
Overexpression of GFP-Exo70 stimulated focal degradation in MDA-MB-231
(Y527F c-Src) cells (upper panel) and more invadopodia were formed compared with
GFP-transfected cells. (D) Quantification of degradation areas in cells overexpressing
Exo70. Three independent measurements (~50 cells each) for each treatment were
carried out. Error bars, SD. “*” indicates p < 0.01. Scale bar = 5 µm.
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role in invadopodia formation and invasion.
I also noticed that cells transfected with siRNA against Exo70 have weaker
F-actin staining comparing with control cells or SEC8 siRNA-treated cells. The effect
of Exo70 on actin organization will be discussed later.
The localization of the exocyst at invadopodia-forming sites
Next, I tested the localization of exocyst components in the parental
MDA-MB-231 cells using confocal microscopy. In addition to the staining in the
cytosol, endogenous Exo70 was also enriched in invadopodia as well as the plasma
membrane (Figure 3.3A). Part of the Exo70 staining colocalized with actin puncta at
sites of matrix degradation. I have also examined the localization of GFP-tagged
Exo70, expressed at low levels using the pJ3-GFP vector, in MDA-MB-231 cells. As
shown in Figure 3.3B, there are partial overlaps of Exo70 and the degrading foci.
Sec8 has a similar pattern of localization to the invasion sites (data not shown;
Sakurai-Yageta et al., 2008). The exocyst components were not always concentrated
at the degradation sites. This may be due to the highly dynamic nature of invadopodia
and the mobility of the cells. Indeed, as previously demonstrated, F-actin and its
regulatory proteins were not always detected at sites of degradation (Artym et al.,
2006). It is possible that the localization of the exocyst at invadopodia-forming sites
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Figure 3.3 Localization of Exo70 at the focal degradation sites. (A)
MDA-MB-231 cells were plated on fluorescent Alexa 568–labeled gelatin (red) for
20 h. After fixation, cells were stained for Exo70 (green). Individual and merged
images are shown. Endogenous Exo70 showed colocalization with the "degradation
holes" in the fluorescent gelatin matrix, along with some enrichment at the plasma
membrane. (B) The localization of GFP-tagged Exo70 expressed at low levels using
the pJ3-GFP vector was examined in MDA-MB-231 cells. GFP-tagged Exo70
partially overlapped with the degrading spots. Higher magnification views of the
boxed areas are shown underneath each image. Arrows, colocalization of Exo70 and
invadopodia-forming sites. Scale bars, 5 µm.
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is transient. Some of the focal degradation sites may represent regions where the
exocyst completed their function.
The exocyst is required for the secretion of MMPs in MDA-MB-231 (Y527F
c-Src) cells
The exocyst is involved in the tethering of secretory vesicles at the plasma membrane
for exocytosis. It is thus likely that the exocyst mediates the secretion of MMPs at
invadopodial sites. To test this hypothesis, I examined the effect of siRNA
knockdown of EXO70 and SEC8 on the secretion of MMP-2 and MMP-9 in
MDA-MB-231 (Y527F c-Src) cells. Substrate zymography was performed to detect
the secretion levels of MMP-2 and MMP-9. Zymography is the most sensitive and
widely used assay for the detection of various forms of gelatinases on the basis of
their different molecular weights (Mott and Werb, 2004). It involves the incorporation
of substrate gelatin in polyacrylamide gels, the digestion of the “in gel” gelatin by
separated gelatinases after electrophoresis, and the detection of gelatinases by the
appearance of white areas on a blue background after Coomassie blue staining (Van
den Steen et al., 2002). Gelatin zymography analyses of cell culture media detected a
dramatic decrease in the levels of MMP-2 and MMP-9 in both EXO70 and SEC8
knockdown cells (Figure 3.4A). I have also tested whether the expression of rat
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Exo70 is able to rescue the secretion defect in siEXO70-treated MDA-MB-231
(Y527F c-Src) cells. The expression of rat Exo70 did not affect EXO70 siRNA
knockdown efficiency on endogenous Exo70 (Figure 3.4A). I found that the MMP
secretion defects in siEXO70(1)-treated cells was restored by the expression of rat
Exo70 (Figure 3.4A). In all treatments, quantification of secreted MMP levels was
carried out based on three independent experiments for each group. The levels of
secreted MMP were represented as normalized secretion index, which was calculated
as the percentage of secreted MMP relative to that of the control cells. As shown in
Figure 3.4B, in EXO70 and SEC8 siRNA-treated cells, secretion of MMP-2 decreased
to 8% for siEXO70(1), 18% for siEXO70(2), and 17% for siSEC8; secretion of
MMP-9 decreased to 10% for siEXO70(1), 19% for siEXO70(2), and 18% for siSEC8.
The expression of rat Exo70 in siEXO70-treated cells largely rescued the secretion
defect of MMPs in these cells (72% for MMP-2 and 81% for MMP-9). These
findings strongly suggest that the exocyst complex plays a critical role in the
secretion of the key invadopodia-associated MMPs.
Our lab has previously shown that Exo70 directly interacts with
phosphatidylinositol 4,5-bisphosphate (PI(4,5)P2) through its C-terminus, and that
this interaction is important for the association of Exo70 with the plasma membrane
and the targeting of post-Golgi secretory vesicles to the plasma membrane
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Figure 3.4 The exocyst is required for the secretion of MMPs in MDA-MB-231
(Y527F c-Src) cells. (A) Secretion of MMP-2 and MMP-9 was examined in
MDA-MB-231 (Y527F c-Src) cells with Exo70 or Sec8 RNAi knockdown. Cell
culture media were collected and concentrated. MMP-2 and MMP-9 activities were
analyzed by gelatin zymography. Knockdown of Exo70 or Sec8 led to dramatic
decreases of MMP-2 and MMP-9 secreted in the media (top panel). Expression of rat
Exo70 rescued MMP secretion in Exo70 knockdown cells (siEXO70(1) + rExo70).
The amounts of endogenous Exo70 and Sec8, and the amount of extragenically
expressed GFP-tagged rat Exo70 were detected by Western blot using anti-Exo70 and
Sec8 monoclonal antibodies (middle panel). Actin was used as the loading control
(bottom panel). A and B are duplicates of each experiment. (B) Quantification of
secreted MMP levels was performed based on three independent experiments for each
group. Secreted MMPs were represented as normalized secretion index, which was
calculated as the amount of secreted MMP relative to that of the control cells. Error
bars, SD. *p < 0.01.
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(He et al., 2007; Liu et al., 2007). Disrupting the interaction between Exo70 and the
plasma membrane by overexpressing a C-terminal truncated version of Exo70
(GFP-Exo70∆C, a.a. 1-408) would be expected to impair the secretion of MMPs. As
shown in Figure 3.5, A and B, expression of Exo70∆C caused a significant decrease
in the amounts of MMP-2 and MMP-9 secreted in the medium (81% decrease for
MMP-2 and 82% decrease for MMP-9) compared with GFP-transfected cells. I also
examined whether the overexpression of full-length Exo70 (GFP-Exo70) is able to
promote the secretion of MMPs. Gelatin zymography detected elevated levels of
secreted MMP-2 (1.6-fold) and MMP-9 (1.7-fold) in cells transfected with
GFP-Exo70 (Figure 3.5, A and B). The observation that overexpression of Exo70∆C
led to decreased level of secreted MMPs suggests that the plasma-membrane
targeting of the exocyst is crucial for the secretion of MMPs.
The interaction between Exo70 and the Arp2/3 complex is required for
invadopodia formation
Invadopodia
formation
involves
membrane
protrusions
driven
by
Arp2/3-mediated actin polymerization (Yamaguchi et al., 2005). Studies from our lab
have shown that Exo70 directly interacts with the Arpc1 subunit of the Arp2/3
complex and that this interaction is important for actin-based membrane protrusion
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Figure 3.5 Zymography analyses of MMP-2 and MMP-9 secreted from
MDA-MB-231 (Y527F c-Src) cells overexpressing Exo70∆C (amino acids 1–408)
or full-length Exo70. (A) After transfection with GFP-tagged Exo70∆C or Exo70 for
18 h, cell culture media were collected and analyzed by gelatin zymography.
Overexpression of Exo70∆C caused a dramatic decrease in the amounts of MMP-2
and MMP-9 secreted in the media. Overexpression of Exo70 stimulated the secretion
of MMPs (bottom panel). Cell lysates were collected for the detection of
extragenically expressed GFP-tagged Exo70 proteins (top panel). Actin staining was
shown as a loading control (middle panel). (B) Quantification of MMP secretion.
Error bars, SD. *p < 0.01.
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and cell migration (Zuo et al., 2006). To investigate whether the exocyst-Arp2/3
interaction also plays a role in invadopodial activity, I first examined this interaction
in carcinoma cells with different levels of invasiveness. It has been well demonstrated
that the CA domain from the WASP family proteins directly interacts the Arp2/3
complex. I then used a recombinant GST-CA fusion protein to pull down the Arp2/3
complex from cells with various levels of invasiveness (HeLa cells, the parental
MDA-MB-231 cells, and the Y527F c-Src cells) and examined the interaction of the
exocyst with Arp2/3 in these cells. As shown in Figure 3.6A, a much greater amount
of Exo70 was pulled down by GST-CA from Y527F c-Src cells than from parental
MDA-MB-231 cells, and less Exo70 was pulled down from HeLa cells than from
parental MDA-MB-231 cells. As a control, similar amounts of Arp3 were pulled
down. On the other hand, neither Arp3 nor Exo70 was pulled down by GST. In
addition, I did not detect any direct interaction between Exo70 and CA or VCA
(Figure 3.6B). This observation indicated that the Exo70-Arp2/3 interaction was
much stronger in Y527F c-Src cells than in parental MDA-MB-231 cells. As the
Y527F c-Src cells are more invasive because of Src activation, this result suggests
that the exocyst-Arp2/3 interaction is up-regulated in cells with high invasive
potential.
Our lab has previously shown that the exo70 mutant, Exo70∆628–630, is
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Figure 3.6 The interaction between Exo70 and the Arp2/3 complex is required
for invadopodia formation. (A) The interaction between Exo70 and the Arp2/3
complex was examined in cells with different levels of invasiveness. GST-CA
pulldown assay was performed in HeLa cells, MDA-MB-231 parental and c-Src
(Y527F)-transfected cells as described in Materials and Methods. Although
GST–CA–Sepharose (the CA domain of N-WASP) pulled down similar amounts of
Arp2/3 in HeLa, MDA-MB-231 parental and c-Src (Y527F)-transfected cells, more
Exo70 bound to Arp2/3 in c-Src (Y527F)-transfected cells than in MDA-MB-231
parental cells and less Exo70 bound to Arp2/3 in HeLa cells than in MDA-MB-231
parental cells. The inputs and the bound Exo70 and Arp2/3 were analyzed by Western
blot using anti-Exo70 monoclonal and anti-Arp3 polyclonal antibodies, respectively.
GST alone was used as a negative control in the binding assay. (B) The interaction
between Exo70 and CA/VCA was tested by in vitro GST pulldown assay. Glutathione
Sepharose conjugated with 20 µg of GST or GST-Exo70 (labeled on the top) was
incubated with either 15 µg of Hisx6-tagged CA or VCA (labeled at the bottom). The
input (1/20 of the total) and bound Hisx6-CA or Hisx6-VCA proteins were analyzed
by western blot using an anti-Hisx6 monoclonal antibody. The molecular weights
(“MW”) are marked to the left (in kDa). (C) Matrix degradation assay was performed
in MDA-MB-231 (Y527F c-Src) cells transfected with GFP-tagged Exo70∆628–630 or
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GFP alone. After transfection with GFP-tagged Exo70∆628–630 or GFP for 18 h, cells
were cultured on Alexa 568–labeled gelatin for 4 h and then processed for microscopy.
Individual and merged images of GFP-Exo70 fluorescence (green), F-actin (blue),
and gelatin (red) were shown. Overexpression of GFP-tagged Exo70∆628–630 in Y527F
c-Src cells largely inhibited matrix degradation, whereas transfection with GFP did
not affect invadopodia formation. (D) Quantification of percentages of cells with
different degradation levels in each treatment in B. Error bars, SD. *p < 0.01.
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specifically deficient in interacting with the Arp2/3 complex (Zuo et al., 2006). I thus
tested whether the overexpression of Exo70∆628–630 would affect invadopodia
formation. As shown in Figure 3.6C, overexpression of GFP-tagged Exo70∆628–630
(GFP-Exo70∆628–630) in Y527F c-Src cells largely impaired matrix degradation,
whereas transfection with GFP did not affect invadopodia formation. The percentage
of
cells
without
any
invadopodia
was
substantially
increased
in
Exo70∆628–630-transfected cells (Figure 3.6D, 66% for GFP-Exo70∆628–630-transfected
cells and 22% for GFP-transfected cells). Overall, the above results suggest that the
interaction between the exocyst and the Arp2/3 complex plays an important role in
the formation of invadopodia.
Next, I directly tested whether Exo70 affects Arp2/3-mediated actin
polymerization in cell lysates using pyrene actin assay (Kouyama and Mihashi, 1981).
In this assay, a fraction of actin monomers were labeled with pyrenyl iodoacetamide
on Cys-374. Because the fluorescence of pyrene increases 20 times upon actin
polymerization, the fluorescence intensity will be proportional to the concentration of
actin polymers. Thus, the kinetics of actin polymerization can be assessed by
measuring the fluorescence intensity in real time using a fluorometer.
The lysates of Y527F c-Src cells with various siRNA treatments were collected
and mixed with pyrenyl-actin in the presence of the VCA domain of mammalian
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N-WASP. siRNA knockdown efficiency as well as the amounts of actin and Arp2/3 in
cell lysates was examined in all siRNA-treated samples (Figure 3.7A). Similar
amounts of actin and Arp3 were observed for all the samples. As shown in Figure
3.7B, actin polymerization was inhibited in lysates from Exo70 knockdown cells
compared with control siRNA-treated cells, as revealed by longer lag phase and
decreased actin polymerization rate. As a control, cell lysates had little actin
polymerization activity in the absence of VCA. SEC8 siRNA-treated cells had a much
smaller effect on actin assembly compared with EXO70 siRNA-treated cells. I also
examined the final levels of actin polymerization in all siRNA-treated samples after
24 h. As shown in Figure 3.7D, the final level of actin polymerization is similar
among all the samples. Moreover, I checked the amount of available actin in each
reaction. As shown in Figure 3.7E, the amounts of actin in the pellet (polymerizable
actin) and supernatant (unpolymerizable actin) fractions were at the same level in
each treatment. This result suggests that the differences in initial actin polymerization
rates are not likely due to the difference in the levels of polymerization-competent
actin. Quantification was carried out based on normalized polymerization rate.
Polymerization rate was represented as the slope of the linear approximation line of
each curve, and the normalized polymerization rate was calculated as the rate of each
treatment relative to control siRNA-treated cells. As shown in Figure 3.7C, EXO70
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Figure 3.7 Exo70 is involved in Arp2/3-mediated actin polymerization in the cell.
(A) Lysates were prepared from MDA-MB-231 (Y527F c-Src) cells transfected with
indicated siRNA oligos. Exo70 and Sec8 knockdown levels were examined by
Western blot, along with the amounts of actin and Arp3 in cell lysates. (B) The ability
of the lysates to stimulate actin polymerization was analyzed by pyrene actin assay in
the presence or absence of the VCA domain of N-WASP as described in Materials
and Methods. The initial rate of actin polymerization, an indicator of actin nucleation
by Arp2/3, was two fold lower in Exo70 knockdown cells than in the control cells.
Sec8 knockdown slightly decreased the initial rate of actin polymerization. Cell
lysates in the absence of VCA had little actin polymerization activity. (C) Normalized
initial polymerization rates in each treatment in A were calculated by dividing the
actin polymerization rate of each treatment by that of the control siRNA-treated cells.
Three independent measurements for each treatment were carried out. Error bars, SD.
*p < 0.01. (n = 3). (D) The final level of actin polymerization in exocyst knockdown
and GFP-Exo70 overexpression samples. Cell lysates from all the treatments were
ultracentrifuged and mixed with pyrenyl-actin for the actin polymerization reaction as
described in MATERIALS AND METHODS. After 24 hours of incubation at room
temperature, the samples were transferred into a cuvette and the fluorescence
intensity was read in the fluorometer. The final extent of actin polymerization was
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shown to be identical for all the samples tested. (E) The amounts of available actin in
the reactions of all the samples. Actin polymerization reactions were set up as
described above. After 24 hours of incubation for the reactions to reach the steady
state, the samples were ultracentrifuged and the pellet / supernatant fractions of each
sample were collected. The fractions were then subjected to SDS-PAGE and stained
with SYPRO-Red. The amounts of actin in pellet and supernatant fractions were at
the same level in each treatment. (F) Actin polymerization kinetics was analyzed in
lysates from MDA-MB-231 (Y527F c-Src) cells transfected with GFP-Exo70 and
GFP-Exo70∆628–630 mutant. The levels of Exo70, actin, and Arp3 in cell lysates were
detected by Western blot. (G) Lysates from cells expressing GFP-Exo70∆628–630 had a
lower initial actin polymerization rate compared with GFP-transfected cells. Cells
overexpressing GFP-Exo70 had an increased actin polymerization rate compared with
the control cells. (H) Quantification of normalized polymerization rate of each
treatment in D. Error bars, SD. *p < 0.01; n = 3.
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siRNA treatment reduced the normalized actin polymerization rate by more than two
fold compared with control siRNA treatment.
I also tested whether overexpression of Exo70 promotes Arp2/3-mediated actin
polymerization in cell lysates. As shown in Figure 3.7, G and H, lysates from cells
overexpressing GFP-Exo70 showed a decreased lag phase and an increased actin
polymerization rate compared with GFP-transfected cells (1.38-fold that of
GFP-transfected cells). In contrast, lysates from cells overexpressing the Arp2/3
binding-deficient mutant of Exo70, GFP-Exo70∆628–630, had an increased lag phase
and a lower actin polymerization rate compared with control cells (Figure 3.7, G and
H, with a normalized polymerization rate of 0.52). The final levels of actin
polymerization were identical in Exo70 overexpression samples (Figure 3.7, C and D).
The amounts of actin and Arp3 were similar for all the samples (Figure 3.7F).
Collectively, these results suggest that Exo70 plays a positive regulatory role in
Arp2/3-mediated actin polymerization in addition to its function in vesicle tethering
and exocytosis during invadopodia formation.
3.3. Discussion
Invadopodia are actin-based membrane protrusive structures formed by tumor
cells that degrade the extracellular matrix for invasion. Investigation of the molecular
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mechanisms of invadopodia formation and regulation is important for the
understanding of tumor invasion and metastasis. Here, I demonstrate that the exocyst
plays a critical role in invadopodia formation and invasion. The exocyst is enriched at
focal degrading sites, and disruption of its function results in a reduction of
invadopodial activity.
Focal degradation of the ECM barrier is a key feature of invadopodia, which is
achieved by the secretion of proteases that degrade the basement membrane
surrounding the tumor. A number of MMPs, including MMP-2 and MMP-9, have
been shown to play an important role in degrading ECM during tumor invasion (Boyd,
1996; Chen, 1996; Chen and Wang, 1999; Deryugina and Quigley, 2006; Itoh and
Seiki, 2006; Furmaniak-Kazmierczak et al., 2007). MMPs are delivered to the surface
of the tumor cells through exocytosis, and components of membrane traffic
machinery have been implicated in tumorigenesis (Palmer et al., 2002; Cheng et al.,
2004; Steffen et al., 2008; Sakurai-Yageta et al., 2008). The exocyst is involved in
tethering post-Golgi secretory vesicles to the plasma membrane for exocytosis. Here,
using RNAi and dominant-negative mutants to inhibit exocyst function, I demonstrate
that the exocyst is required for the secretion of MMP-2 and MMP-9. Our results are
also in agreement with recent study showing the involvement of the exocyst in the
secretion
of
MT1-MMP,
a
transmembrane protease
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that
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(Sakurai-Yageta et al., 2008). Together, these results indicate that the exocyst
mediates the secretion of different classes of MMPs during tumor invasion.
Although the principal function of the exocyst is to tether secretory vesicles for
secretion, recent studies suggest that the exocyst is also involved in actin-based
membrane protrusion. It was shown that RalA and RalB, members of the Ras family
of small GTP-binding proteins, regulate exocyst function (Brymora et al., 2001;
Moskalenko et al., 2002; Sugihara et al., 2002; Polzin et al., 2002), and the
Ral-exocyst interaction induces filopodia formation through a mechanism that is
independent of exocytosis (Sugihara et al., 2002). The exocyst is involved in cell
migration (Zuo et al., 2006; Rosse et al., 2006; Spiczka and Yeaman, 2008).
Furthermore, Exo70 was found to directly interact with the Arpc1 subunit of the
Arp2/3 complex; EGF, which promotes cell membrane protrusion, stimulates the
interaction between Exo70 and the Arp2/3 complex in HeLa cells (Zuo et al., 2006).
The Arp2/3 complex is the core machinery that nucleates actin for the generation of
the branched actin network underneath the leading edges of the plasma membrane for
membrane protrusion (Pollard and Borisy, 2003). It has been well established that the
Arp2/3 complex is essential for invadopodia formation (Buccione et al., 2004; Lorenz
et al., 2004; Yamaguchi et al., 2005). RNAi-mediated knockdown of members of the
Arp2/3 complex or N-WASP, the activator of Arp2/3, inhibited invadopodia
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formation (Lorenz et al., 2004; Yamaguchi et al., 2005). Microscopy analyses
demonstrated that the Arp2/3 complex is enriched at invadopodia together with
N-WASP (Yamaguchi et al., 2005). Other regulators of the Arp2/3 complex such as
Nck1, Cdc42, cortactin, and WIP have also been shown to be involved in invadopodia
formation (Yamaguchi et al., 2005; Clark et al., 2007). In the present study, we found
that the exocyst has stronger interaction with the Arp2/3 complex in Src-activated
cells compared with parental cells. Overexpression of the exo70 mutant that is
defective in its interaction with Arp2/3 inhibited invadopodia formation. How Src
kinase regulates the Exo70-Arp2/3 interaction is still under investigation. Because
GTPases such as Rac and Rho function downstream of Src kinase, these GTPases
could be potential candidates for this process.
Using the pyrene actin assay, I have found that lysates from Y527F c-Src
MDA-MB-231 cells are capable of stimulating Arp2/3-mediated actin polymerization
in the presence of VCA. I further found that cells with Exo70 knockdown by RNAi or
cells with overexpression of the exo70 mutant deficient in Arp2/3-binding were less
potent in Arp2/3-mediated actin polymerization, whereas lysates prepared from cells
with Exo70 overexpression were more potent in stimulating actin polymerization.
These data are consistent with the fluorescence microscope observation that there
were fewer and dimmer F-actin foci in the Exo70 knockdown cells (Figure 3.1).
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These findings suggest that Exo70 plays a positive regulatory role in Arp2/3-mediated
actin polymerization in cells. This is consistent with the observation that Exo70
overexpression led to extensive membrane protrusions in many types of cultured cells
(Wang et al., 2004; Xu et al., 2005; Zuo et al., 2006).
Overall, my study suggests that the exocyst is involved in invadopodia through
mediating MMP secretion and regulating Arp2/3-mediated actin polymerization. The
results suggest a coordination of protease secretion and cytoskeleton dynamics during
tumor invasion. Future studies will focus on the molecular mechanisms by which
Exo70 regulates actin dynamics and how the exocyst is regulated by upstream
signaling molecules in the cell.
3.4. Materials and Methods
Plasmids and Antibodies
Full-length rat Exo70 (rExo70 FL) cDNA was cloned in-frame in pEGFP-C1
and pJ3-GFP vectors for expression as enhanced green fluorescent protein (EGFP) or
GFP fusions. Exo70∆C (amino acids 1-408) was cloned in-frame in pEGFP-C1. The
exo70 mutant, Exo70∆628–630, was generated using the QuikChange site-directed
mutagenesis kit (Stratagene, La Jolla, CA) with GFP-Exo70 in pEGFP-C1 as a
template (Zuo et al., 2006). Constructs were confirmed by restriction enzyme analysis
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and sequencing. Monoclonal antibodies against Exo70 and Sec8 were generously
provided by Dr. Shu-Chan Hsu (Rutgers University, Piscataway, NJ). Mouse
anti-actin mAb (MAB 1501, clone C4) was purchased from Chemicon, Millipore
(Bedford, MA).
Cell Culture and RNA Interference Treatment
Human breast carcinoma MDA-MB-231 cells and stable lines of parental cells
transfected with Y527F constitutively active c-Src were maintained at 37°C in
DMEM (Invitrogen, Carlsbad, CA) supplemented with 10% FBS, 2 mmol/L
L-glutamine, 100 U ml–1 penicillin, and 100 µg ml–1 streptomycin in a 5% CO2
incubator. Cell transfections were carried out using Lipofectamine 2000 (Invitrogen,
Carlsbad, CA). For RNA interference (RNAi), cells were grown to 50% confluence
and transfected with small interfering RNA (siRNA) duplexes using Lipofectamine
2000.
The
human
EXO70(1)
siRNA
target
sequence
is
5'-GGTTAAAGGTGACTGATTA-3'. The human EXO70(2) siRNA target sequence is
5'-GACCTTCGACTCCCTGATA-3'. The human SEC8 siRNA target sequence is 5'AGAACCTGCTTTCATGCAA-3'. The control Luciferase GL2 siRNA target
sequence
is
5'-AACGTACGCGGAATACTTCGA-3'.
knockdown was determined by Western blot.
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Fluorescent Gelatin Degradation Assay
AlexaFluor 568–conjugated gelatin was prepared by labeling porcine gelatin
(Sigma, St. Louis, MO) with AlexaFluor 568 (Molecular Probes, Eugene, OR)
according to the manufacturer's instructions. Coverslips (18 mm) were precleaned
with 20% nitric acid for 30 min followed by extensive washing and ethanol
sterilization. The coverslips were coated with 50 µg/ml poly-L-Lysine (Sigma) for 20
min at room temperature, washed with PBS, and fixed with 0.5% glutaraldehyde (Ted
Pella, Irvine, CA) for 15 min followed by extensive washing. The coverslips were
then inverted on a drop of gelatin matrix (0.2% gelatin and AlexaFluor 568–labeled
gelatin at an 8:1 ratio) and incubated for 10 min at room temperature. After washing
with PBS, coverslips were incubated in 5 mg/ml sodium borohydride for 15 min,
washed three times in PBS, and finally incubated in 2 ml of DMEM for 2 h before
adding the cells.
To examine the ability of cells to form invadopodia and degrade matrix, 4 x
105 cells were plated on coverslips coated with AlexaFluor 568 and incubated at
37°C for 4 h. Cells were then fixed and prepermeabilized with 10% Formalin/0.1%
Triton X-100 in PBS for 15 min at room temperature. After three washes, cells were
postpermeabilized with 0.5% Triton X-100 for 5 min. Cells were then washed with
PBS, labeled with primary antibodies for 2 h, and followed by labeling with
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secondary fluorochrome-conjugated antibodies for 1 h. Actin filaments were
visualized with Alexa-phalloidin (Molecular Probes). Cells were imaged with the
Leica DM IRB microscope (Deerfield, IL; 100xobjective), a high-resolution CCD
camera (model ORCA-ER, Hamamatsu Photonics, Bridgewater, NJ) and the Leica
TCS SL laser-scanning confocal microscope (63x objective, Deerfield, IL). Images
were processed with Adobe Photoshop (Adobe Systems, San Jose, CA; ver. 7.0). For
quantification of degradation, percentage of cells with different degradation levels
was calculated. Degradation levels of individual cells are reported as the total area of
the degraded zones per cell relative to the area of the whole cell. The area of degraded
matrix
in
the
fields
was
measured
using
ImageJ
1.73v
software
(http://rsb.info.nih.gov/ij/). Dark spots on the bright, fluorescent gelatin matrix were
inverted and thresholded into black dots on white background, followed by automatic
outlining of the dots (Figure 3.1D). The area of the whole cell was measured by
manually outlining the cell boundary (Figure 3.1D).
Zymography
The "in-gel" zymography was used for the detection of MMPs on the basis of
their different molecular weights (Van den Steen et al., 2002; Mott and Werb, 2004).
After treatments, cells were cultured in serum-free DMEM for 48 h. Cell culture
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media were concentrated 50 times by filtration on Microcon concentrators (Millipore).
Samples were mixed with SDS loading buffer (10% SDS, 50% glycerol, 0.4 M Tris,
pH 6.8, and 0.1% bromophenol blue) and separated on 8% polyacrylamide/0.3%
gelatin gels. Gels were then washed in 2.5% Triton X-100, 30 min each time, and
incubated in reaction buffer (50 mM Tris, pH 8.0, and 5 mM CaCl2) at 37°C for
24–48 h. After the reaction, gels were stained with staining buffer (0.12% Coomassie
blue R-250, 50% methanol, and 20% acetic acid) for 1 h and destained overnight with
destaining buffer (22% methanol and 10% acetic acid). Gels were scanned using
CanoScan 4400F. Gel loadings were normalized to total protein measured with a
Bio-Rad Protein Assay (Richmond, CA).
Glutathione S-Transferase-CA (Cofilin and Acidic Domains of N-WASP)
Pulldown Assay
MDA-MB-231 parental and c-Src (Y527F)-transfected cells were lysed in the
lysis buffer (20 mM Tris-HCl, pH 7.5, 25 mM KCl, 1 mM MgCl2, 0.5 mM EGTA, 1
mM DTT, 0.5% Triton X-100, and protease inhibitors). A high-speed centrifugation
(12,000 rpm, 15 min) was carried out, and 1 ml of precleared cell lysates (1.5 mg
total protein) was mixed with 20 µl (50% vol/vol) of glutathione Sepharose
conjugated with 10 µg glutathione S-transferase (GST)-CA (cofilin and acidic
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domains of N-WASP) proteins. After incubation at 4°C overnight, the beads were
washed five times with the lysis buffer, and the bound proteins were analyzed by
Western blot using antibodies against Exo70 and Arp3. GST alone was used as a
negative control in the experiment.
Pyrene Actin Assay
Cell lysates were collected in buffer B (20 mM Tris-HCl, pH 7.5, 25 mM KCl,
1 mM MgCl2, 0.5 mM EGTA, 0.1 mM ATP, and protease inhibitor cocktail [Sigma
P8340, 4-(2-aminoethyl) benzenesulfonyl fluoride, pepstatin A, E-64, bestatin,
leupeptin, and aprotinin], and 1 mM DTT) and spun successively at 16,000 x g for 15
min and 80,000 rpm in a Beckman TLA-100.3 rotor (Fullerton, CA) for 20 min at
4°C. The resulting high-speed supernatant (HSS) was used for later experiments.
Pyrenyl-actin was dissolved in column buffer (TEA, 0.3 mM CaCl2, 0.1 mM EDTA,
0.7 mM ATP, and 6.25 mM NaN3) for 1 h, spun at 80,000 rpm in a Beckman
TLA-100.3 rotor for 20 min at 4°C to remove F-actin, and mixed with Mg2+
converting buffer for 5 min to convert Ca2+-actin to Mg2+-actin. Mg2+-pyrenl-actin
was then diluted in the polymerization buffer (60 mM KCl, 2.5 mM NaCl, 0.6 mM
MgCl2, 5 mM Tris-HCl, pH 7.5, 2.5 mM HEPES, pH 7.1, 0.5 mM EGTA, 30 µM
CaCl2, 0.2 mM ATP, and 0.3 mM NaN3) to a final concentration of 1.5 µM and
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immediately mixed with HSS, which contained 2.4–2.6 µM unlabeled G-actin as
estimated by Western blot (data not shown) in the presence of 0.2 mM ATP and 50
nM GST-tagged verprolin, cofilin, and acidic (VCA) domain of mammalian N-WASP.
The mixture was quickly transferred into a cuvette, and the fluorescence intensity was
read every 5 s in a fluorometer. Three independent measurements were carried out for
each treatment. Polymerization curves and rates were obtained using Excel
(Microsoft, Redmond, WA). The polymerization rate was represented as the maximal
slope of the elongation phase of each curve, and the normalized polymerization rate
was calculated as the rate of each treatment relative to the control treatments. Each
polymerization curve was first smoothed using Sigmaplot software to elicit trends
from noisy data. Then the slope of each point on the curve was calculated as the slope
of the line between this point and the point 60 s ahead. The slopes of all the points on
each curve were used to create a new plot. The maximal value of this slope curve is
regarded as the actin polymerization rate.
* This work has been published in Molecular Biology of the Cell in June 2009.
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The role of Exo70 in Arp2/3-mediated actin assembly
during cell migration
Abstract
Efficient cell migration requires the coordination of actin reorganization and
membrane trafficking. The Arp2/3 complex is a key nucleating factor for the
generation of branched actin network at the leading edge of migrating cells. The
exocyst is an octameric protein complex essential for tethering secretory vesicles to
specific domains of the plasma membrane for exocytosis. It has been shown that the
exocyst component Exo70 interacts with the Arp2/3 complex via Arpc1 (a subunit of
the Arp2/3 complex) and plays an important role in cell migration. However, the
molecular implication of Exo70-Arp2/3 interaction is unclear. In this study, I found
that Exo70 enhances Arp2/3-mediated actin polymerization in concert with WAVE2.
TIRFM examination of actin filament dynamics further suggests that Exo70 acts by
promoting Arp2/3-mediated F-actin branching in the presence of WAVE2. In
addition, I have examined how the Exo70-Arp2/3 interaction is regulated in the cell
downstream of EGF signaling. Overall, these results suggest that Exo70 synergizes
with WAVE2 to promote dendritic actin nucleation through a direct interaction with
the Arp2/3 complex, which may reveal a novel mechanism for regulating actin
polymerization and branching during cell migration.
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4.1. Introduction
Cell migration plays critical roles in the development and maintenance of living
organisms. Cell migration is a highly integrated process that includes a series of steps:
establishment of cell polarity, extension of membrane protrusions at the leading edge,
formation of the adhesion with extracellular matrix (ECM), contraction of the cell
body and release from the ECM at the cell rear (Ridley et al., 2003; Sheetz et al.,
1999). The completion of efficient cell migration requires the coordination of actin
dynamics and membrane trafficking. However, how they are functionally coupled is
still under investigation.
To migrate, a cell first forms membrane protrusions at the leading edge, which
is driven by the assembly of a branched network of actin filaments. The Arp2/3
complex plays a crucial role in the initial nucleation step for the generation of
branched actin networks, a rate-limiting step in actin polymerization. The Arp2/3
complex is thought to mimic an actin dimer to function as the “seed” for the initiation
of a new actin filament that emerges from an existing filament in a 70° y-branch
configuration.
Purified Arp2/3 complex has low actin nucleation activity, but can be activated
by a number of nucleation-promoting factors (NPFs). The major NPFs in mammals
are the WASP and SCAR/WAVE family proteins, which include the two WASP
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family proteins WASP and N-WASP, and the three WAVE family proteins WAVE1,
WAVE2 and WAVE3. All of them share a conserved carboxyl-terminal VCA
(verprolin homology, central, and acidic) domain, which is the region required for
Arp2/3 activation. The V and C regions bind G-actins, and the CA region mediates a
direct interaction with the Arp2/3 complex which promotes a conformational change
that activates the complex. The activated Arp2/3 complex along with the actin
monomer delivered by NPF forms an Arp2-Arp3-actin trimer that functions as the
nucleation core for the elongation of the new daughter filament. As a result, the
daughter filament is anchored to the side of the mother filament as a branch.
The organization of the N-terminal region, which consists of domains that are
thought to provide a link with regulatory proteins, is quite different among
WASP/WAVE family proteins. WASP and N-WASP contain a WASP homology 1
(WH1) domain, also known as an Ena/VASP homology 1 (EVH1) domain, and a
CRIB domain, which binds to the small GTPase Cdc42. The WH1 domain interacts
with the WASP-interacting protein (WIP) family of proteins, which is thought to
inhibit the activity of WASP or N-WASP. By contrast, the SCAR/WAVE proteins
contain a SCAR homology domain (SHD), and do not contain any type of
GTPase-binding motif. Both WASPs and SCARs/WAVEs contain a basic region,
which mediates the binding to PtdIns(4,5)P2 and PtdIns(3,4,5)P3, respectively, and
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localize the proteins to the plasma membrane (Rohatgi et al., 2000; Oikawa et al.,
2004). Thus, the N-terminal domains determine the regulation and subcellular
localization of WASP and SCAR/WAVE proteins.
In resting cells, WASP and N-WASP are predominantly found in an
auto-inhibited conformation in which intramolecular interactions between GBD and
the C region obscure the regions that are required for Arp2/3 activation. This
auto-inhibition is released by the competitive binding of the small GTPase Cdc42 and
PI (4,5)P2 (Kim et al., 2000; Rohatgi et al., 2000; Prehoda et al., 2000). Unlike
WASPs, WAVE proteins are not auto-inhibited and they form a complex with four
other proteins – PIR121, Nap1, Abi and HSPC300 in the cell (Eden et al., 2002).
Upon stimulation, the small GTPase Rac associates with the complex via direct
interaction with the PIR121 subunit and leads to WAVE activation (Ibarra et al.,
2006). Activation of the WAVE2 complex requires simultaneous interactions with
prenylated Rac-GTP and acidic phospholipids, as well as a specific state of
phosphorylation (Lebensohn and Kirschner, 2009).
WASP and WAVE family proteins are implicated in distinct cellular functions.
WASP is specifically expressed in hematopoietic cells and plays a role in
phagocytosis. N-WASP is universally expressed, and functions in a variety of cellular
processes, including endocytosis, organelle motility, invadopodia formation, and
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vesicular trafficking (Merrifield et al., 2004; Lommel, et al., 2001; Yanagawa et al.,
2001; Yamaguchi et al., 2005; Gasman et al., 2004). WAVE2 and WAVE3 are
essential for lamellipodia formation and directional cell migration, whereas WAVE1
is required for the formation of dorsal ruffles and functions to stabilize lamellipodia
protrusions (Suetsugu et al., 2003; Yamazaki et al., 2003 & 2005).
In addition to regulation by WASP and WAVE proteins, recent studies have
indicated that the Arp2/3 complex is phosphorylated by serine/threonine and tyrosine
kinases. For example, the p21-activated kinase (PAK) phosphorylates Arpc1 on
Thr21 (Vadlamudi et al., 2004); the MARK-activated protein kinase 2 phosphorylates
Ser77 in Arpc5 (Singh et al. 2003); mass spectrometry revealed phosphorylated
Thr237 and Thr238 in Arp2 (LeClaire et al., 2008). Although phosphorylation of the
Arp2/3 complex has been shown to regulate its actin nucleating activity and increase
cell motility, the molecular mechanisms downstream of this phosphorylation are still
unknown.
The p21-activated kinases (PAK) were the first Rho family GTPase-regulated
kinases to be identified in a screen for specific Rho GTPase binding partners in rat
brain cytosol (Manser et al., 1994). Pak kinases are serine/threonine protein kinases
that function as important effectors of Rac and Cdc42 GTPases in response to
extracellular stimuli such as EGF. Paks play fundamental roles in a wide range of
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cellular processes, including cytoskeletal dynamics, cell motility, gene transcription,
cell-cycle progression, cell death and survival signaling (Sells et al., 1997;
Slack-Davis et al., 2003; Beeser et al., 2005; Eblen et al., 2002). The most
well-characterized function of the Pak kinases is its regulation of cytoskeletal
organization and cell motility. Immunofluorescence analysis using Pak1-specific
antibody have shown that PAK1 redistributed from the cytosol into cortical actin
structures after cell stimulation (Dharmawardhane et al., 1997). Overexpression of
Pak induced the formation of lamellipodia, filopodia and membrane ruffles (Sells et
al., 1997). The molecular basis for these activities is not completely understood, but a
number of known Pak substrates may account for the effects of Pak on the
cytoskeletal structure. Arpc1 is a newly discovered substrate of PAK1 kinase and the
phosphorylation of Arpc1 by PAK1 was indicated to be required for both constitutive
and growth-factor-induced cell motility (Vadlamudi et al., 2004). However, the exact
molecular mechanism of how the Pak1-phosphorylation on Arpc1 regulates actin
dynamics is still unclear.
In addition to actin reorganization, cell migration also requires polarized
exocytosis for the localized delivery of membrane components to the front of the cell.
The exocyst is an evolutionarily conserved octameric protein complex that mediates
the targeting of post-Golgi and endocytic recycling vesicles at specific domains of the
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plasma membrane for exocytosis (Guo et al., 2000; Hsu et al., 2004). The exocyst
complex is required for secretion, thus is involved in many cellular processes that
require polarized exocytosis including yeast budding, cell-cell adhesion, cytokinesis
and neurite extension (TerBush and Novick, 1995; Matern et al., 2001; Hazuka et al.,
1999; Chen et al., 2006). Furthermore, the exocyst has also been implicated in several
cellular events through mechanisms that are beyond its role in exocytosis. Recently,
the exocyst has been shown to be involved in cell migration through its direct
interaction with the Arp2/3 complex (Zuo et al., 2006). The exocyst component
Exo70 was shown to directly bind to Arpc1, a component of the Arp2/3 complex.
Arpc1 is an essential subunit for the nucleating activity of Arp2/3, which interacts
with both the CA region of NPFs and the F-actin filaments during the formation of
actin branches (Kelly et al, 2006; Rouiller et al, 2008). The Exo70-Arpc1 interaction
is regulated by epidermal growth factor (EGF) signaling and is important for the
regulation of actin assembly at the leading edge of migrating cells. However, the
molecular function and the cellular regulation of the Exo70-Arpc1 interaction are still
under investigation.
Here, I have identified Exo70 as a positive regulator for the activity of Arp2/3
and WAVE2 in dendritic actin nucleation. It suggests a potential molecular
connection between machineries that control exocytosis and actin organization. I have
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also examined the possible mechanisms of the molecular basis of the stimulatory
effect of Exo70 in actin assembly. In addition, I have examined how the
Exo70-Arp2/3 interaction is regulated in the cell downstream of EGF signaling.
4.2. Results
rExo70 enhances Arp2/3-mediated actin polymerization
Exo70 has a direct interaction with the Arp2/3 complex. Moreover, Exo70 has
been shown to colocalize with Arp2/3 at the leading edge of the migrating cells and is
required for the formation of lamellipodia (Zuo et al., 2006). Based on these
observations, I hypothesize that Exo70 promotes the nucleation activity of Arp2/3 in
actin assembly. The activity of Arp2/3 and its activators in actin polymerization was
examined using the standard pyrene actin assay (Kouyama and Mihashi, 1981). In
this assay, a fraction of actin monomers were labeled with pyrenyl iodoacetamide on
Cys-374. Because the fluorescence of pyrene increases 20 times upon actin
polymerization, the fluorescence intensity will be proportional to the concentration of
actin polymers. Thus, the kinetics of actin polymerization can be assessed by
measuring the fluorescence intensity in real time using a fluorometer.
Mammalian Arp2/3 complex was purified from cell lysates by affinity
chromatography
using
recombinant
GST-CA
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immobilized
on
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Sepharose-4B (Figure 4.1A). rExo70 was purified from E. coli as a GST fusion
protein and then treated with Prescission Protease to cleave the GST tag.
Recombinant GST-WAVE2 was purified from E. coli (Figure 4.1A). To examine
actin assembly, Arp2/3 and proteins to be tested were mixed and pre-incubated at 4°C
for 30 min prior to addition to the actin assembly mix. As shown in Figure 4.1B, the
purified Arp2/3 has a low activity in promoting actin polymerization. Addition of
WAVE2 enhanced the actin nucleation activity of Arp2/3 as previously reported. In
contrast, addition of rExo70 alone did not affect the activity of the Arp2/3 complex.
rExo70 does not promote or inhibit the spontaneous actin nucleation (Figure 4.1B).
Thus, Exo70 neither nucleates actin polymerization like an actin nucleator, nor
functions like NPFs to activate the Arp2/3 complex.
Next, I examined whether Exo70 affects the activity of Arp2/3 in the presence
of WAVE2. As shown in Figure 4.1B, addition of rExo70 in the presence of Arp2/3
and WAVE2 further promoted actin assembly, as indicated by a shortened lag phase
and increased actin polymerization rate compared to samples without Exo70. As a
control, GST had no effect on the kinetics of actin polymerization mediated by Arp2/3
and WAVE2 (Figure 4.1B). The effect of Exo70 on actin assembly was quantified by
measuring the time needed to reach half maximal polymerization and the rate of the
actin polymerization at the elongation phase (Figure 4.1C). And the effect of rExo70
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Figure 4.1 rExo70 enhances actin polymerization mediated by the Arp2/3
complex and WAVE2. (A) Proteins used in the pyrene actin assay. The molecular
weights (“MW”) are marked to the left (in kDa). (B) Actin polymerization kinetics
for 2 µM monomeric rabbit muscle actin (20% pyrenyl-labeled) in the presence of
proteins to be tested including Arp2/3, WAVE2, GST and rExo70. The amounts of
various proteins added in the reactions were indicated below the curve. (C) Diagram
showing the time to reach half maximal polymerization (left panel) or the rate of actin
polymerization during the elongation phase (right panel) for reactions described in
(B). Error bars represents standard deviation. n=3. (D) Dose-dependent enhancement
of the Arp2/3 activity by rExo70. The graph shows the assembly of 2 µM G-actin in
the presence of 15 nM Arp2/3, 25 nM WAVE2, and 0-2000 nM rExo70. The amounts
of rExo70 were indicated below the curve.
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in Arp2/3-WAVE2 mediated actin polymerization was dose-dependent, which
saturated at an Exo70 concentration below 2 µM (Figure 4.1D).
Exo70 promotes actin nucleation in synergy with Arp2/3 and WAVE2
Since Exo70 synergizes with WAVE2 in activating Arp2/3, examining the
morphology of the filaments, such as the length of filaments, or the number of
branches, may provide insights to the molecular mechanism of the action of Exo70.
In order to directly visualize the dynamics of individual actin filaments in real time,
total internal reflection fluorescence microscopy (TIRFM) was applied. With TIRFM,
only filaments captured at the surface of the flow chamber will be illuminated, while
the background fluorescence from the solution was largely eliminated (Amann and
Pollard, 2001; Kuhn and Pollard, 2005). Experiments were carried out in flow
chambers treated with NEM-myosin to capture the actin filaments at the surface of
the coverslip. Arp2/3 and proteins to be tested were mixed in the actin polymerization
buffer and incubated at 4°C for 30 min. G-actin (8% Rhodamine labeled) was then
mixed with the pre-incubated samples which contain (1) Arp2/3 + WAVE2, (2)
Arp2/3 + WAVE2 + rExo70 and immediately transferred into the flow chamber for
TIRF microscopic observation. Images were recorded in real time at 1.14 sec per
frame after the polymerization started. As shown in Figure 4.2, A and B, more actin
branches were formed in the presence of rExo70 (marked by red dots). Quantification
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Figure 4.2. Elongation and branching of actin filaments visualized by TIRFM.
(A, B) G-Actin (0.8 µM, 8% rhodamine-labeled) was polymerized in fluorescence
buffer (60 mM KCl/2.5 mM NaCl/0.6 mM MgCl2/5 mM Tris, pH 7.5/2.5 mM
HEPES, pH 7.1/0.5 mM EGTA/30 µM CaCl2/0.2 mM ATP/0.3 mM NaN3/1%
glucose/100 µg/ml glucose oxidase/20 µg/ml catalase/0.5% Methylcellulose) in a
flow chamber with 6 nM Arp2/3, 10 nM WAVE2, in the presence (B) or absence (A)
of 1 µM rExo70. The coverslip of the flow chamber was coated with 6 nM
NEM-myosin for 1.5 min, and blocked with 1% BSA for 4 min before use. Exposures
of 1 sec were collected every 1.14 sec for 10 min. Shown are frames representing the
time course of elongation and branching. Red dots indicate the branch points. (C)
Images captured for samples with (right panel) or without (left panel) rExo70 at
663sec after the polymerization reaction started. Actin branches are labeled with red
dots. (D) Quantification of branch numbers in each treatment. Three movies were
analyzed for each category. Error bars stand for standard errors. (E) Total length of
F-actin filaments was measured for each treatment. Three movies were analyzed for
each category. Error bars stand for standard errors.
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of the branch numbers was carried out based on three independent experiments for
each group. Each branch nucleation event of every 100 seconds was counted during
the first ~600 seconds from the beginning of the polymerization reaction. As shown
in Figure 4.2, C and D, the branch numbers of samples with rExo70 was 2-3-fold
higher than samples without rExo70. The total length of F-actin filaments in the field
was also measured for each treatment (Materials and Methods). As shown in Figure
4.2E, the sample with rExo70 has similar amount of total F-actin filaments as
compared to the sample without rExo70, which suggests that the difference in branch
numbers between the treatments is not likely due to dfferent amount of total F-actin
filaments in each treatment. As a control, GST had no effect on actin branching in the
experiment (data not shown). Overall, these observations suggest that Exo70
facilitates Arp2/3-WAVE2 mediated actin polymerization by promoting the
generation of branched actin filaments.
Examine whether Exo70 directly interacts with WAVE2
Since Exo70 promotes actin assembly synergistically with Arp2/3 and WAVE2, it
is possible that Exo70 directly binds to WAVE2 and regulates its activity through direct
interaction. Recombinant GST-WAVE2 was conjugated to Glutathione Sepharose and
incubated with HeLa cell lysates or the cell lysates expressing GFP-rExo70 or
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Figure 4.3 GST-WAVE2 pulled down endogenous and GFP/myc-tagged rExo70
from HeLa cells. HeLa cell lysates alone or the cell lysates transfected with
GFP-rExo70 or myc-rExo70 were incubated with glutathione Sepharose conjugated
with GST-WAVE2. The inputs and the bound endogenous, GFP and myc-tagged
Exo70 were analyzed by Western blot using anti-Exo70 (A), anti-GFP or anti-myc (B)
monoclonal antibodies, respectively. GST alone was used as a negative control in the
binding assay.
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myc-rExo70. The bound proteins were analyzed by Western blot using antibodies
against Exo70, GFP or myc tag. As shown in Figure 4.3, GST-WAVE2, but not GST
alone, was able to pull-down endogenous (Figure 4.3A), GFP-Exo70 and myc-Exo70
(Figure 4.3B) from HeLa cell lysates, respectively. Further investigations on
Exo70-WAVE2 interaction will be discussed in the Discussion part.
Examine whether Pak1 phosphorylation stimulates the interaction between
Exo70 and Arpc1 using the in vitro binding assay.
While the interaction between Exo70 and the Arp2/3 complex has been shown
to be regulated by epidermal growth factor (EGF) signaling, the signaling pathway
downstream of EGF that mediates the interaction still remains unclear. Since PAK1
kinase phosphorylates Arpc1 and is downstream of EGF signaling, it is likely that
Pak1 phosphorylation promotes Exo70-Arpc1 interaction in response to EGF stimuli.
To test whether Pak1 phosphorylation promotes Exo70-Arpc1 interaction, an
Arpc1 mutant, in which the site of Pak1 phosphorylation (threonine 21) is mutated to
glutamic acid (T21E) was used in the experiment. Threonine 21 is the only site in
Arpc1 that is phosphorylated by Pak1, and the Arpc1T21E mutant has been shown to
functionally mimic the phosphorylated form of Arpc1 in promoting cell motility
(Vadlamudi et al., 2004). In vitro binding assay was carried out using GST-tagged
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Figure 4.4. Examine the in vitro interaction of GST-tagged rExo70 and
His(6)-tagged Arpc1 wt or Arpc1 (21E) mutant. Glutathione Sepharose conjugated
with GST or GST-rExo70 was incubated with either His(6)-tagged Arpc1 wt or
Arpc1(T21E) mutant. The input and the bound Arpc1 proteins were analyzed by
western blot using anti-His(6) monoclonal antibody.
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rExo70 and His-tagged Arpc1 T21E or wild type Arpc1, which were all purified from
E.coli. GST-rExo70 was conjugated to the glutathione Sepharose and then incubated
with increasing concentrations of His-tagged Arpc1 T21E or wild type Arpc1. The
bound proteins were analyzed by Western blot using antibodies against His tag. As
shown in Figure 4.4, Arpc1 (T21E) mutant bound slightly stronger to Exo70 than the
wild type Arpc1. Further investigations on the regulation of Exo70-Arpc1 interaction
by Pak1 in vivo are included in the Discussion part.
4.3. Discussion and future perspectives
Exo70 is an essential component of the exocyst complex, which is the key
regulator of the tethering step in exocytosis mediating the initial contact of the
secretory vesicles with the plasma membrane. Exo70 has also been implicated in cell
migration through direct interaction with the Arp2/3 complex. However, the
molecular basis of its function in cell migration is unknown. In this study, I found that
in addition to functioning in exocytosis, Exo70 also functions in regulating
Arp2/3-mediated actin polymerization. Even though Exo70 is not able to promote
actin nucleation on its own or activate Arp2/3 like an NPF, it enhances Arp2/3
activation in concert with WAVE2. TIRFM examination of actin filament dynamics
suggests that Exo70 acts by promoting the generation of branched actin filaments in
Arp2/3-WAVE2-mediated actin polymerization. Overall, these results suggest that
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Exo70 synergizes with WAVE2 to promote dendritic actin nucleation through a direct
interaction with the Arp2/3 complex, which may reveal a novel mechanism for
regulating actin dynamics.
4.3.1. Examine the molecular mechanisms for the positive effect of Exo70 in
Arp2/3-WAVE2-mediated actin polymerization
The stimulatory effect of Exo70 in Arp2/3-mediated actin polymerization
suggests that Exo70 is a novel positive regulator of the Arp2/3 complex. Despite its
direct interaction with Arp2/3, Exo70 does not activate Arp2/3 on its own, which is
distinct from typical NPFs. Instead, Exo70 functions in synergy with WAVE2 to
activate Arp2/3. Exo70 acts, at least partially, by promoting the generation of actin
branches. However, how Exo70 facilitates WAVE2 in Arp2/3 activation still remains
elusive.
Since Exo70 cannot promote actin assembly on its own, it is unlikely that
Exo70 activates actin polymerization by directly increasing the rate of actin
nucleation or accelerating the barbed end elongation, or severing the newly formed
actin filaments, which generates more barbed ends for elongation. Rather, since the
positive effect of Exo70 on actin polymerization is mediated by Arp2/3 and WAVE2,
it may function through the following mechanisms:
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1. Exo70 may promote the interaction between Arp2/3 and WAVE2, which in turn
facilitates the activation of Arp2/3 by WAVE2.
To test whether Exo70 regulates the function of WAVE2 through direct
interaction, I examined the interaction between Exo70 and WAVE2 as the first step.
By GST pulldown assay, I found that recombinant GST-WAVE2 could pull down
Exo70 from the cell lysates, which suggests that Exo70 interacts with WAVE2.
However, it is possible that this association is through other proteins. To further test
whether there is a direct interaction between Exo70 and WAVE2, an in vitro binding
assay using purified proteins will be carried out. If Exo70 directly interacts with
WAVE2, the domain of Exo70 that binds to WAVE2 will be further mapped.
Furthermore, the in vivo functional implication of Exo70-WAVE2 interaction will be
examined. Since WAVE2 and Exo70 are both required for lamellopodia formation,
the interaction between Exo70 and WAVE2 will be disrupted by a dominant-negative
exo70 mutant that does not bind WAVE2 and the formation of lamellopodia will be
examined.
To directly test whether Exo70 promotes the interaction between Arp2/3 and
WAVE2, I will examine the interaction in the presence or absence of Exo70.
GST-tagged WAVE2 purified from E.coli will be immobilized on Sepharose 4B and
tested for binding with purified Arp2/3 complex in the presence or absence of Exo70.
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2. Exo70 may enhance the association between Arp2/3 and F-actin
Exo70 may directly bind to F-actin and function as an additional connection
between Arp2/3 and F-actin filaments to reinforce their interaction. I have tested this
by measuring the affinity between Exo70 and F-actin. rExo70 was mixed with
preformed F-actin and centrifuged at 80,000 rpm in a TLA 100.3 rotor for 20 min.
Only negligible amounts of Exo70 were found to co-precipitate with F-actin (data not
shown).
Alternatively, Exo70 may facilitate the binding between Arp2/3 and F-actin
without direct interacting with F-actin. Thus, I will directly test the effect of Exo70
on the binding of activated Arp2/3 to the F-actin filament. Purified Arp2/3 and
WAVE2 will be mixed with F-actin in the presence or absence of Exo70, followed by
ultra-speed centrifugation to precipitate F-actin. The amount of Arp2/3 associated
with F-actin in the pellet will be determined by Western blot.
3.
Exo70 may directly bind to G-actin and facilitate the association of G-actin with
the Arp2/3 complex
Exo70 may directly bind to G-actin and facilitate the association of G-actin with
the Arp2/3 complex thus promote the formation of nucleation core. GST pull-down
assay was carried out to test the interaction between Exo70 and G-actin. GST-rExo70
and GST alone were conjugated to Glutathione Sepharose and then incubated with
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0.05µM G-actin, which is below the critical concentration for barbed filament ends.
Beads were washed and the bound proteins were analyzed by Western blot using
anti-actin antibody. GST-rExo70 did not pull down any G-actin (data not shown).
However, it is possible that the conditions used in the experiment did not favor the
binding of Exo70 to G-actin. Therefore, the binding assay will be repeated in the
future, in which a positive control, such as the VCA domain of N-WASP, will be
included.
4.
Exo70 may function in stabilizing the existing actin branch structures
Whether Exo70 stabilizes actin branches can be tested by the following
debranching assay, which was used before to characterize the role of Cortactin in
stabilizing actin branches (Weaver et al, 2001). Actin will be polymerized in the
presence of Arp2/3 and WAVE2 to generate branched actin filaments. Right after
polymerization reaches plateau, Exo70 or the control buffer will be added to the actin
mix. Aliquots will be taken after various periods of time and immediately incubated
with fluorescently-labeled phalloidin to stabilize actin filaments and stop debranching.
Phalloidin decorated F-actin will be diluted and visualized under fluorescence
microscope. The effect of Exo70 on actin branch stability can be determined by
quantifying the rate of debranching in the presence or absence of Exo70.
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Overall, these studies will be informative for clarifying the molecular basis of
the stimulatory effect of Exo70 on Arp2/3-WAVE2-mediated actin polymerization,
and may reveal novel mechanisms for Arp2/3 activation.
4.3.2. Determine the specificity of the effect of Exo70 on different NPFs
In addition to functioning synergistically with WAVE2, Exo70 may also be
involved in the activation of Arp2/3 in the presence of other NPFs. Therefore, it is
necessary to determine whether the stimulatory effect of Exo70 is specific to WAVE2.
I have tested the effect of Exo70 in Arp2/3-mediated actin polymerization in the
presence of the VCA domain of rat N-WASP or ∆EVH1 N-WASP, an N-WASP
truncation mutant lacking the WH1/EVH1 domain that exhibits increased
autoinhibition. Exo70 did not show a clear stimulatory effect in Arp2/3 activation
with either of them (data not shown). In addition, rExo70 has no effect on N-WASP
activation of Arp2/3 as well (unpublished data from Dr. Jeff Peterson). These results
suggest that there is some selectivity in the effect of Exo70 on NPFs. Because
WAVE2 plays a critical role in lamellipodia formation and cell migration, whereas
N-WASP is largely involved in endocytosis, it strongly suggests that Exo70 is
specifically involved in Arp2/3-mediated actin dynamics during cell migration.
The interaction between Exo70 and Arpc1 (Arc40 in yeast) is conserved in
yeast (unpublished data from Guo lab). Furthermore, it was found that the yeast
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Exo70 also promotes the nucleation of branched actin filaments mediated by Arp2/3
and Las 17, the sole WASP homologue in budding yeast (Li, 1997; Madania et al,
1999; Winter et al, 1999). Interestingly, the stimulatory effect of yeast Exo70 in
Arp2/3-mediated actin polymerization is more potent than that of the mammalian
counterpart (data not shown). Additional regulatory factors may be lacking in actin
reactions with mammalian Exo70.
4.3.3. Regulation and functional implications of the Exo70-Arpc1 interaction
1. Further examine whether the Exo70-Arp2/3 interaction is regulated by PAK1
phosphorylation on Arpc1.
The in vitro binding result suggests that PAK1 phosphorylation on Arpc1 may
enhance its interaction with Exo70. I also tested the interaction between Exo70 and
Arpc1/Arpc1 (T21E) using in vivo binding assays. HeLa cells expressing myc-tagged
Arpc1/Arpc1(T21E) were lysed and incubated with GST-CA conjugated to
Glutathione Sepharose beads. The bound proteins were analyzed by Western blot
using antibodies against the myc tag and Exo70. Endogenous Exo70 bound to
Arpc1(T21E) is at similar level compared with that bound to wt Arpc1 (data not
shown). In addition, I tested the interaction between myc-Arpc1/Arpc1(T21E) and
overexpressed GFP-Exo70. The binding affinity of GFP-Exo70 and ArpC1(T21E) is
comparable to that of GFP-Exo70 and ArpC1 (data not shown). Moreover, the
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recombinant GST-rExo70 purified from E.coli was used to pull-down myc-Arpc1/
Arpc1(T21E) from HeLa cells. And the bound myc-Arpc1/ Arpc1(T21E) was
detected by Western blot. The interaction between GST-Exo70 and ArpC1(T21E) is at
similar level compared with that between GST-Exo70 and Arpc1 (data not shown).
From these results, I did not see a clear enhancement of Exo70-Arpc1 interaction
upon the phosphorylation of Arpc1.
To further examine the regulation of Exo70-Arpc1 interaction by Pak1
phosphorylation, Arpc1 (T21A) mutant will also be used. The Pak1 phosphorylation
site was mutated to alanine in this mutant, which completely abolishes Pak1
phosphorylation (Vadlamudi et al., 2004). In vitro and in vivo binding assays will be
carried out to test the interaction between Exo70 and Arpc1/Arpc1(T21A).
It is also possible that Pak1 indirectly regulates Exo70-Arpc1 interaction.
Therefore, the NIH-3T3 variant S2-6 cell lines inducibly expressing various forms of
Pak1 will be used here. The exocyst-Arp2/3 interaction will be examined in the cells
expressing active Pak1(E423) by GST-CA pull-down assay before and after the
induction. As a control, the kinase-dead form of Pak1 (Pak1R299) will also be used in
the experiments. On the other hand, the Pak auto-inhibitory peptide (“PID”, amino
acid 83-149), which has been shown to effectively inhibit the activity of Paks in cells
(Beeser et al., 2005) can be used in the experiments as well. As a control, an
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inactivated form of PID (the Phe107 mutant) (Zhao et al., 1998; Beeser et al., 2005)
will be included.
2. Determine whether the positive effect of Exo70 in actin assembly is
curvature-dependent
Dynamic rearrangements of membrane shape occur frequently during the
formation of cell protrusions and cell migration. It has been shown that rExo70 can
induce tubulated membranes invaginations when it binds to PI(4,5)P2-rich liposomes
in vitro (unpublished data from our lab). However, the functional implication of the
role of Exo70 in generating membrane curvature is still unknown. Recent work from
Lappalainen Pekka’s lab discovered that “I-BAR” proteins, which are known to be
involved in the Arp2/3-mediated actin filament assembly, can bend membranes and
further couple actin polymerization and membrane deformation during cell migration
(Mattila et al., 2007; Saarikangas et al., 2009). Thus, it would be intriguing to
examine whether the effect of Exo70 in Arp2/3-mediated actin assembly is curvature
dependent. In pyrene actin assay and TIRFM, rExo70 and PI(4,5)P2-containing
liposomes with different sizes will be simultaneously added to the actin
polymerization reaction mixture containing Arp2/3, WAVE2 and actin. The obtained
stimulatory effect will be compared with the samples with either rExo70 or the
liposomes alone. The effects between different sizes of liposomes will also be
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compared. Liposomes with different composition, such as PC alone, or PC/PS will
also be included as controls. If membrane curvature can further enhance the
stimulatory effect of Exo70 in Arp2/3-WAVE2-mediated actin polymerization, I will
further examine its molecular basis. One of the most plausible mechanisms for
curvature-dependent actin polymerization by rExo70 is the increased interaction
between Exo70 and the Arp2/3 complex. Thus, Exo70-Arp2/3 interaction will also be
tested in the presence or absence of PI(4,5)P2-containing liposomes.
3. Examine whether the interaction with GTPases regulates the function of Exo70 in
Arp2/3-mediated actin assembly
A number of small GTP-binding proteins interact with components of the
exocyst and regulate the assembly, localization, and function of this complex. It has
been shown that TC10 directly interacts with Exo70 (Inoue et al., 2003). However,
the cellular function of this interaction is still unclear. It is possible that TC10
regulates the function of Exo70 in actin assembly. In pyrene actin assay and TIRFM,
GTP-bound TC10 will be added in the reaction mixture containing Arp2/3, WAVE2,
Exo70 and actin. GDP-bound or wild type TC10 will be included as controls. I will
also examine whether TC10 regulates the interaction between Exo70 and the Arp2/3
complex.
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Overall, these studies will largely help to elucidate the molecular mechanisms
of the function of Exo70 in actin assembly.
4.4. Materials and Methods
Purification of mammalian Arp2/3 complex
Mammalian Arp2/3 complex was purified from cell lysates extracted from
mammalian cells by affinity chromatography using recombinant GST-CA
immobilized on glutathione Sepharose 4B beads. Cell lysates with 20-25mg total
proteins were collected and spun successively at 16,000 g for 10 min and 80, 000 rpm
in a Beckman TLA-100.3 rotor (Beckman Coulter Inc., Fullerton, CA) for 15 min.
The resulting high speed supernatant (HSS) was mixed with GST-CA beads (-10 mg
proteins) for 1 hr at 4°C. The beads were transferred to a column and washed
successively with 20 ml Buffer B (25 mM KC1, 1 mM MgCl2, 0.5 mM EGTA, 0.1
mM ATP, and 1 mM DTT) and 20 ml Buffer B plus 200 mM KC1. Additional
washes were performed, if necessary, until no proteins were detected in the flow
through. The bound Arp2/3 proteins were eluted from the beads with 5 ml Buffer B
plus 200 mM MgCl2. The 1.5 ml fractions with peak protein concentration were
pooled and dialyzed against Buffer B. Finally, the Arp2/3 proteins were concentrated
to 0.5 ml, supplied with 0.2 M sucrose, snap frozen in liquid nitrogen and stored at
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-80°C.
Actin polymerization assay
Kinetics of actin filament polymerization was monitored by pyrene
fluorescence with excitation of 370 nm and emission at 410 nm. Proteins to be tested
for each reaction were mixed and diluted in 90µl polymerization buffer (60 mM KC1,
2.5 mM NaCl, 0.6 mM MgCl2, 5 mM Tris-HCl, pH 7.5, 2.5 mM HEPES, pH 7.1, 0.5
mM EGTA, 30 uM CaCl2, 0.2 mM ATP, and 0.3 mM NaN3), then pre-incubated for
30min at 4°C. 11.5µl G-actin (20.9 µM, 20% pyrenyl-labeled) in G-actin buffer were
converted to Mg -G-actin by mixing with 18.5µl Mg2+ converting buffer (1.6 mM
Tris-HCl, pH8, 0.18 mM EGTA, and 0.51 mM MgCl2) for 5 min. The converted actin
mix was then diluted into 90µl protein mix, and immediately transferred into a
cuvette and monitored for the increase in fluorescence at 410 nm in a fluorescence
spectrophotometer. The pyrenyllabeled and unlabeled rabbit skeletal actin used in the
assay was from Cytoskeleton (Cytoskeleton Inc., Denver, CO). Three independent
measurements were carried out for each treatment. Polymerization curves and rates
were obtained using Excel (Microsoft, Redmond, WA). The polymerization rate was
represented as the maximal slope of the elongation phase of each curve.
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Visualization of actin polymerization in real time by TIRFM
Experiments were carried out in 10-20µl flow chambers assembled by
mounting a precleaned coverslip to a standard glass slide using two strips of
double-sided adhesive tape as spacers. Before use, the flow chambers were coated
with NEM-myosin by flowing in 50 nM NEM-myosin in high salt buffer (500 mM
KC1) and incubating for 1.5 min. The NEM-myosin functions to capture the actin
filaments at the surface of the chamber for TIRF illumination. The flow chambers
were then treated with 1% BSA for 4 min to prevent the nonspecific binding to the
surface. Arp2/3 and proteins to be tested were mixed in the actin polymerization
buffer and incubated at 4°C for 30 mins. 0.8µM G-actin (8% Rhodamine labeled) was
converted to Mg-G-actin, then mixed with the pre-incubated samples which contain
(1)6 nM Arp2/3 + 10 nM WAVE2, or (2) 6 nM Arp2/3 + 10 nM WAVE2 + 1 uM
Exo70, and immediately transferred into the flow chamber for TIRF microscopic
observation. Total internal reflection excitation was generated on a Nikon TE2000
inverted microscope by projecting a laser beam at 532 nm (Crystal Laser) on the back
focal plane of a Nikon 100x, 1.49 NA objective lens. Fluorescence emission was
collected by the objective and imaged on an EMCCD camera (Cascade 11:512,
Photometries). Images were recorded for 10 min at 1.14 sec per frame after the
polymerization started. The total length of F-actin filaments was measured using
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ImageJ 1.73v software (http://rsb.info.nih.gov/ij/). White filaments in the dark field
were inverted and thresholded into black filaments in the white field. All the particles
were then skeletonized and the total length of filaments was obtained.
GST pulldown assay
HeLa cells alone or the cells expressing GFP-rExo70 or myc-rExo70 were
scraped from 10cm culture plates and lysed in the lysis buffer (20 mM Tris-HCl, pH
7.5, 25 mM KCl, 1 mM MgCl2, 0.5 mM EGTA, 1 mM DTT, 0.5% Triton X-100, and
protease inhibitors). A high-speed centrifugation (12,000 rpm, 15 min) was carried
out, and 1 ml of precleared cell lysates (1.5 mg total protein) was mixed with 20 µl
(50% vol/vol) of glutathione Sepharose conjugated with 10 µg GST-WAVE2. After
incubation at 4°C overnight, the beads were washed five times with the lysis buffer,
and the bound proteins were analyzed by Western blot using antibodies against Exo70,
GFP or myc tag. GST alone was used as a negative control in the experiment.
In vitro protein binding assays
His(6)-tagged Arpc1 wt and Arpc1(T21E) mutant were expressed as
His(6)-tagged fusion proteins. rExo70 was expressed as GST-tagged fusion protein
(GST-rExo70). The recombinant fusion proteins were purified from E. coli. and GST
or GST-rExo70 (3 µg) was immobilized on glutathione Sepharose 4B beads. The
GST or GST-rExo70 beads were incubated with His(6)-tagged Arpc1 wt or
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Arpc1(T21E) mutant with various amounts (0 - 7.2 µg) for 2 hrs at room temperature
in the binding buffer containing 20 mM Tris-HCl, pH 7.4, 100 mM NaCl, 1 mM
EGTA, 1 mM DTT, 10 mM MgCl2 and 1% Triton X-100. The beads were then
washed five times with the binding buffer and the HIS-Arpc1 fusion proteins bound
to the beads were detected by Western blot with anti-HIS monoclonal antibody.
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Chapter 5. Discussion and Future Perspectives
5.1. Impact of my research in the field
My studies have revealed crucial roles of the exocyst in exocytosis and tumor
cell migration. Firstly, I have elucidated a molecular mechanism by which the
exocyst directly interacts with the PM that is critical for vesicle tethering and
exocytosis. I identified a direct interaction between the exocyst component Exo70
and PI(4,5)P2; and demonstrated that this interaction is essential for the recruitment of
the exocyst to the PM. Furthermore, disruption of this interaction impaired later
stages of exocytosis of post-Golgi secretory vesicles at the PM. Secondly, I have
found that the exocyst plays a pivotal role in the formation of invadopodia, a
degradative membrane protrusive structures formed by tumor cells. Blocking the
exocyst function inhibits invadopodial formation. RNAi knockdown of the exocyst
components abolished the secretion of MMPs, whereas the overexpression of Exo70
promoted MMP secretion. In addition, the exocyst-Arp2/3 interaction is important for
actin assembly during invadopodia formation. Finally, I have found that Exo70
promotes the generation of branched actin filaments in Arp2/3-WAVE2-mediated
actin polymerization, which suggests that Exo70 is a novel positive regulator of the
exocytosis and actin reorganization for effective cell migration and cell invasion.
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5.2. The molecular mechanism of exocyst targeting to the plasma membrane
The exocyst components are recruited to designated regions of the plasma
membrane where active exocytosis and membrane expansion occur. However, in
most cell types, PI(4,5)P2 distributes throughout the whole plasma membrane.
Therefore, there must be additional factors that contribute to the polarized localization
of the exocyst. The exocyst is a downstream effector of a variety of small GTPases
(see Chapter 1). In mammalian cells, growth factor signaling involving small
GTPases may mediate translocation of the exocyst from intracellular compartments to,
or activation of the exocyst at, the PM (Sugihara et al., 2002; Moskalenko et al., 2002,
2003; Inoue et al., 2003; Takaya et al., 2004). Phopshoinositides and small GTPases
may coordinate to regulate the recruitment of the exocyst to specific sites on the
plasma membrane. The observed polarization of the exocyst may in fact be a result of
both physical recruitment and kinetic reinforcement. Future work will be focused on
the identification and characterization of proteins that spatially regulate exocyst
components using different eukaryotic systems.
Similar to its mammalian counterpart, yeast Exo70 also interacts with PI(4,5)P2
through conserved regions (He et al., 2007b). Other tethering factors, such as the
HOPS complex that functions in yeast homotypic vacuolar fusion, have also been
found to interact with phosphoinositides (Stroupe et al., 2006). It may be a
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generalized mechanism that phosphoinositides function to recruit the tethering
proteins to specific membrane compartments for vesicle tethering.
5.3. A crosstalk between actin cytoskeleton and exocytosis machineries during
cell migration
The relationship between actin dynamics and exocytosis has been widely
studied in different systems. In several neuron cells such as hippocampal neurons, the
actin cytoskeleton has inhibitory roles in neurotransmitter release (Morales et al.,
2000; Ohnishi et al., 2001). In their study, they showed that latrunculin A but not
cytochalasin D increased neurotransmission (Morales et al., 2000). However, cell
migration requires the coordination of actin reorganization and membrane remodeling.
The leading edges of the migrating cells are generated by a branched network of
filamentous actin that pushes the membrane. To accommodate actin “pushing”,
exocytosis is involved in the recycling of membranes and proteins to the leading
edges. How are actin remodeling and secretion linked during cell migration? In my
study, I found that the exocyst directly interacts with the Arp2/3 complex and
promotes actin polymerization in addition to its fundamental role in exocytosis. It
thus functionally couples actin reorganization and membrane addition at the leading
edges for effective directional cell migration.
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Chapter 5
5.4. The functions of the exocyst in many cellular processes
The exocyst plays important roles in a variety of cellular processes including
cell migration, epithelial polarity establishment, cytokinesis and ciliogenesis. Why is
the exocyst involved in so many cellular processes? Exocytosis is one of the most
fundamental cellular events in the cell. It is involved in a number of processes that
requires secretion. This may explain the universal role of the exocyst in the cell.
5.5. Future perspectives
Recent decade has witnessed exciting progresses toward our understanding of
the exocyst. However, a number of important questions remain unsolved: What are
the kinetics of exocyst assembly and disassembly? How are the exocyst components
associated with secretory vesicles? While it is thought that the exocyst serves as a
vesicle tether, can the tethering step be observed and characterized by microscopy? Is
the exocyst also involved in the early steps of vesicular trafficking such as vesicle
budding and cargo sorting at the donor compartments? Recent studies demonstrate
the roles of the exocyst in a number of developmental and physiological processes.
Future studies are needed to elucidate how the cellular functions of the exocyst are
manifested at the multicellular organismal level.
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References
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