26 Aug 2003
20:13
AR
AR197-CB19-12.tex
AR197-CB19-12.sgm
LaTeX2e(2002/01/18)
P1: GCE
10.1146/annurev.cellbio.19.111401.093127
Annu. Rev. Cell Dev. Biol. 2003. 19:287–332
doi: 10.1146/annurev.cellbio.19.111401.093127
c 2003 by Annual Reviews. All rights reserved
Copyright °
ACTIN ASSEMBLY AND ENDOCYTOSIS: From Yeast
to Mammals
Annu. Rev. Cell Dev. Biol. 2003.19:287-332. Downloaded from arjournals.annualreviews.org
by University of Pittsburgh on 08/17/07. For personal use only.
Åsa E.Y. Engqvist-Goldstein and David G. Drubin
Department of Molecular and Cell Biology, University of California, Berkeley,
California 94720-3202; email: [email protected]
Key Words clathrin, dynamin, Arp2/3 complex, caveolae, macropinocytosis
■ Abstract Internalization of receptors, lipids, pathogens, and other cargo at the
plasma membrane involves several different pathways and requires coordinated interactions between a variety of protein and lipid molecules. The actin cytoskeleton is
an integral part of the cell cortex, and there is growing evidence that F-actin plays a
direct role in these endocytic events. Genetic studies in yeast have firmly established
a functional connection between actin and endocytosis. Identification of several proteins that may function at the interface between actin and the endocytic machinery
has provided further evidence for this association in both yeast and mammalian cells.
Several of these proteins are directly involved in regulating actin assembly and could
thus harness forces produced during actin polymerization to facilitate specific steps in
the endocytic process. Recent microscopy studies in mammalian cells provide powerful evidence that localized recruitment and polymerization of actin occurs at endocytic
sites. In this review, we focus on progress made in elucidating the functions of the actin
cytoskeleton in endocytosis.
CONTENTS
INTRODUCTION . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
INSIGHTS FROM YEAST . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Genetic Identification of Proteins Important for the Internalization
Step of Endocytosis Reveals a Strict Requirement for Actin . . . . . . . . . . . . . . . . .
Actin Organization in Yeast: Are Cortical Actin Patches Sites of
Endocytosis? . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Protein-Protein Interactions Between Components Important for
Internalization: How is the Actin Machinery Recruited to Endocytic
Sites? . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Several Yeast Proteins Important for Endocytic Internalization Can
Promote Actin Assembly . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
ENDOCYTIC FUNCTION OF THE ACTIN CYTOSKELETON IN
MORE COMPLEX EUKARYOTES . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Clathrin-Mediated Endocytosis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Caveolae-Mediated Endocytosis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Macropinocytosis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
1081-0706/03/1115-0287$14.00
288
289
289
290
295
300
302
303
307
309
287
26 Aug 2003
20:13
288
AR
AR197-CB19-12.tex
ENGQVIST-GOLDSTEIN
¥
AR197-CB19-12.sgm
LaTeX2e(2002/01/18)
P1: GCE
DRUBIN
PROTEINS AT THE INTERFACE BETWEEN THE ACTIN
CYTOSKELETON AND THE CLATHRIN-MEDIATED
ENDOCYTOSIS MACHINERY . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Links Between Coat Components and Actin . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Dynamin and the Actin Cytoskeleton . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
CONCLUDING REMARKS . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
311
311
315
320
Annu. Rev. Cell Dev. Biol. 2003.19:287-332. Downloaded from arjournals.annualreviews.org
by University of Pittsburgh on 08/17/07. For personal use only.
INTRODUCTION
As the interface between the intracellular and extracellular environment, the plasma
membrane is critically important for vital functions such as cell growth regulation, cell polarity establishment, cell motility, nutrient absorption, defense against
pathogens and toxins, and ion homeostasis. Endocytosis is important for all these
plasma membrane–associated functions because of its role in control of the protein
and lipid composition of the plasma membrane, regulation of signaling pathways,
control of cell surface area, and uptake of nutrients and pathogens. In mammalian
cells, wherein endocytosis is most thoroughly characterized, several distinct endocytic pathways for internalization exist. These pathways include the clathrindependent pathway, the caveolar pathway, a clathrin- and caveolae-independent
pathway, macropinocytosis, and phagocytosis. Clathrin-mediated endocytosis is
the major pathway for internalization of proteins and lipids from the plasma membrane, and it is important for the recycling of synaptic vesicles at nerve terminals
(Brodin et al. 2000, Brodsky et al. 2001, Jarousse & Kelly 2001). Internalization
via caveolae is much less well understood but appears to have a role in cholesterol
homeostasis, recycling of glycosyl-phosphatidylinositol (GPI)-anchored proteins,
glycosphingolipid transport, transcytosis of serum components, and uptake of certain viruses (Razani & Lisanti 2001). Macropinocytosis involves the engulfment
of extracellular fluid, whereas phagocytosis involves engulfment of particles such
as invading bacteria (Dramsi & Cossart 1998, Galán 2001).
Although each of these pathways uses distinct machinery for internalization,
all require remodeling of the cell cortex during the internalization step. For the
clathrin- and caveolae-dependent pathways, invaginations of the plasma membrane
are formed, whereas for macropinocytosis and phagocytosis, membrane protrusions are formed. Subsequent steps involve vesicle fission, transport of vesicles
away from the plasma membrane, vesicle uncoating, fusion to other membrane
compartments, and sorting of cargo for degradation or recycling. The actin cytoskeleton is an integral part of the cell cortex, and there is growing evidence
that F-actin directly participates in internalization events (Munn 2001, Qualmann
& Kessels 2002, Schafer 2002). Genetic studies in yeast show a tight coupling
between actin assembly and endocytosis, and the biochemical mechanisms upon
which these phenotypes are based are now being revealed (D’Hondt et al. 2000,
Munn 2001, Shaw et al. 2001). In mammalian cells, it is clear that actin is involved
in the formation of membrane protrusions for macropinocytosis and phagocytosis
(May & Machesky 2001, Welch & Mullins 2002). Furthermore, recent studies of
caveolae-mediated endocytosis suggest that actin dynamics play an essential role
26 Aug 2003
20:13
AR
AR197-CB19-12.tex
AR197-CB19-12.sgm
LaTeX2e(2002/01/18)
Annu. Rev. Cell Dev. Biol. 2003.19:287-332. Downloaded from arjournals.annualreviews.org
by University of Pittsburgh on 08/17/07. For personal use only.
ACTIN AND ENDOCYTOSIS
P1: GCE
289
during internalization (Pelkmans & Helenius 2002). Although actin may not play
an obligatory role in clathrin-mediated internalization in all cells, there is increasing evidence that actin is involved. A number of proteins that can regulate actin
dynamics [e.g., Arp2/3 activators, motor proteins, F-actin crosslinking proteins,
and a Cdc42 guanine-nucleotide exchange factor (GEF)] have been identified as
bridges between actin and endocytic components (McPherson 2002, Qualmann
& Kessels 2002, Schafer 2002). Furthermore, there is now evidence that actin
polymerization occurs at coated pits (Merrifield et al. 2002). This review focuses
on recent advances in understanding the roles of the actin cytoskeleton during
endocytic internalization at the plasma membrane.
INSIGHTS FROM YEAST
Genetic Identification of Proteins Important for the
Internalization Step of Endocytosis Reveals a Strict
Requirement for Actin
More than 10 years ago, Riezman and colleagues began isolating end mutants
defective in receptor-mediated endocytosis (RME) (D’Hondt et al. 2000, Munn
2001). The Riezman laboratory specifically looked for mutants defective in αfactor (pheromone) internalization. α-factor is a small peptide secreted by yeast
of the α mating type, and it is internalized after binding to the Ste2 receptor (a G
protein–coupled serpentine receptor) on cells of the a mating type. The α-factor
and Ste2 are then transported to the vacuole (yeast lysosome) where they are
degraded (Dulic et al. 1991). Internalization and degradation of Ste2p occurs in
the absence of α-factor (constitutive endocytosis), but the rate increases five- to
tenfold upon α-factor binding (ligand-induced endocytosis) (Jenness & Spatrick
1986). The measuring of α-factor uptake is the most commonly used assay for internalization, although more recently, internalization of the a-factor receptor, transporters, and permeases have been investigated (Munn 2001). Emr and colleagues
isolated dim mutants defective in internalization of the lipophilic dye FM4-64 as
a second type of screen for endocytic mutants (Wendland et al. 1996). In addition,
several mutants have been identified to be defective in fluid-phase endocytosis as
measured by uptake of the membrane-impermeant fluorescent dye lucifer yellow
(LY) (Riezman 1985). It should be noted that LY uptake assays do not distinguish
between internalization defects and defects in subsequent trafficking steps (Dulic
et al. 1991, Riezman 1985).
Unexpectedly, some of the first mutants identified to be defective in α-factor uptake were in ACT1 (END7), the only yeast actin gene; SAC6, encoding the F-actin
cross-linking protein fimbrin; and SLA2 (END4), encoding an actin-binding protein important for polarization of the actin cytoskeleton (Kubler & Riezman 1993,
Munn et al. 1995, Raths et al. 1993). At approximately the same time, deletion mutants and temperature-sensitive mutants of CHC1, encoding clathrin heavy chain,
were also found to be defective in α-factor uptake (Payne et al. 1988, Tan et al.
1993). Since then, a number of laboratories have contributed to the isolation of
26 Aug 2003
20:13
Annu. Rev. Cell Dev. Biol. 2003.19:287-332. Downloaded from arjournals.annualreviews.org
by University of Pittsburgh on 08/17/07. For personal use only.
290
AR
AR197-CB19-12.tex
ENGQVIST-GOLDSTEIN
¥
AR197-CB19-12.sgm
LaTeX2e(2002/01/18)
P1: GCE
DRUBIN
approximately 50 genes that are important for receptor internalization (see RME in
Table 1). The majority of these genes encode proteins that either have homologues
in mammalian cells or have domains that share homology with domains present
in mammalian proteins (Table 1). As we discuss below, many of these related
proteins are involved in endocytosis and/or in actin functions in mammalian cells.
The severity of the endocytic defects for different mutants varies from mild (∼30%
reduction) to showing an essentially complete block in α-factor uptake. As shown
in Table 1, the majority of mutants isolated, including mutants of CHC1 and
CLC1, show defects in the organization of the actin cytoskeleton. The severity of
the endocytic defect is generally correlated with the severity of the actin phenotype. Exceptions to this rule are certain mutants of proteins involved in ergosterol
(sterol) biosynthesis (e.g., Erg2p, Erg3p, Erg6p) that do not have apparent actin
defects (Heese-Peck et al. 2002, Munn et al. 1999). Specific sterols are necessary
for α-factor-dependent receptor hyperphosphorylation, a prerequisite for internalization (Heese-Peck et al. 2002). In addition, it is clear that only a subset of proteins
important for normal actin organization are important for endocytic internalization.
This is an important finding because it shows that defects in the actin cytoskeleton are not a secondary effect of blocking endocytic internalization or vice versa.
Rather, these results point to a specific role for actin during internalization.
Proteins important for endocytic internalization (Table 1) can be placed broadly
into five groups based on their activities and properties: (a) activities affecting
actin organization/dynamics, (b) activities regulating membrane lipid composition (sterols, phospholipids, and sphingolipids), (c) activities affecting posttranslational modifications of receptors and/or other proteins important for internalization (e.g., phosphorylation and ubiquitination), (d) activities regulating
clathrin assembly (including clathrin), and (e) lipid and/or receptor binding involving adaptor proteins that link endocytic components to the plasma membrane.
Approximately one third of the endocytic proteins directly regulate actin assembly
and/or bind to actin (Group a). Considered together with the observation that most
endocytic mutants also have actin defects, these observations strongly suggest that
actin is required for endocytic internalization in yeast. This requirement is likely
to reflect a direct involvement of actin because temperature-sensitive mutants affecting actin are blocked rapidly for endocytic internalization upon a shift to the
restrictive temperature (Kubler & Riezman 1993). A role for actin in endocytosis
is also supported by the fact that yeast cells treated with latrunculin-A (Lat-A)
have endocytic defects (Ayscough et al. 1997). Lat-A sequesters G-actin, which
then leads to rapid disassembly of F-actin (Ayscough et al. 1997).
Actin Organization in Yeast: Are Cortical Actin Patches
Sites of Endocytosis?
Yeast cells contain three types of actin filamentous structures: actin cables, an actinmyosin contractile ring, and cortical actin patches (Pruyne & Bretscher 2000a,b).
Actin cables are bundles of actin filaments aligned with the mother-bud axis of
AP−
Arp2/3
complex
Clathrin
HC and LC
Calmodulin
Cofilin
Eps15j
Eps15R
EH-containing
Epsin
Arp2p, Arp3p,
Arc4p, Arc15p,
Arc18p, Arc19p,
Arc35p
Chc1p, Clc1p
Cmd1p
Cof1p
Ede1p
End3p
Ent1p, Ent2pk
−
−
ND
ND
−
−
−
ND
−
−
−
−
−
−
−
−
−
−
−
−
ND
ND
ND
ND
ND
+i
−
ND
ND
No defects detected
Larger and depolarized
APs
Larger and depolarized
APs
Mild depolarization
of APs
Larger and
depolarized APs
Depolarized APs
Larger and
depolarized APs
Abnormal and
depolarized APs
Actin clumps,
depolarized APs
ND
Essential
b
e?
?
e?
a
a?
d
a
c
c
a
a
ACTIN AND ENDOCYTOSIS
(Continued )
Enzymes involved in ergosterol
biosynthesis
Binds ubiquitin via UIM
motif; putative adaptor
Inhibits phosphorylation
of Pan1p by Prk1p
Binds ubiquitin via UBA motif
Severs F-actin and stimulates
F-actin disassembly
Binds to Myo3/5 and
the Arp2/3 complex
Coat protein
Nucleates F-actin assembly;
branches actin filaments
Ser/Thr kinases; phosphorylate
Pan1p, Sla1p, Ent1/2p
Palmitoyl transferase;
palmitoylates Yck2p
Assembles into F-actin
Binds F-actin; activates Arp2/3
complex
FG
LaTeX2e(2002/01/18)
Peroxisomal
fraction
AP−
ND
No defects detected
Known activities
AR197-CB19-12.sgm
ND
Multiple
localization
including APs
TGN
AP
−
−
−d
ND
Actin defects
AR197-CB19-12.tex
Erg2pk
AP
AAK1f
BIKeg GAKh
Ark1p, Prk1pk
AP
AP, AC, CR
Cortical
localization
Actin
Hip14e
ND
+c
FM464
AR
Akr1p
AP
ABP1b
Abp1
LY
RME
20:13
Act1p/End7p
Localization
Related
proteinsa
Yeast protein
TABLE 1 Yeast proteins implicated in endocytic internalization∗
Annu. Rev. Cell Dev. Biol. 2003.19:287-332. Downloaded from arjournals.annualreviews.org
by University of Pittsburgh on 08/17/07. For personal use only.
26 Aug 2003
P1: GCE
291
AP, AP−
Cytosolic, ER
Cortical
Localization
AP−
AP
WASPm
SPTLC1n
PIP5K1o
Type 1
myosin
Eps15j
Eps15R
PDK1p
Nedd4
Amphiphysin
Amphiphysin
Fimbrin
Las17p
Lcb1p/End8p
Mss4p
Myo3p, Myo5pk
Pan1p
Pkh1p, Pkh2pk
Rsp5p
Rvs161p/End6p
Rvs167p
Sac6p
AP, AC
−
−
−
−
−
−
−
−
−
−
−
−
−
−
−
−
−
−
−
−
ND
ND
ND
ND
ND
−
ND
ND
ND
−
−
Depolarized APs
Depolarized APs
Depolarized APs
No defects in actin
organizationq
Depolarized APs
Actin clumps,
depolarized APs
Depolarized APs
Larger and
depolarized APs
Abnormal and
depolarized APs
Larger and depolarized
APs
Depolarized APs
No defects detected
Crosslinks F-actin
Unknown, amphiphysin tubulates
liposomes
Unknown, amphiphysin tubulates
liposomes
E3 ubiquitin ligase ubiquitinates
receptors and transporters
Ser/Thr kinases, phosphorylate
Ypk1p and Ypk2
Activates Arp2/3 complex
F-actin motor protein, may activate
Arp2/3 complex
Phosphatidylinositol(4)-phosphate
5-kinase; regulates cellular
PtdIns(4,5)P2 levels
Involved in sphingolipid biosynthesis
Binds G-actin; activates Arp2/3
complex
Inositol polyphosphate-5 phosphatases;
regulate cellular PtdIns(4,5)P2 and
PtdIns(3,5)P2 levels
Enzymes involved in ergosterol
biosynthesis
Enzymes involved in ergosterol
biosynthesis
a
?
?
c
b,c
a
a
b
b
a
b
b
b
FG
LaTeX2e(2002/01/18)
AP
−
−
−
No defects detected
Known activities
¥
Cytosolic
−
−
−
Actin defects
ENGQVIST-GOLDSTEIN
AR197-CB19-12.sgm
PM and prevacuole
sites
−
−
FM464
AR197-CB19-12.tex
Nucleus, peripheral
patches
Cytosolic; upon
osmotic stress
localize to AP
Synaptojanin
Inp51p/Sjl1pk
Inp52p/Sjl2p
Inp53p/Sjl3p
Microsomal
fraction
LY
RME
292
AR
Cytosolic, ER
SC5DLl
Erg3p
Localization
20:13
Erg6p
Related
proteinsa
Yeast protein
TABLE 1 (Continued )
Annu. Rev. Cell Dev. Biol. 2003.19:287-332. Downloaded from arjournals.annualreviews.org
by University of Pittsburgh on 08/17/07. For personal use only.
26 Aug 2003
P1: GCE
DRUBIN
AP−
AP−
AP, AP−
Cytosolic, ER
AP−
PM and intracellular
membranes
Cortical localization,
cytosolic
Intersectin?
Hip1r
Hip1R
CAPs
Auxilin,
GAK
WIPu
AP180
Casein
kinase 1
SGKv
Sla1p
Sla2p/End4p
Srv2p/End14p
Swa2p/Aux1p
Vrp1p/End5p
yAP180Ap,
yAP180Bp
Yck1p, Yck2pk
Ypk1p
−
−
+
ND
−
+
+
−
−
−
+t
−
−
+
−
−
ND
ND
Depolarized APs
ND
No defects detected
Depolarized APs
ND
Depolarized APs
Depolarized APs
Larger and depolarized
APs
Larger and depolarized
APs
Ser/Thr kinase involved in sphingolipid
signaling
Ser/Thr kinases; candidates for
phosphorylating Ste2p
Unknown, AP180 promotes clathrin
assembly
Binds to actin (2-hybrid)
Required for clathrin function and
disassembly
Adenylate-cyclase associated protein;
binds G-actin
Binds F-actin, Hip1/R promotes clathrin
assembly
Adaptor for NPFX(1,2)D-mediated
endocytosis
Unknown, Sla2p localization is
dependent on Scd5p
b,c
c
e
a
d
a
a,e?
e
?
∗
AR197-CB19-12.sgm
Yeast proteins implicated in endocytic internalization. AC, localizes to actin cables; CR, localizes to cytokinetic ring; AP, localizes to actin patches (tight colocalization);
AP−, partial localization to actin patches; RME, LY, FM4-64 and actin defects refer to phenotypes associated with mutants of the indicated genes. RME, receptor-mediated endocytosis (majority of studies have looked at α-factor uptake); LY, lucifer yellow uptake (fluid-phase endocytosis); −, defective; +, normal; ND, not determined; FG, functional groups (see text).
AR197-CB19-12.tex
ND
ND
ND
ND
−
−
ND
AR
a
Related proteins in mammalian cells or conserved domains; bactin binding protein 1; csla21coil1 + abp11SH3 is defective in α-factor uptake; dnot defective in ligand-mediated Ste2p
uptake; ehuntingtin interacting protein 14; fadaptor-associated kinase 1; gBMP-2-inducible kinase; hcyclin G-associated kinase; iFM4-64 is less bright in arp2 mutants; jEGFR pathway
substrate clone 15/EGFR pathway substrate clone 15 related; kproteins have redundant functions; lsterol-C5-desaturase-like; mWiscott-Aldrich syndrome protein; nserine palmitoyl
transferase long chain; ophosphaditylinositol-4-phosphate 5-kinase type 1; p3-phosphoinositide-dependent protein kinase-1; qrsp5 mutants are Lat-A resistant; rhuntingtin-interacting
protein 1/huntingtin-interacting proteins 1 related; sadenylyl cyclase-associated protein 1; tsla21coil1 + srv21 are defective in α-factor uptake; uWASP-interacting protein; vserum
glucocorticoid regulated kinase.
−
−
20:13
AP−
AP−
Scd5p
Annu. Rev. Cell Dev. Biol. 2003.19:287-332. Downloaded from arjournals.annualreviews.org
by University of Pittsburgh on 08/17/07. For personal use only.
26 Aug 2003
LaTeX2e(2002/01/18)
P1: GCE
ACTIN AND ENDOCYTOSIS
293
26 Aug 2003
20:13
Annu. Rev. Cell Dev. Biol. 2003.19:287-332. Downloaded from arjournals.annualreviews.org
by University of Pittsburgh on 08/17/07. For personal use only.
294
AR
AR197-CB19-12.tex
ENGQVIST-GOLDSTEIN
¥
AR197-CB19-12.sgm
LaTeX2e(2002/01/18)
P1: GCE
DRUBIN
yeast cells. They are involved in polarized secretion and organelle and mRNA transport. The actin-myosin contractile ring forms transiently at the mother-daughter
neck and is important for cytokinesis. Cortical actin patches are the least understood
of these structures. They appear by fluorescence microscopy as cortical puncta,
and they localize near regions of exocytosis in growing cells (Adams & Pringle
1984). Actin patches are highly dynamic structures that are constantly turning over.
They persist for approximately 10 s, and they assume either highly mobile states,
moving rapidly along the cell cortex (velocities up to 1.9 µm/s), or stationary
states (Ayscough et al. 1997, Doyle & Botstein 1996, Smith et al. 2001, Waddle
et al. 1996). Ultrastructure analysis of actin patches suggested that these patches
might associate with membrane invaginations (finger-like membrane structures)
(Mulholland et al. 1994), but how actin is organized around these structures is not
known. A host of actin-binding proteins and regulatory proteins are components of
actin patches (Goode & Rodal 2001, Pruyne & Bretscher 2000a). It is interesting
that many proteins important for endocytic internalization either are tightly associated with cortical actin patches or colocalize partially with cortical actin patches
(Table 1). Furthermore, as mentioned above, the majority of endocytic mutants have
defects in organization of the actin cytoskeleton, wherein cortical actin patches are
depolarized and often appear larger than in wild-type cells (Table 1). Components
of other actin structures (e.g., actin cables and the actin-myosin contractile ring) do
not appear to be involved in endocytosis, although not all components have been
tested for endocytic function (Pruyne & Bretscher 2000a). Taken together, these
observations strongly suggest that actin patches are involved specifically in endocytosis. This leads to the question of whether actin patches are sites of endocytosis.
As mentioned above, actin patches localize near sites of exocytosis, suggesting that
they perform compensatory endocytosis, thereby recycling plasma membrane and
protein machinery delivered to the plasma membrane during exocytosis. Inhibition of endocytosis could prevent downregulation of cell wall synthesis machinery
and prevent recycling of plasma membrane. Consistent with this possibility, many
mutants of actin patch components (e.g., act1-1, myo31myo51, sla11, pan1-4,
end31, sla21) have defects in cell wall assembly (thickened and/or layered cell
wall), and they grow larger than wild-type cells (Ayscough et al. 1999, Goodson
et al. 1996, Mulholland et al. 1997, Tang et al. 2000). It is not clear, however,
whether a block in endocytosis causes any of these defects. For example, depolarized secretion and/or upregulation of machinery for cell wall synthesis may also
cause the thickened cell wall phenotype (Li et al. 2002).
Whether endocytosis occurs at cortical actin patches is unclear. Compared with
mammalian cells, much less is known about the ultrastructure of endocytic internalization sites in yeast. Coat proteins such as clathrin have not been detected
at the budding-yeast plasma membrane (Baggett & Wendland 2001). Mulholland
et al. (1999) showed by electron microscopy that Ste2p receptors were concentrated
at furrow-like membrane invaginations distinct from the cortical actin patches that
they identified by virtue of their labeling with cofilin and actin antibodies. These
furrow-like invaginations contained some actin but less than is present in cortical
15 Sep 2003
13:10
AR
AR197-CB19-12.tex
AR197-CB19-12.sgm
LaTeX2e(2002/01/18)
Annu. Rev. Cell Dev. Biol. 2003.19:287-332. Downloaded from arjournals.annualreviews.org
by University of Pittsburgh on 08/17/07. For personal use only.
ACTIN AND ENDOCYTOSIS
P1: GCE
295
patches (Mulholland et al. 1999). This finding raises the possibility that different
types of cortical actin patches exist, consistent with the observation that many
proteins (e.g., Sla2p, Sla1p, Pan1p) localize to cortical puncta that do not appear
to contain actin-binding protein 1 (Abp1) or actin (Tang & Cai 1996, Warren et al.
2002, Yang et al. 1999). These cortical puncta may contain actin that cannot easily
be detected by light microscopy. Therefore, only a subset of cortical actin patches
may represent sites of internalization.
An alternative hypothesis is that cortical actin patches form transient interactions with endocytic sites. When cortical actin patches move rapidly along the cell
cortex (Doyle & Botstein 1996, Waddle et al. 1996), they may form short-lived
connections to endocytic components. In support of the hypothesis that interactions
between different patch components can be transient, Warren et al. (2002) showed
by fluorescence resonance energy transfer analysis of Sla1p-YFP and Abp1p-CFP
that these proteins can associate with each other at the cell cortex but that there
are also puncta that contain only Sla1p or Abp1p. In addition, the motility and
relative proportion of puncta that move were different for Sla1p versus Abp1p.
These experiments clearly demonstrate that there are distinct subpopulations of
cortical patches, and they suggest that individual patch components may come
together in a transient fashion.
For future studies in yeast, it is critically important to define cortical patch
motility and protein composition in relation to internalization events. Key questions
include whether patch motility is required for internalization, whether a subset of
cortical actin patches localize to endocytic sites, and whether distinct patches with
distinct protein compositions have distinct endocytic functions or cargo.
Protein-Protein Interactions Between Components
Important for Internalization: How is the Actin
Machinery Recruited to Endocytic Sites?
Genetic screens led to the identification of a number of yeast proteins important for endocytic internalization from the plasma membrane. The challenge now
is to elucidate the specific role of each of these proteins and to understand at
the molecular level how the actin cytoskeleton participates in the internalization
event. Biochemical studies are beginning to reveal activities for these proteins and
to provide information on how they interact in the cell. Figure 1 illustrates some
of the interactions that have been identified between proteins important for internalization and certain biochemical activities. For an excellent review detailing
some of these interactions, see Goode & Rodal (2001). The interactions shown are
not necessarily direct because many were detected by coimmunoprecipitation and
two-hybrid experiments. However, the cartoon provides a working model for how
these proteins may assemble into modular complexes and how these complexes
might induce actin polymerization at endocytic sites.
A critical issue is how the endocytic machinery associates with receptors,
transporters, and membranes destined to be endocytosed. As shown in Figure 1,
26 Aug 2003
20:13
Annu. Rev. Cell Dev. Biol. 2003.19:287-332. Downloaded from arjournals.annualreviews.org
by University of Pittsburgh on 08/17/07. For personal use only.
296
AR
AR197-CB19-12.tex
ENGQVIST-GOLDSTEIN
¥
AR197-CB19-12.sgm
LaTeX2e(2002/01/18)
P1: GCE
DRUBIN
endocytosis in yeast has been classified into two main pathways based on cargo,
RME and fluid-phase endocytosis, although whether these pathways are really
distinct is not clear. RME can further be divided into ubiquitin-dependent internalization (see Figure 1a) (Hicke 2001) and NPFX(1,2)D-dependent internalization
(see Figure 1b) (Howard et al. 2002, Tan et al. 1996); this division depends on the
signal for internalization. Unexpectedly, most mutants defective in α-factor RME
are also defective in fluid-phase endocytosis, suggesting that these pathways share
some of their protein machinery (Table 1). A functional actin cytoskeleton may be
critically important for both pathways. One prediction is that proteins specifically
involved in receptor phosphorylation and ubiquitination would not be important for
fluid-phase endocytosis. This is the case for the redundant casein kinases, Yck1p
and Yck2p, which are candidates to phosphorylate Ste2 (Table 1) (Feng & Davis
2000, Hicke et al. 1998). On the other hand, mutants of RSP5, which encodes an
E3 ubiquitin ligase involved in mono-ubiquitination of receptors, have defects in
both pathways (Dunn & Hicke 2001), suggesting that Rsp5p needs to ubiquitinate
machinery required for fluid-phase endocytosis in addition to the proteins targeted
for endocytosis. Actin cytoskeleton components are candidate targets because rsp5
mutants are Lat-A resistant and show genetic interactions with genes important
for actin organization (e.g., ARP2, END3, SLA2) (Kaminska et al. 2002).
The main pathway for RME in yeast is the ubiquitin-dependent pathway (see
Figure 1a). G protein–coupled receptors, permeases, and transporters are ubiquitinated at the cell surface to signal internalization (Munn 2001). The G-coupled
receptor Ste2p has been studied most extensively, and ligand binding stimulates
phosphorylation followed by ubiquitination of specific residues in the cytoplasmic
tail (Hicke & Riezman 1996, Hicke et al. 1998, Reneke et al. 1988). The sequence
motif SINNDAKSS in Ste2p, wherein the lysine is mono-ubiquitinated and the
serines are phosphorylated, is sufficient, although not necessary, for internalization (Rohrer et al. 1993). Outside of this motif, multiple additional lysines that get
ubiquitinated are important for internalization (Terrell et al. 1998).
Although we have limited knowledge regarding the sequential steps for internalization in yeast, we attempt to order these events based on mutant phenotypes,
genetic epistasis, and some speculation (Figure 2). In step 1, ligand binds to the receptor and triggers phosphorylation of several serine and threonine residues in the
Ste2p cytosplasmic tail (step 2). Receptor hyperphosphorylation is a prerequisite
for ubiquitination. The redundant casein kinase I homologues Yck1p and Yck2p
are candidates for this step. In yck-ts cells, the Ste2p receptor is not internalized owing to lack of phosphorylation and ubiquitination (Feng & Davis 2000, Hicke et al.
1998). The requirement for specific ergosterols (sterols) appears to be coincident
with this step or earlier in the pathway (Heese-Peck et al. 2002, Munn et al. 1999).
Similar to yck-ts mutants, erg (sterol) mutants are defective in hyperphosphorylation and ubiquitination of the Ste2p receptor. As mentioned above, these ergosterols
may not be important for a functional actin cytoskeleton (Heese-Peck et al. 2002).
Akr1p (ankyrin-repeat protein) is also likely to act at this step because it is required
for the recruitment of Yck2p to the plasma membrane (Feng & Davis 2000).
26 Aug 2003
20:13
AR
AR197-CB19-12.tex
AR197-CB19-12.sgm
LaTeX2e(2002/01/18)
Annu. Rev. Cell Dev. Biol. 2003.19:287-332. Downloaded from arjournals.annualreviews.org
by University of Pittsburgh on 08/17/07. For personal use only.
ACTIN AND ENDOCYTOSIS
P1: GCE
297
Figure 2 Steps in ubiquitin-dependent endocytosis in yeast. This flow chart illustrates
putative steps during ligand-induced internalization of the Ste2p receptor (see text for
details). The requirement for different proteins and lipids, when known, is indicated
on the right side of the figure.
In step 3, the E3 ubiquitin ligase Rsp5p ubiquitinates receptors and possibly
other proteins required for internalization (Dunn & Hicke 2001, Hicke & Riezman
1996). In step 4, adaptor proteins bind to the ubiquitinated receptor. These adaptor
proteins might recruit the machinery required for actin polymerization (see step
5). Candidates to perform this adaptor function are Ent1p and Ent2p (Ent1/2p),
which have redundant functions in endocytosis and in actin cytoskeletal organization (Wendland 2002). Ent1/2p are related to epsins in mammalian cells, which are
important for clathrin-mediated endocytosis (Chen et al. 1998). Ent1/2p contain
ubiquitin-interacting motifs (UIM) that bind to mono-ubiquitin in vitro (Shih et al.
2002). As shown in Figure 1, Ent1/2p interact with Pan1p, an Arp2/3 activator, and
with Chc1p (clathrin heavy chain) (Duncan et al. 2001, Wendland & Emr 1998,
Wendland et al. 1999). Therefore, one can speculate that Ent1/2p recruits Pan1p
and clathrin to the receptors via interactions with ubiquitin (Wendland 2002). In
support of the possibility that Ent1/2p function as adaptors, it has been shown
26 Aug 2003
20:13
Annu. Rev. Cell Dev. Biol. 2003.19:287-332. Downloaded from arjournals.annualreviews.org
by University of Pittsburgh on 08/17/07. For personal use only.
298
AR
AR197-CB19-12.tex
ENGQVIST-GOLDSTEIN
¥
AR197-CB19-12.sgm
LaTeX2e(2002/01/18)
P1: GCE
DRUBIN
that the UIMs in Ent1/2p are important for internalization of Ste2p, although this
function may be redundant with that of Ede1p, another ubiquitin-binding protein
(Shih et al. 2002). Furthermore, the UIMs in Ent1p are important for binding to
crude yeast membrane fractions (Aguilar et al. 2003). Ent1/2p also contain epsin
N-terminal homology (ENTH) domains that interact with phosphatidylinositol4,5-biphosphate [PtdIns(4,5)P2] (Aguilar et al. 2003). This interaction may further
anchor the endocytic complex to the plasma membrane (Aguilar et al. 2003).
Studies in mammalian cells suggest that binding to PtdIns(4,5)P2 in the plasma
membrane is important for targeting proteins to endocytic sites (Gaidarov & Keen
1999). In yeast, mutants of proteins that regulate PtdIns(4,5)P2 levels, such as inositol polyphosphate-5 phosphatase (Inp51p/Sjl1p, Inp52p/Sjl2p, and Inp53p/Sjl3p)
and phosphatidylinositol(4)-phosphate 5-kinase (Mss4p), are defective in endocytic internalization and in the organization of the actin cytoskeleton (Table 1)
(Desrivieres et al. 2002, Singer-Kruger et al. 1998).
Other candidates that may function as adaptors are Sla2p and yAP180a and
yAP180b (yAP180a/b). These proteins are related to proteins involved in clathrinmediated internalization in mammalian cells, huntingtin interacting protein 1
(Hip1)/huntingtin interacting protein 1 related (Hip1R) and AP180/CALM, respectively (McMahon 1999, McPherson 2002). Similar to Ent1/2p, Sla2p and
yAP180a/b can bind to Pan1p and clathrin and are predicted to interact with
PtdIns(4,5)P2 via their ENTH-like domains [renamed AP180 N-terminal homology (ANTH) domain] (Figure 1) (Henry et al. 2002, Wendland & Emr 1998;
M.J. Cope, Y. Sun, J. Toshima & D.G. Drubin, unpublished data). Additionally,
yAP180a/b have been reported to interact with Ede1p (B. Wendland, personal
communication). Sla2p and yAP180a/yAP180b may not interact with receptors
because they do not appear to contain ubiquitin-interacting domains. However, they
could help tether endocytic complexes to the plasma membrane. Sla2p is required
for internalization of α-factor and is required for actin assembly in a permeabilized cell assay (Li et al. 1995), suggesting that it might function at the interface
between actin and the endocytic machinery. Sla2p contains an actin-binding domain (I/LWEQ module) (McCann & Craig 1997). However, it is currently not
clear whether this domain is important for endocytosis (Wesp et al. 1997, Yang
et al. 1999). It has been shown that the localization of Sla2p to punctate structures
at the cell cortex is dependent on clathrin but independent of F-actin (Ayscough
et al. 1999, Henry et al. 2002). Furthermore, Sla1p is required for correct localization of Sla2p (Ayscough et al. 1999). Because Sla1p is a putative adaptor for
NPFX(1,2)D-mediated internalization (see below), we speculate that the cortical
patches containing Sla2p are sites of endocytosis. The fact that clathrin is required
to properly localize Sla2p to the cell cortex may also explain why clathrin mutants
have actin defects; Sla2p is required for normal actin cytoskeleton organization.
In contrast to sla2 mutants, yap180a/b mutants do not have endocytic defects. It
seems likely, however, that these proteins perform functions redundant with other
proteins (e.g., Ent1/2 p or Sla2p).
It is not clear whether AP-2-related proteins in yeast function in endocytosis
because deletion mutants of the genes encoding subunits of this complex do not
26 Aug 2003
20:13
AR
AR197-CB19-12.tex
AR197-CB19-12.sgm
LaTeX2e(2002/01/18)
Annu. Rev. Cell Dev. Biol. 2003.19:287-332. Downloaded from arjournals.annualreviews.org
by University of Pittsburgh on 08/17/07. For personal use only.
ACTIN AND ENDOCYTOSIS
P1: GCE
299
have endocytic defects (Huang et al. 1999, Yeung et al. 1999). Again, it is possible
that this function is redundant with the functions of Ent1p, Ent2p, Sla2p, or other
proteins that operate as adaptors. In the future, determining which proteins function
as adaptors in ubiquitin-dependent internalization in yeast will be important.
In step 5, the machinery required for actin polymerization is recruited to endocytic sites via binding to adaptor proteins. This can then lead to rapid F-actin
polymerization at these sites to provide a force during vesicle formation. Mutants of
Sla2p (end4-1), Vrp1p (vrp11), and Rvs167p (rvs1671) accumulate hyperphosphorylated and ubiquitinated Ste2p and/or Ste3p receptor at the plasma membrane,
showing that these earlier steps are not blocked when endocytosis is blocked in
mutants of actin-associated proteins (Feng & Davis 2000, Heese-Peck et al. 2002).
These proteins are required for a functional actin cytoskeleton (Bauer et al. 1993,
Donnelly et al. 1993, Yang et al. 1999). Vrp1p binds to Las17p and Myo3/5p and
may regulate their ability to activate the Arp2/3 complex (Evangelista et al. 2000,
Lechler et al. 2000, Naqvi et al. 1998). Rvs167p forms multiple interactions with
other proteins important for endocytic internalization, which suggests that it may
function as a scaffold protein in endocytosis. Further support for the conclusion
that actin functions after receptor phosphorylation and ubiquitination comes from
studies on Pan1p and on End3p (Shih et al. 2000), which is a protein important for
localization of Pan1p to the cortex (Tang et al. 1997). Mutants of Pan1p (pan1-20)
and End3p (end3-1) block internalization of a Ste2p chimera, in which an in-frame
fusion to the ubiquitin amino acid sequence has been added to a truncated cytoplasmic tail (Shih et al. 2000). This Ste2p receptor bypasses the normal requirement
for phosphorylation and ubiquitination for internalization.
In the future, utilizing electron microscopy to visualize receptors (e.g., Ste2p) in
mutants blocked at different endocytic steps will be critical. For example, do endocytic sites (labeled with Ste2p) look different in yck mutants (blocks step 2) compared with the way they look in sla2/end4 mutants (blocks after step 3)? There is
precedence for the observation of intermediate endocytic structures in yeast cells by
electron microscopy. Wendland et al. (1996) spheroplasted yeast cells and labeled
them with an endocytic marker (cationized ferritin) that is often used in mammalian
cells to label endocytic pathways. Interestingly, alleles of PAN1 (pan1-3) and SLA2
(end4-1) showed an increase in the number of plasma membrane invaginations
labeled with cationized ferritin (Wendland et al. 1996). These may represent endocytic intermediates that failed to pinch off from the plasma membrane. Membrane
invaginations were also observed in sjl11/sjl21 double mutants (synaptojaninlike proteins) (Srinivasan et al. 1997). Because synaptojanin-like proteins in yeast
control the concentration and the cellular distribution of PtdIns(4,5)P2, we speculate that the recruitment of Ent1/2p is affected in sjl11/sjl21 double mutants.
This could in turn affect the recruitment of Pan1p and the actin polymerization
machinery to endocytic sites.
The other pathway for RME is NPFX(1,2)D-mediated internalization (Figure 1).
The NPFX(1,2)D motif was originally discovered in the cytosplasmic tail of the
furin-like protease Kex2p, although Kex2p is not thought to be internalized from
the plasma membrane (Tan et al. 1996). This motif is also found in the Ste3p
26 Aug 2003
20:13
Annu. Rev. Cell Dev. Biol. 2003.19:287-332. Downloaded from arjournals.annualreviews.org
by University of Pittsburgh on 08/17/07. For personal use only.
300
AR
AR197-CB19-12.tex
ENGQVIST-GOLDSTEIN
¥
AR197-CB19-12.sgm
LaTeX2e(2002/01/18)
P1: GCE
DRUBIN
receptor, and a similar motif (GPFAD) is found in the Ste2p receptor (Howard et al.
2002). The NPFX(1,2)D motif is sufficient for internalization of the Ste2p receptor
lacking most of the cytoplasmic tail. This motif, however, is not necessary for
internalization, which suggests that the ubiquitin internalization signal is masking
the requirement for the NPFX(1,2)D motif (Howard et al. 2002). Sla1p was recently
shown to bind to the NPFX(1,2)D motif, suggesting that Sla1p may serve as an
adaptor (Howard et al. 2002) (Figure 1). Furthermore, SLA1 disruption severely
inhibited NPFX(1,2)D-mediated internalization of Ste2p, but it only marginally
affected ubiquitin-mediated internalization. Sla1p is important for normal actin
organization, and it interacts with several proteins required for both endocytosis
and actin functions in yeast (Figure 1) (Ayscough et al. 1999, Ayscough et al. 1997,
Holtzman et al. 1993, Warren et al. 2002). Coimmunoprecipitation and/or yeast
two-hybrid experiments show that Sla1p can interact with Pan1p, End3p, Las17p,
Rvs167p, and Abp1p (Drees et al. 2001, Li 1997, Tang et al. 2000, Warren et al.
2002). Because both Pan1p and Las17p can activate the Arp2/3 complex (Duncan
et al. 2001, Winter et al. 1999), Sla1p is an excellent candidate for directing actin
polymerization to sites of endocytosis.
During endocytosis, assembly and disassembly of the endocytic complex are
likely to be tightly regulated. Evidence is emerging that this regulation is accomplished through cycles of phosphorylation and dephosphorylation of key components of the endocytic machinery. Ark1p and Prk1p, two redundant serine/threonine
kinases, have been implicated in this process. Ark1p and Prk1p phosphorylate
Sla1p, Pan1p, and Ent1/2p, and overexpression of Prk1p leads to disruption of the
interaction between Pan1p and Sla1p (Watson et al. 2001, Zeng & Cai 1999, Zeng
et al. 2001). Because Pan1p functions to promote F-actin assembly, possibly at
endocytic sites, one might speculate that Ark1p/Prk1p negatively regulates actin
polymerization at endocytic sites. In support of this possibility, lack of both Ark1p
and Prk1p inhibits endocytosis and causes formation of large actin clumps containing proteins that are important for both actin polymerization and internalization
(e.g., Ent1p, Ent2p, Sla2p, Pan1p, Cof1p, and Sac6p) (Cope et al. 1999, Watson
et al. 2001).
Several Yeast Proteins Important for Endocytic
Internalization Can Promote Actin Assembly
Actin polymerization occurs preferentially at free (growing) barbed ends of filaments. Because the rate-limiting step in actin filament formation is nucleation, the
generation of free barbed ends controls where and when polymerization occurs.
There are three known mechanisms for generating free barbed ends: de novo nucleation, severing of existing filaments, and uncapping of filaments. At least two
of these activities (de novo nucleation and severing) may be important for endocytic internalization. To date, approximately one third of the proteins important
for endocytic internalization can directly bind to actin or affect its assembly or
organization (Table 1). The activities of these proteins include actin nucleation
26 Aug 2003
20:13
AR
AR197-CB19-12.tex
AR197-CB19-12.sgm
LaTeX2e(2002/01/18)
Annu. Rev. Cell Dev. Biol. 2003.19:287-332. Downloaded from arjournals.annualreviews.org
by University of Pittsburgh on 08/17/07. For personal use only.
ACTIN AND ENDOCYTOSIS
P1: GCE
301
and generation of branched actin networks (Arp2/3 complex), Arp2/3 activation
(Las17p, Pan1p, Abp1p, and possibly Myo3p/Myo5p), motor activities (Myo3p
and Myo5p), F-actin severing and depolymerization (Cof1p), and F-actin crosslinking (Sac6p) (Goode & Rodal 2001, Welch & Mullins 2002) (Figure 1). In
addition, Vrp1p, Sla2p, and Srv2p bind to actin, but their actin-related activities,
if any, are not known (Figure 1) (Freeman et al. 1995, McCann & Craig 1997,
Vaduva et al. 1997).
The Arp2/3 complex contains seven highly conserved subunits (Arp2p, Arp3p,
Arc40p, Arc35p, Arc19p, Arc18p, and Arc15p), wherein Arp2p and Arp3p are
actin-related proteins (Goode & Rodal 2001, Welch & Mullins 2002). Conditional
alleles of ARP2 (arp2-1) and ARC35 (end9) are defective in the uptake of uracil
permease and α-factor, respectively, providing evidence that the Arp2/3 complex
is required for endocytic internalization (Moreau et al. 1996, Moreau et al. 1997,
Munn & Riezman 1994). Because overexpression of Las17p (yeast WASP) suppresses the actin and the endocytic defect of arp2-1 (in this case LY uptake was
measured) (Madania et al. 1999), these experiments suggest that actin nucleation
by the Arp2/3 complex is required for internalization. The mammalian Arp2/3
activator N-WASP was recently implicated in endocytosis, suggesting that this
Arp2/3-related function is conserved in mammalian cells (Kessels & Qualmann
2002, Zhang et al. 1999).
In yeast, two additional proteins, Pan1p and Abp1p activate the Arp2/3 complex
(Duncan et al. 2001, Goode et al. 2001). In addition, type I myosins, Myo3p and
Myo5p, bind to the Arp2/3 complex, and genetic and biochemical data suggest
that they activate the Arp2/3 complex (Evangelista et al. 2000, Geli et al. 2000,
Lechler et al. 2000). This mechanism has not yet been shown directly, although
fission yeast type I myosins do activate the Arp2/3 complex (Lee et al. 2000).
Pan1p is required for endocytosis and normal actin organization in yeast, and it
interacts with other proteins important for internalization (Figure 1) (Tang & Cai
1996, Tang et al. 1997, Wendland & Emr 1998, Wendland et al. 1996, Zoladek
et al. 1997). Similar to las17 and pan1 mutants, mutants of Myo3/5p have severe
defects in endocytic internalization and in actin organization (Geli & Riezman
1996). In contrast, abp11 cells show no apparent defect in α-factor uptake or actin
organization (Kubler & Riezman 1993). Abp1p may share some redundant functions with Sla2p in endocytosis because combining deletions of certain domains of
these proteins (sla21coil and abp11SH3) causes defects in α-factor uptake (Wesp
et al. 1997). In addition, there is genetic evidence that the role of Abp1p as an actin
nucleation factor might be different from those of Las17p and Pan1p (Goode &
Rodal 2001). As shown in Figure 1, Pan1p, Las17p, Abp1p, and Myo3/5p form a
network of interactions with other proteins important for internalization and could
thereby get recruited to sites of internalization.
The observation that several Arp2/3 activators are required for endocytic internalization suggests that actin polymerization driven by distinct activators might
be required during different steps in internalization. It was surprising to find that
a mutant of Las17p lacking the activation domain (acidic motif) does not have an
26 Aug 2003
20:13
Annu. Rev. Cell Dev. Biol. 2003.19:287-332. Downloaded from arjournals.annualreviews.org
by University of Pittsburgh on 08/17/07. For personal use only.
302
AR
AR197-CB19-12.tex
ENGQVIST-GOLDSTEIN
¥
AR197-CB19-12.sgm
LaTeX2e(2002/01/18)
P1: GCE
DRUBIN
endocytic defect and has only a mild actin cytoskeleton defect (Duncan et al. 2001,
Winter et al. 1999). Similarly, point mutations in the activation domain of Pan1
(pan1-101) did not cause an endocytic defect, although a larger deletion including
the activation domain (pan11855-1480) caused endocytic defects (Duncan et al.
2001). Combining alleles of PAN1 and LAS17 lacking the activation domains does
cause more severe growth and actin phenotypes (Duncan et al. 2001). This suggests
that Pan1p and Las17p each perform a unique function in endocytic internalization.
However, they may also share a distinct function related to activation of the Arp2/3
complex. Myo3/5p also share functions with Las17p in actin polymerization and
possibly in endocytosis (Evangelista et al. 2000, Geli et al. 2000, Lechler et al.
2000). Deletion of the acidic Arp2/3 activation motifs in both Las17p and Myo3p
abolished actin nucleation in permeabilized myo3 myo51 yeast cells, whereas
single mutants had no effect (Lechler et al. 2000). These particular mutants have
not yet been tested for defects in endocytosis. It will be important to dissect the
specific roles of these activators in mediating endocytic internalization.
In addition to a requirement for de novo nucleation of actin filaments and/or
generation of branched actin networks, there is also evidence that actin filament
turnover is required for endocytic internalization. Jasplakinolide (Jas), a drug that
stabilizes F-actin, inhibits endocytosis in yeast (Ayscough 2000). Furthermore,
Cof1p (cofilin), which severs and depolymerizes F-actin, is critically important
for LY and α-factor internalization (Idrissi et al. 2002, Lappalainen & Drubin
1997). In a finding consistent with these results, Belmont & Drubin (1998) showed
that an actin mutant (V159N) defective in actin filament disassembly was also
defective in LY uptake. Because these conditions (drugs and mutants) limit the
available pool of G-actin, it is currently not clear whether the requirement for actin
turnover in endocytosis is due to block of new assembly, block of disassembly, or
both.
ENDOCYTIC FUNCTION OF THE ACTIN CYTOSKELETON
IN MORE COMPLEX EUKARYOTES
In mammalian cells, there are several distinct pathways for endocytic internalization: the clathrin-dependent pathway, the caveolar pathway, a clathrin- and
caveolae-independent pathway, macropinocytosis, and phagocytosis. These pathways are distinct, but it has become evident that they share some of the same
machinery. Components of the actin cytoskeleton (e.g., the SCAR/WASP proteins) and dynamin, a protein that functions during vesicle fission (see below),
have been implicated in several of these pathways (Orth & McNiven 2003, Welch
& Mullins 2002). Furthermore, there is now evidence that localized polymerization
of F-actin occurs at endocytic sites in caveolae-mediated endocytosis (Pelkman
et al. 2002), clathrin-mediated endocytosis (Merrifield et al. 2002), macropinocytosis (Merrifield et al. 1999), and phagocytosis (May & Machesky 2001). These
observations of mammalian endocytosis, considered with the mounting evidence
that the actin polymerization machinery is required for yeast endocytosis, suggest
26 Aug 2003
20:13
AR
AR197-CB19-12.tex
AR197-CB19-12.sgm
LaTeX2e(2002/01/18)
ACTIN AND ENDOCYTOSIS
P1: GCE
303
Annu. Rev. Cell Dev. Biol. 2003.19:287-332. Downloaded from arjournals.annualreviews.org
by University of Pittsburgh on 08/17/07. For personal use only.
that actin polymerization plays a role in most, if not all, endocytic pathways, and
it may play a similar role during these different internalization events. However,
differences in sensitivity to actin poisons suggest that the importance of this function may vary for different organisms and cell types. Because the link between
actin polymerization and phagocytosis is well established and has been reviewed
previously (May & Machesky 2001, Welch & Mullins 2002), it is not discussed
in detail here.
Clathrin-Mediated Endocytosis
Clathrin-mediated endocytosis depends on structural components of the coat and
a growing list of accessory factors (Brodsky et al. 2001). Structural components
of the coat (e.g., clathrin and AP-2) are highly enriched in clathrin-coated vesicle
(CCV) preparations, whereas most of the accessory factors form more transient
interactions with clathrin-coated pits (CCPs) and CCVs. Actin and proteins important for actin polymerization (e.g., the Arp2/3 complex) are either absent or are
present at low levels in CCV preparations (P.S. McPherson, personal communication). This observation is consistent with ultrastructure and real-time microscopy
analyses, which suggest that the actin polymerization machinery may form only
transient interactions with CCVs (Fujimoto et al. 2000, Merrifield et al. 2002).
The clathrin coat, however, does contain actin-binding proteins. Both Hip1R and
a spliced variant of Myosin VI are enriched in CCV preparations and appear to
stably associate with the clathrin coat (see Links Between Coat Components and
Actin, below).
A role for actin in clathrin-dependent endocytosis has not been as definitively established as that for other types of endocytosis (e.g., macropinocytosis and phagocytosis). Cytochalasin-D (CD), a drug that destabilizes actin filaments, inhibited
apical endocytosis in polarized epithelial cells but had no detectable effect on basolateral endocytosis (Apodaca 2001). In other cell types, actin-depolymerizing
drugs have had varied effects on RME (Lamaze et al. 1996, 1997; Salisbury et al.
1980; Sandvig & van Deurs 1990; Wolkoff et al. 1984). In an extensive study,
using several different actin-perturbing drugs (Lat-A, CD, and Jas), Fujimoto
et al. (2000) found that the ability of these drugs to inhibit clathrin-mediated endocytosis depended on cell type and growth conditions. It is important to note that the
assay used in this study measured sealing of vesicles and not subsequent endocytic
events. Therefore, in those cases in which an effect was not observed, F-actin may
have played a role subsequent to vesicle sealing, such as detachment and transport
of vesicles away from the cell cortex. In fact, recent live-cell-microscopy studies in
mammalian cells provided evidence that actin polymerization occurs concomitant
with late stages of CCV formation (Merrifield et al. 2002). Taken together, these
various observations suggest that actin functions in clathrin-mediated endocytosis,
although this function may not always be obligatory for CCV formation.
What are the specific functions of the actin cytoskeleton in CCV formation,
and at which stages in CCV formation is actin involved? Genetic approaches
are less practical in mammalian cells than in yeast for example. Therefore, few
26 Aug 2003
20:13
Annu. Rev. Cell Dev. Biol. 2003.19:287-332. Downloaded from arjournals.annualreviews.org
by University of Pittsburgh on 08/17/07. For personal use only.
304
AR
AR197-CB19-12.tex
ENGQVIST-GOLDSTEIN
¥
AR197-CB19-12.sgm
LaTeX2e(2002/01/18)
P1: GCE
DRUBIN
studies in mammalian cells have looked at loss-of-function phenotypes for proteins
implicated in endocytosis. Instead, functional studies often depend on less-thanideal overexpression and dominant-negative phenotypes. Studies of mammalian
cells, however, have some advantages over studies of yeast cells. For example, the
ultrastructure of endocytic sites (e.g., CCPs) in mammalian cells is well described
(Higgins & McMahon 2002). Furthermore, permeabilized cell assays for endocytic
internalization (Schmid & Smythe 1991) and assays to reconstitute initial stages
of CCV formation (Higgins & McMahon 2002) have been developed. Based on
the results from these assays, microinjection studies (e.g., antibodies, domains of
proteins, or peptides), and overexpression studies (e.g., use of dominant-negative
forms of the protein), clathrin-mediated internalization in mammalian cells has
been divided into several distinct stages. These stages include coat assembly on
membranes, invagination, fission, movement of vesicles away from the plasma
membrane, and finally, uncoating (Brodsky et al. 2001, Jarousse & Kelly 2001).
Genetic approaches in Drosophila melanogaster and Caenorhabditis elegans, and
studies using the lamprey reticulospinal synapse, have also contributed to our
understanding of CCV formation.
Clathrin is a triskelion-shaped cytoplasmic protein having three heavy chains
and three light chains. According to current models, adaptor proteins (e.g., AP-2)
recruit and promote the assembly of clathrin triskelia on the plasma membrane
to form clathrin lattices (Brodsky et al. 2001). The recruitment of AP-2 to the
plasma membrane is thought to involve interactions with phospholipids, tyrosineand dileucine-based endocytic sorting motifs present in the cytoplasmic tails of receptors, synaptotagmin, and accessory factors (e.g., Eps15) (Brodsky et al. 2001).
Other proteins (e.g., β-arrestin) can also recruit receptors to clathrin lattices, although these proteins do not appear to promote clathrin assembly (Goodman et al.
1997). AP180/CALM, epsins, and possibly Hip1/Hip1R may also function as adaptors because, similar to AP-2, they bind to clathrin and phospholipids, and they
promote clathrin assembly in vitro (Ford et al. 2002, Ford et al. 2001, Wendland
2002).
The recruitment of adaptors to the plasma membrane may also involve Eps15, a
protein involved in clathrin-mediated endocytosis (Benmerah et al. 1998, Carbone
et al. 1997). Because Eps15 binds to other endocytic proteins including AP-2, epsin,
intersectin, and synaptojanin (Benmerah et al. 1995, Chen et al. 1998, Haffner et al.
1997, Sengar et al. 1999), it might be part of a core endocytic complex that is required for clathrin-mediated endocytosis. As discussed above, a related complex
in yeast is required for endocytic internalization and normal actin organization
(Figure 1). The yeast Arp2/3 activator Pan1p is related to Eps15; the putative
adaptor proteins Ent1/2p are related to epsins; the inositol polyphosphatase-5
phosphatases (Inp51p/Sjl1p, Inp52p/Sjl2p, and Inp53p/Sjl3p) are related to synaptojanins; and the Sla1p/End3p complex shares conserved domains with intersectin.
Eps15 has not been reported to activate the Arp2/3 complex. However, intersectinl, which binds to Eps15, recently was shown to regulate actin assembly via Cdc42
and N-WASP (see below) (Hussain et al. 2001).
15 Sep 2003
13:11
AR
AR197-CB19-12.tex
AR197-CB19-12.sgm
LaTeX2e(2002/01/18)
Annu. Rev. Cell Dev. Biol. 2003.19:287-332. Downloaded from arjournals.annualreviews.org
by University of Pittsburgh on 08/17/07. For personal use only.
ACTIN AND ENDOCYTOSIS
P1: GCE
305
According to current models, after clathrin lattices are formed, they invaginate
to form CCPs. Endophilin, which is a lysophosphatic acid transferase, has been
implicated in this step as well as in fission and vesicle recycling (Gad et al. 2000,
Ringstad et al. 1999, Schmidt et al. 1999). After deeply invaginated coated pits
are formed, the neck narrows to form constricted pits. These subsequently pinch
off from the plasma membrane as CCVs. The large GTPase dynamin has been
convincingly implicated in these steps (Hinshaw 2000). The temperature-sensitive
mutant shibire in D. melanogaster, which has a mutation in the dynamin gene, is
blocked in clathrin-mediated endocytosis when switched to the restrictive temperature (Koenig & Ikeda 1989, Kosaka & Ikeda 1983). Visualization of the nerve
terminals in this mutant shows that they accumulate constricted CCPs (Koenig &
Ikeda 1989, Kosaka & Ikeda 1983). Subsequent studies have demonstrated that
dominant-negative forms of dynamin will interfere with clathrin-mediated endocytosis in many different cell types (Hinshaw 2000). It is not clear whether dynamin
acts as a force-generating mechanochemical motor protein, or as a regulator of
the fission event, possibly by recruiting other force-generating proteins, or both
(Sever et al. 2000). Dynamin is a multidomain protein, with a GTPase domain,
a phospholipid-binding pleckstrin-homology domain, a GTPase effector domain,
and a proline-rich domain (PRD). Through its PRD, dynamin interacts with a
number of different src-homology 3 (SH3)-containing proteins (e.g., endophilin,
syndapin, Abp1, cortactin, intersectin, and amphiphysin). As discussed in the next
section, many of these SH3-containing proteins associate with the actin cytoskeleton either directly or indirectly.
After CCVs pinch off, they move away from the plasma membrane, shed their
coats, and fuse with other vesicles to form early endosomes. Biochemical studies
and imaging of clathrin-DsRed in live cells suggest that uncoating occurs soon
after CCVs pinch off from the plasma membrane (Gaidarov et al. 1999, Merrifield
et al. 2002). Auxilin and cyclin G–associated kinase have uncoating activities and
are thought to recruit Hsc70 to CCVs to disassemble the clathrin coat (Lemmon
2001). Synaptojanin, a protein that hydrolyzes several phosphoinositide species,
including PtdIns(4,5)P2 and PtdIns(3,4,5)P2, is also thought to function during
vesicle uncoating (Cremona & De Camilli 2001).
Actin may function at several distinct stages of clathrin-mediated endocytosis.
First, evidence is emerging that actin is involved in specifying sites of coatedpit formation on the plasma membrane (Figure 3). Bennett et al. (2001) recently
showed that AP-2 is organized in linear arrays that colocalize with actin stress
fibers. This colinearity was disrupted by treatment of cells with CD, which disassembles actin (Bennett et al. 2001). The linear AP-2 staining patterns were also
disrupted in cells that were induced to express the Hub fragment of clathrin, which
acts as a dominant-negative inhibitor of clathrin-dependent endocytosis (Bennett
et al. 2001). Hip1R, a protein that binds to clathrin and F-actin, also dissociated
from coated pits when Hub fragment was overexpressed (Bennett et al. 2001), suggesting that Hip1R may be important for proper association between clathrin and
actin.
26 Aug 2003
20:13
Annu. Rev. Cell Dev. Biol. 2003.19:287-332. Downloaded from arjournals.annualreviews.org
by University of Pittsburgh on 08/17/07. For personal use only.
306
AR
AR197-CB19-12.tex
ENGQVIST-GOLDSTEIN
¥
AR197-CB19-12.sgm
LaTeX2e(2002/01/18)
P1: GCE
DRUBIN
Further support for the hypothesis that actin acts as a scaffold for assembly
or anchoring of the endocytic machinery comes from a study in which clathrin
dynamics were observed in live cells. Gaidarov et al. (1999) found that treatment
with the actin monomer-sequestering drug Lat-B increased the lateral movement
of CCPs in the plane of the plasma membrane. Actin may therefore localize the
endocytic machinery to certain domains of the plasma membrane and function in
CCV formation. Localization of the endocytic machinery to certain sites on the
plasma membrane may be important to organize the cell cortex and/or to establish
and maintain cell polarity. This function may be especially important in polarized
cells such as neurons, epithelial, and migrating cells where lipids and cargo need
to be internalized at particular sites on the plasma membrane. The concept that
coated pits are formed at particular sites on the plasma membrane is supported
by the observations that clathrin-GFP (green fluorescent protein) puncta in live
Cos-7 cells disappear and then reappear many times at the same site (Gaidarov
et al. 1999). Furthermore, there is evidence in D. melanogaster that coated-pit
formation is initiated at specific, restricted sites (endocytic zones) during synaptic
vesicle recycling at the neuromuscular junction (Roos & Kelly 1999). Several
observations also indicate that actin filaments are enriched in endocytic zones at
the synapse (Dunaevsky & Connor 2000, Gad et al. 2000, Shupliakov et al. 2002),
where actin may play a role in recycling of synaptic vesicles (see below).
Second, several lines of evidence suggest that the actin cytoskeleton functions
during late events in coated-pit formation (at coated-pit neck constriction, fission,
and/or detachment of CCVs) (Figure 3: 1b–c). Lamaze et al. (1997) showed that
treatment of A431 cells with Lat-A blocked RME at the stage of invaginated coated
pits before vesicle closing (Lamaze et al. 1997). Consistent with a role for actin
at this stage, actin polymerization occurs at endocytic sites during late stages of
CCV formation. In an elegant study, Merrifield et al. (2002) used evanescent field
microscopy to observe the dynamics of CCV formation in live cells. By comparing
fluorescence intensities of clathrin-DsRed using both epifluorescence and evanescent field illumination, they detected the movement of clathrin-coated structures
(CCSs) away from the plasma membrane. After being stationary in the plane of the
plasma membrane for tens of seconds, CCSs moved approximately 100 nm deeper
into the cytoplasm, away from the plasma membrane, in a process that lasted 30–
60 s. It is currently not clear whether at the end of this process the CCVs were still
connected to the plasma membrane via an extended neck or whether the endpoint
represented release of CCVs (Figure 3: 1c versus 1c0 ). An alternative interpretation
is that the inward movement of clathrin represents invagination of the clathrin coat
(Santini & Keen 2002). Subsequently, the clathrin structures disappeared, perhaps
reflecting CCV uncoating. It is interesting to note that the movement away from
the plasma membrane occurred immediately after a brief burst of dynamin recruitment and was accompanied by a transient increase in actin association with
the CCSs. This work represents the first direct demonstration that actin assembly
occurs at CCPs associated with the plasma membrane, and it raises the possibility that actin polymerization provides forces driving endocytic internalization
26 Aug 2003
20:13
AR
AR197-CB19-12.tex
AR197-CB19-12.sgm
LaTeX2e(2002/01/18)
Annu. Rev. Cell Dev. Biol. 2003.19:287-332. Downloaded from arjournals.annualreviews.org
by University of Pittsburgh on 08/17/07. For personal use only.
ACTIN AND ENDOCYTOSIS
P1: GCE
307
(Figure 3: 1b). These forces may sometimes function redundantly with dynamin or
other accessory proteins during vesicle constriction and/or during vesicle fission
because actin does not appear to play an obligatory role in the formation of CCVs
in all cell types under all conditions. In addition, these experiments suggest that
actin may function to propel CCVs away from the cell cortex (Figure 3: 1c0 ). Another, nonmutually exclusive possibility is that the actin structures formed at CCSs
provide tracks upon which myosin VI motor protein can transport vesicles and/or
can provide a force during vesicle formation. However, the transient nature of these
actin structures makes such a role seem unlikely. As we discuss below, myosin VI
may be involved in clathrin-mediated endocytosis in polarized epithelial cells that
contain specialized actin structures such as microvilli.
The observation that dynamin recruitment precedes the transient appearance
of an actin-plume proximal to CCSs supports the model that dynamin recruits
machinery required for actin polymerization to endocytic sites. Using this type of
visualization analysis, it should now be possible to test this hypothesis using, for
example, dominant-negative dynamin and RNA interference. In the future, it will
also be important to establish the ultrastructure of the actin plumes to determine
which proteins are required for their formation and whether actin plumes are
required for endocytosis.
Further support for a role of actin in CCV fission and transport of vesicles
away from the plasma membrane comes from studies in polarized cells. CD treatment of MDCK and Caco-2 cells inhibited RME (transferrin uptake) and led to
accumulation of invaginated CCPs at the apical surface of the plasma membrane
(∼twofold increase in the number of CCPs) (Apodaca et al. 2001). The CCPs were
often connected to the apical surface by long narrow necks (Gottlieb et al. 1993).
Furthermore, in a recent study by Shupliakov et al. (2002) using the lamprey giant
reticulospinal synapse, recycling of synaptic vesicles was shown to be associated
with expansion of a cytoskeletal matrix containing actin-like filaments at the sites
of endocytosis. Compounds interfering with actin functions caused accumulation
of aggregates of synaptic vesicles near the endocytic zone, which suggests that
recycling of synaptic vesicles was impaired (Shupliakov et al. 2002).
In summary, there is evidence that actin functions during several distinct stages
of CCV formation (Figure 3). First, actin may provide a scaffold for the assembly
and anchoring of the endocytic machinery during CCV formation (Figure 3: 1a).
Second, actin polymerization may provide a force during constriction and/or fission
of CCVs (Figure 3: 1b,c). Third, actin may propel newly released CCVs away from
the plasma membrane (Figure 3: 1c0 ).
Caveolae-Mediated Endocytosis
It has been recognized for some time that there are clathrin-independent pathways for endocytic internalization, although our understanding of these endocytic
processes is in its infancy relative to our understanding of clathrin-dependent pathways. One of these pathways is caveolae-mediated endocytosis, which has been
26 Aug 2003
20:13
Annu. Rev. Cell Dev. Biol. 2003.19:287-332. Downloaded from arjournals.annualreviews.org
by University of Pittsburgh on 08/17/07. For personal use only.
308
AR
AR197-CB19-12.tex
ENGQVIST-GOLDSTEIN
¥
AR197-CB19-12.sgm
LaTeX2e(2002/01/18)
P1: GCE
DRUBIN
implicated in the internalization of certain membrane components (glycosphingolipids and GPI-anchored proteins), extracellular ligands (folic acid, albumin,
autocrine motility factor), bacterial toxins (cholera toxin, tetanus toxin), and several nonenveloped viruses (Simian virus 40, Polyoma virus) (Razani et al. 2001,
Pelkmans & Helenius 2002). Additionally, caveolae are involved in transcytosis
and cell signaling. Caveolae typically appear as flask-shaped plasma membrane
invaginations (50–80 nm wide), although their composition, appearance, and function are cell-type dependent (Razani et al. 2001). These membrane invaginations
share features with lipid rafts. Caveolae and lipid rafts are both enriched in cholesterol and sphingolipids and can be purified in a Triton-X insoluble fraction (Simons
& Toomre 2000). Caveolae, however, are distinct from lipid rafts in that they contain a protein coat composed primarily of caveolin-1 and caveolin-2 (caveolin-3
in muscle cells) (Rothberg et al. 1992).
The detailed mechanism of caveolae-mediated internalization is poorly understood. However, several experiments suggest that dynamin functions during the
fission step, similar to its role in clathrin-mediated internalization. Expression of
dominant-negative forms of dynamin, and/or microinjection of anti-dynamin antibodies, inhibited internalization of cholera toxin B and of Simian virus 40 (SV40)
particles (Henley et al. 1998, Oh et al. 1998, Pelkmans et al. 2002). Furthermore,
dynamin is necessary and sufficient for budding of caveolae in a cell-free budding
assay (Oh et al. 1998). Dynamin is concentrated at the neck of caveolae (Henley
et al. 1998, Oh et al. 1998). The recruitment of dynamin to caveolae, however, appears to be regulated. By visualizing internalization of SV40 particles, Pelkmans
et al. (2002) showed that dynamin-YFP associates with caveolae (caveolin-CFP)
at the cell cortex only after an SV40 particle was bound (Figure 3: 2b).
Pelkmans et al. (2002) showed that the actin cytoskeleton is important for
SV40 internalization via caveolae and that actin transiently associates with SV40containing caveolae at the stage when dynamin is recruited (Figure 3). Lat-A and
Jas reduced virus internalization by ∼60% and inhibited infection by the virus to a
similar extent. By visualizing caveolin 1-CFP (coat component) and YFP-actin in
live cells, Pelkmans et al. (2002) were able to determine the stage in internalization
during which actin appears to function. Caveolae, similar to CCSs, show very little
mobility in the plane of the membrane (Pelkmans et al. 2002, Thomsen et al. 2002).
Although Lat-A did not inhibit the association between SV40 particles and caveolae, it did cause the caveolae to move laterally in the plane of the plasma membrane,
similar to what was seen with CCPs. This suggests that actin may function as a scaffold to restrict the lateral mobility of both caveolae and CCPs (Figure 3: 2a). Actin
is also likely to function in later stages of caveolae internalization, possibly during
fission and/or during transport of vesicles away from the plasma membrane (Figure
3: 2b,c). Ten to 20 minutes after adding SV40 particles to cells, dramatic changes
in the actin cytoskeleton were observed. Stress fibers depolymerize with a concomitant recruitment of F-actin structures (patches and tails) to SV40-containing
caveolae at the cell cortex. The actin tails were on average 1.3 µm long, wherein
SV40 and caveolin 1 localized to one end of these tails. We speculate that these
26 Aug 2003
20:13
AR
AR197-CB19-12.tex
AR197-CB19-12.sgm
LaTeX2e(2002/01/18)
Annu. Rev. Cell Dev. Biol. 2003.19:287-332. Downloaded from arjournals.annualreviews.org
by University of Pittsburgh on 08/17/07. For personal use only.
ACTIN AND ENDOCYTOSIS
P1: GCE
309
actin structures are similar to those described above as associating with CCSs. The
caveolae-associated tails are, however, larger and are less transient in nature. It will
be important to determine which proteins are involved in promoting this F-actin tail
assembly. The Arp2/3 complex, N-WASP, and cortactin are prime suspects. Another important question concerns which proteins are involved in recruiting these
cytoskeletal proteins to caveolae. SV40 particles induce tyrosine phosphorylation
of proteins at virus-loaded caveolae, and inhibition of tyrosine kinases inhibits
internalization (Pelkmans et al. 2002). Therefore, it is possible that tyrosine phosphorylation of caveolae-associated proteins triggers recruitment of components
required for F-actin assembly. Finally, it is important to determine whether F-actin
is required for internalization of other types of cargo via the caveolae pathway.
Macropinocytosis
Macropinocytosis is a specialized form of endocytosis wherein extracellular fluid
is engulfed to form large vesicles (0.5–5 µm) called pinosomes. Macropinocytosis
may be either constitutive or stimulated by growth factors (e.g., epidermal growth
factor and platelet-derived growth factor). Our understanding of this endocytic
pathway in mammalian cells is fragmented because there are few molecular markers to identify pinosomes in vivo. Therefore, large vesicles containing a fluid-phase
marker (e.g., rhodamine-labeled dextran) are generally referred to as pinocytic
vesicles. However, different types of pinocytic vesicles and pathways may exist, depending on cell type, growth conditions, etc. Macropinocytosis, however,
has been studied quite extensively in Dictyostelium discoideum (Cardelli 2001,
Maniak 2002). A number of different proteins required for macropinocytosis have
been identified. Among these are several proteins that control actin organization
(e.g., the WASP-related protein Scar, coronin, profilin, DAip1, and type I myosin)
(Cardelli 2001, Maniak 2002). In addition, a number of proteins that may play regulatory roles in macropinocytosis have been identified. These regulators include a
member of the Ras family of GTPases (RasS), a GEF for Ras (RasGEFB), a member of the Rho family of GTPases (Rac1), and phosphatidylinositol kinase (PI
3-K) (Cardelli 2001, Maniak 2002). Actin is likely to function during formation
and closure of plasma membrane protrusions to form macropinosomes (Figure
3: 3a). This process is morphologically similar to Fcγ receptor–mediated phagocytosis, and it has been speculated that in both cases the extension of the plasma
membrane is mechanistically related to lamellipodium extension. In support of this
possibility, macropinocytosis appears to occur near membrane ruffles. In addition,
actin and components important for F-actin polymerization in lammelipodia (e.g.,
WASP-related proteins) are also important for phagocytosis and macropinocytosis in D. discoideum (Hacker et al. 1997, Insall et al. 2001, Lorenzi et al. 2000).
Furthermore, the Arp2/3 complex and Scar localize to sites of macropinocytic and
phagocytic internalization (Insall et al. 2001, Seastone et al. 2001).
Similar to clathrin-mediated endocytosis and caveolae-mediated endocytosis,
actin tails or plumes have been observed to associate with newly forming pinocytic
26 Aug 2003
20:13
Annu. Rev. Cell Dev. Biol. 2003.19:287-332. Downloaded from arjournals.annualreviews.org
by University of Pittsburgh on 08/17/07. For personal use only.
310
AR
AR197-CB19-12.tex
ENGQVIST-GOLDSTEIN
¥
AR197-CB19-12.sgm
LaTeX2e(2002/01/18)
P1: GCE
DRUBIN
vesicles at the cell cortex of mammalian cells (Figure 3: 3c). These actin tails have
been observed under physiological conditions (Kaksonen et al. 2000, Orth et al.
2002), but they are rare and transient in nature. Therefore, the majority of studies
have artificially induced the formation of pronounced actin tails (e.g., La3+ and
Ni2+ treatment, hyperosmolar media, overexpression of type 1 PIP kinase, and
overexpression of Arf6 mutants) (Heuser & Morisaki 1992, Lee & De Camilli
2002, Merrifield et al. 1999, Orth et al. 2002, Rozelle et al. 2000, Schafer et al.
2000). Tail-like actin structures in tissue culture cells and in Xenopus oocytes or
Xenopus egg exstracts have also been observed to associate with endosomes, lysosomes, GLUT4-positive vesicles, and Golgi-derived CCVs (Frischknecht et al.
1999, Kaksonen et al. 2000, Kanzaki et al. 2001, Rozelle et al. 2000, Taunton
et al. 2000). These actin structures resemble actin comet tails that propel intracellular pathogens such as Listeria monocytogenes through the cytoplasm of a
host cell (Taunton 2001). This phenomenon of endosomal “rocketing” was first
observed in intact, La3+- and Ni2+-treated cells (Heuser & Morisaki 1992). More
recently, Merrifield et al. (1999) characterized the association between actin and
pinocytic vesicles by observing GFP-actin in rat basophilic leukemia cells undergoing macropinocytosis in a mildly hyperosmolar medium. In these cells, actin
appeared to transiently polymerize on pinocytic vesicles (labeled with rhodamine
dextran) as they moved inward from the plasma membrane at a speed of ∼0.2 µm/s.
The actin tails were visible for a few minutes, consistent with observations in
D. discoideum, showing that the Arp2/3 complex dissociates from the macropinosome surface 10–40 s after closure (Insall et al. 2001).
The mechanism by which a membrane protrusion is transformed into a pinosome
is poorly understood. Merrifield et al. (1999) speculated that pinosomes acquire
the actin polymerization machinery necessary for propulsion from the crest of
the plasma membrane ruffle. This possibility is consistent with the observation
that proteins such as the Arp2/3 complex, N-WASP, and cortactin localize to tails
associated with pinocytic vesicles (Kaksonen et al. 2000, Schafer et al. 2000).
Specific phospholipids concentrated at sites of internalization may recruit and
activate, for example, WASP-related proteins. In support of this possibility, overexpression of phosphatidyl-4-phosphate 5-kinase, which increases the level of
PtdIns(4,5)P2, stimulates formation of vesicles propelled by actin comet tails in
fibroblasts (Rozelle et al. 2000). Several proteins have also been implicated in
regulating macropinocytosis in mammalian cells. These include the Rho family of GTPases (Cdc42 and Rac) (Nobes & Marsh 2000), an effector molecule
of Cdc42 and Rac, p21-activated kinase (Dharmawardhane et al. 2000), and the
ADP-ribosylation factor-6 (Arf6) (Schafer et al. 2000). Studies in dendritic cells
have been particularly fruitful in elucidating the regulation of macropinocytosis. In dendritic cells, macropinocytosis is developmentally regulated. Immature
cells engulf extracellular fluid for the purpose of antigen presentation (Inaba et al.
1993, Sallusto et al. 1995). In contrast, mature dendritic cells lose their ability
to macropinocytose. This switch is thought to be regulated by Cdc42 because
only immature dentritic cells have high levels of GTP-bound Cdc42 and because
26 Aug 2003
20:13
AR
AR197-CB19-12.tex
AR197-CB19-12.sgm
LaTeX2e(2002/01/18)
Annu. Rev. Cell Dev. Biol. 2003.19:287-332. Downloaded from arjournals.annualreviews.org
by University of Pittsburgh on 08/17/07. For personal use only.
ACTIN AND ENDOCYTOSIS
P1: GCE
311
overexpression of a nucleotide exchange factor for Cdc42 induces macropinocytosis in mature cells (Garrett et al. 2000). We suggest that activation of WASP-related
proteins by Cdc42 (Rohatgi et al. 1999) is a mechanism to promote assembly of
F-actin during macropinocytosis.
As discussed above, dynamin functions in both clathrin- and caveolae-mediated
endocytosis. Recently, dynamin was shown to localize to pinosome-associated
actin comet tails in rat hepatocytes, in NIH 3T3 cells, and in cells overexepressing type I PIP kinase, which suggests that dynamin may also function in
macropinocytosis (Lee & De Camilli 2002, Orth et al. 2002). It is interesting
that overexpression of a dynamin 2 mutant harboring a mutation in the GTPase
domain (K44A mutant) resulted in reduction in the number of actin comet tails
observed in cells overexpressing PIP kinase (Lee & De Camilli 2002, Orth et al.
2002). The pinosomes containing actin comet tails that did form also displayed
a reduction in velocity and in their efficiency of movement. These results suggest that dynamin may be involved in regulating actin polymerization during
macropinocytosis. It is currently not clear, however, whether dynamin functions
during the internalization step. Microinjection of inhibitory antibodies against dynamin or expression of dominant-negative forms of dynamin did not appear to
inhibit macropinocytosis (M.A. McNiven, personal communication). However,
disruption of the gene encoding a dynamin homologue in D. discoideum inhibited
macropinocytosis (Wienke et al. 1999). Furthermore, dynamin localizes to phagocytic cups in macrophages, and the K44A dynamin mutant blocks phagocytosis
(Gold et al. 1999).
It is critical to characterize pinocytic vesicles in more detail. Important questions
include the following: Which proteins associate with pinocytic vesicles? What is
the ultrastructure of the actin comet tails? At which stage in macropinocytosis is
dynamin required? How does dynamin influence actin dynamics? How are these
components (e.g., dynamin, actin) recruited to newly forming pinosomes?
PROTEINS AT THE INTERFACE BETWEEN THE ACTIN
CYTOSKELETON AND THE CLATHRIN-MEDIATED
ENDOCYTOSIS MACHINERY
Links Between Coat Components and Actin
Recently, several proteins that may function at the interface between actin and the
clathrin coat have been identified (Table 2), raising the possibility that the endocytic
machinery is physically connected to the actin cytoskeleton at the cell cortex. Two
of these proteins, Hip1R and a spliced variant of myosin VI, are enriched in CCVs
and can interact both with clathrin coat components and with F-actin (Buss et al.
2001, Engqvist-Goldstein et al. 1999, Engqvist-Goldstein et al. 2001). In addition,
ankyrin, which provides a linkage between spectrin-actin networks and the plasma
membrane, binds to clathrin in vitro (Michaely et al. 1999). Finally, two recent
This interaction has only been identified for Hip1.
This interaction is predicted on the basis of homology.
c
LaTeX2e(2002/01/18)
b
Spliced variant of myosin VI.
a
¥
AR197-CB19-12.sgm
Engqvist-Goldstein et al.
1999, 2001; Metzler et al.
2001; Waelter et al. 2001;
Mishra et al. 2001;
Legendre-Guillemin et al.
2002; Gervais et al. 2002
ENGQVIST-GOLDSTEIN
Wells et al. 1999; Buss et al.
2001, 2002; Morris et al.
2002
Michely et al. 1999
Teo et al. 2001; Yang et al.
2001a,b; Lin et al. 2002
References
AR197-CB19-12.tex
Promotes clathrin assembly in vitro;
Hip1R cross-links F-actin and
clathrin cages in vitro;
overexpression of DPF and clathrin
box of Hip1 inhibits endocytosis
(transferrin uptake)
Enriched in CCVs; colocalizes
with clathrin at PM and TGN;
present in some membrane
ruffles
α-adaptinb Clathrin
HC, Clathrin LC,
F-actin, Hippib
PtdIns(4,5)P2c
Hip1R, Hip1
Moves toward the minus end of
actin filaments; overexpression
of tail domain inhibits endocytosis
(transferrin uptake); involved in
cell migration and membrane
dynamics
Enriched in CCVsa colocalizes
with clathrin at PM and TGN;
present in membrane ruffles
MyosinVI
Effector for Cdc42; tyrosine kinase
activity; overexpression of ACK2
inhibits endocytosis (transferrin
uptake) and causes disassembly
of stress fibers and focal adhesions
Dab2, F-actin,
Glut1CBP, SAP97
Clathrin HC,
Spectrin β-subunit,
membrane receptors
Ankyrin
GFP-ACK1 localizes to punctate
structures that show weak or no
clathrin staining; ACK2 Co-IP
with clathrin
Function/activities
AR
Connects the spectrin-actin
cytoskeleton to the membrane;
microinjection of ankyrin repeats
3 and 4 inhibits endocytosis
(LDL uptake)
Cdc42, Clathrin HC,
SH3PX1
ACK1, ACK2
Subcellular localization
20:13
Partially colocalizes with
internalized LDL-receptor
Binding partners
312
Protein
TABLE 2 Links between the clathrin coat and the actin cytoskeleton
Annu. Rev. Cell Dev. Biol. 2003.19:287-332. Downloaded from arjournals.annualreviews.org
by University of Pittsburgh on 08/17/07. For personal use only.
26 Aug 2003
P1: GCE
DRUBIN
15 Sep 2003
13:11
AR
AR197-CB19-12.tex
AR197-CB19-12.sgm
LaTeX2e(2002/01/18)
ACTIN AND ENDOCYTOSIS
P1: GCE
313
Annu. Rev. Cell Dev. Biol. 2003.19:287-332. Downloaded from arjournals.annualreviews.org
by University of Pittsburgh on 08/17/07. For personal use only.
studies reported that human ACK1 (activated Cdc42-associated tyrosine kinase)
and its bovine counterpart (ACK2) are clathrin-binding proteins (Teo et al. 2001;
Yang et al. 2001a,b).
HIP1R AND HIP1 Hip1R is a component of CCPs and CCVs, and it binds to
clathrin and F-actin (Table 2) (Figure 4a). Hip1R belongs to a conserved family
of proteins that includes Sla2p/End4p, a protein essential for both endocytosis and
actin function in yeast (see Insights From Yeast, above) (Holtzman et al. 1993,
Wesp et al. 1997, Yang et al. 1999). Hip1R is closely related to Hip1 (∼50%
overall amino acid sequence identity), a protein implicated in the pathology of
Huntingtons disease (Kalchman et al. 1997, Wanker et al. 1997). Recently, Hip1
was also shown to be enriched in CCVs and to colocalize with markers for RME
(Metzler et al. 2001, Mishra et al. 2001, Waelter et al. 2001). Hip1R is ubiquitously
expressed in mouse tissues (Engqvist-Goldstein et al. 1999), whereas Hip1 was
originally reported to be predominantly expressed in the central nervous system
(Kalchman et al. 1997), although it now appears to be expressed in other tissues
as well (Chopra et al. 2000).
Members of the Sla2/Hip1 family have a conserved domain structure. At the
N terminus, they contain an ANTH domain related to the ENTH domain found in
several proteins implicated in endocytosis, including epsins and AP180/CALM,
as well as their yeast homologues (Kay et al. 1999, McCann & Craig 1999). The
ENTH/ANTH domains of CALM and epsin bind to PtdIns(4,5)P2 at the plasma
membrane (Ford et al. 2001, Itoh et al. 2001). Because all the CALM residues
implicated in PtdIns(4,5)P2 binding are conserved in Hip1R, it is highly likely that
Hip1R also associates with PtdIns(4,5)P2. A coiled-coil region of approximately
300 amino acids containing a short leucine zipper that in yeast Sla2p has been
implicated in dimerization and endocytosis (Wesp et al. 1997, Yang et al. 1999)
follows the N-terminal region. Hip1R, Hip1, and Sla2p interact with clathrin light
chain via the central coiled-coil region (Engqvist-Goldstein et al. 2001, Henry
et al. 2002, Legendre-Guillemin et al. 2002). Finally, at the C terminus, these
proteins contain two shorter predicted coiled coils and a talin-like F-actin-binding
module (I/LWEQ) (Engqvist-Goldstein et al. 1999; McCann & Craig 1997, 1999;
Yang et al. 1999). Hip1R and Sla2p bind to F-actin via the talin-like domain,
whereas Hip1 was reported not to bind to F-actin (Engqvist-Goldstein et al. 1999,
Legendre-Guillemin et al. 2002, McCann & Craig 1997).
In addition to these features common to all family members, Hip1 uniquely
contains an α-adaptin binding motif (DPF) and a clathrin box motif (LMDMD) in
a region preceding the coiled coil (Metzler et al. 2001, Mishra et al. 2001, Waelter
et al. 2001). The DPF motif in Hip1 interacts with α-adaptin of the AP-2 complex,
and the LMDMD interacts with clathrin heavy chain in vitro (Metzler et al. 2001,
Mishra et al. 2001, Waelter et al. 2001).
What is the function of the Hip1/Sla2 family of proteins? A number of experiments suggest that Hip1R is tightly associated with clathrin during CCV formation. First, real-time analysis of Hip1R-YFP and DsRed-clathrin-LC in live cells
26 Aug 2003
20:13
Annu. Rev. Cell Dev. Biol. 2003.19:287-332. Downloaded from arjournals.annualreviews.org
by University of Pittsburgh on 08/17/07. For personal use only.
314
AR
AR197-CB19-12.tex
ENGQVIST-GOLDSTEIN
¥
AR197-CB19-12.sgm
LaTeX2e(2002/01/18)
P1: GCE
DRUBIN
revealed that these two proteins show almost identical temporal and spatial regulation at the cell cortex (Engqvist-Goldstein et al. 2001). Second, the stoichiometry of
Hip1R in CCVs is high (∼10 clathrin:1 Hip1R) (Legendre-Guillemin et al. 2002;
A.E.Y. Engqvist-Goldstein, unpublished observations). Third, at the ultrastructural level, immunogold labeling of unroofed cells showed that Hip1R localizes
to CCPs (lattices and invaginated CCPs) (Engqvist-Goldstein et al. 2001). Hip1R
frequently localizes to the edges of the forming coated pits, where it appears to
connect to filamentous actin at the cortex. Because Hip1R can simultaneously
bind to both F-actin and polymerized clathrin in vitro, Hip1R may be important
in tethering CCPs at the cell cortex (Figure 3: 1a). This is a possibility consistent
with the observation that overexpression of clathrin hub dissociates Hip1R from
CCPs and disrupts the alignment of CCPs with F-actin (Bennett et al. 2001).
Further support for a function of Hip1R at the interface between actin and the
endocytic machinery comes from a recent study from our lab (A.E.Y. EngqvistGoldstein, C.X. Zhang, S. Carreno, J.E. Heuser & D.G. Drubin, manuscript in
preparation). To better understand the function of Hip1R and to gain insights into
the function of the actin cytoskeleton in endocytosis, we used RNA interference to
reduce the expression of Hip1R in HeLa cells. These cells are viable, but they show
defects in the organization of the actin cytoskeleton and in the spatial distribution
of CCSs. Specifically, F-actin and the machinery involved in actin polymerization
(e.g., the Arp2/3 complex and cortactin) are stably associated with a subset of CCSs
at the cell cortex. Many of these actin structures are tail-like. Dynamin, clathrin,
and AP-2 localize to the interface between the F-actin tails and the plasma membrane. Time-lapse video-microscopy of live cells and analysis of unroofed cells
suggest that the actin structures are tethered to the cell cortex via association with
structures containing clathrin, AP-2, and dynamin. This phenotype is rescued by
expressing a Hip1R construct that cannot be targeted by the siRNA, demonstrating
that the observed effect is due to loss of Hip1R function. Taken together with data
of Merrifield et al. (2002) showing that actin transiently associates with clathrin at
endocytic sites, these observations suggest that Hip1R is needed to form a productive association between F-actin and the endocytic machinery, but it is not required
to recruit actin to endocytic sites. Alternatively, Hip1R may negatively regulate
F-actin assembly at CCSs.
Hip1 and Hip1R may also function to promote assembly of clathrin and/or to
help remodel the clathrin coat during CCV formation. Both Hip1R and Hip1 can
promote assembly of clathrin in vitro (Engqvist-Goldstein et al. 2001, LegendreGuillemin et al. 2002). Furthermore, overexpression of the clathrin-binding motif in Hip1 inhibits transferrin uptake in Cos-7 cells, although this inhibition is
mild compared, for example, to the effects of dominant-negative dynamin mutants
(Metzler et al. 2001).
MYOSIN VI Another likely candidate to provide a link between actin and the
clathrin coat is myosin VI (Table 2, Figure 4b). Myosins are motor proteins
that associate with actin filaments, generate forces for membrane protrusion and
26 Aug 2003
20:13
AR
AR197-CB19-12.tex
AR197-CB19-12.sgm
LaTeX2e(2002/01/18)
Annu. Rev. Cell Dev. Biol. 2003.19:287-332. Downloaded from arjournals.annualreviews.org
by University of Pittsburgh on 08/17/07. For personal use only.
ACTIN AND ENDOCYTOSIS
P1: GCE
315
retraction, and propel membrane vesicles through the cytoplasm (Sellers 2000).
Genetic studies in mouse, D. melanogaster, and C. elegans have identified a role
for myosin VI in a range of diverse biological processes such as hearing, cell migration, embryo development, and spermatogenesis (Buss et al. 2002). Recently,
myosin VI has also been implicated in clathrin-mediated endocytosis. Buss et al.
(2001) showed that a spliced variant of myosin VI is enriched in CCVs and colocalizes with clathrin-coat components. Myosin VI binds directly to disabled 2
(Morris et al. 2002), which may recruit myosin VI to CCPs because disabled 2
binds directly to AP-2 and tyrosine-based sorting motifs present in the cytoplasmic
tails of the low-density lipoprotein receptors (Morris & Cooper 2001). The spliced
variant of myosin VI is primarily expressed in microvilli of polarized cells, and it
contains a large insert in the tail domain, which is important for CCV targeting.
Myosin VI without the insert also colocalized with markers for RME but not to
the same extent as the isoform with the insert. Overexpression of the tail domain
containing the insert inhibited transferrin uptake, consistent with a functional role
for myosin VI in clathrin-mediated endocytosis (Buss et al. 2001). Myosin VI is
the only known myosin to move toward the minus (pointed) end of actin filaments
(Wells et al. 1999). Because actin filaments are generally oriented with their plus
(barbed) ends toward the plasma membrane, myosin VI may provide a force during
CCV formation—either deforming the membrane inward or pulling newly formed
vesicles away from the plasma membrane through the cortical actin network (Buss
et al. 2001). Because this spliced variant of myosin VI is primarily expressed in
microvilli of polarized cells, it is an ideal candidate for transporting endocytic
vesicles down microvilli along actin filaments, toward the main body of the cell.
Dynamin and the Actin Cytoskeleton
Dynamin is crucial for the fission step in clathrin-mediated endocytosis (Hinshaw
2000). Recent data also suggest that dynamin is involved in regulating actin dynamics (Orth & McNiven 2003). Overexpression of the dymanin K44A mutant, which
binds GTP with reduced affinity, not only inhibits endocytosis but also affects the
distribution of actin stress fibers and affects cell shape (Damke et al. 1994). It was
not clear from these studies, however, whether these cytoskeletal defects were secondary defects caused by inhibition of endocytosis. Recently, dynamin was more
directly linked to the actin cytoskeleton. In quiescent cells, dynamin 2 predominantly associates with CCVs (McNiven et al. 2000). However, upon treatment with
growth factors to induce cell migration, dynamin 2 became associated with actinrich membrane structures such as lamellipodia (McNiven et al. 2000). In addition,
as discussed above, dynamin 2 has been implicated in regulation of actin dynamics during macropinocytosis, wherein it localizes to actin comet tails (Lee & De
Camilli 2002, Orth et al. 2002). Furthermore, dynamin 2 colocalizes with actin in
podosomes (Ochoa et al. 2000), adhesion structures that are composed of a narrow
tubular invagination surrounded by a dense network of actin filaments. Ochoa et al.
(2000) showed that overexpression of a GFP–dynamin 2 mutant corresponding
26 Aug 2003
20:13
Annu. Rev. Cell Dev. Biol. 2003.19:287-332. Downloaded from arjournals.annualreviews.org
by University of Pittsburgh on 08/17/07. For personal use only.
316
AR
AR197-CB19-12.tex
ENGQVIST-GOLDSTEIN
¥
AR197-CB19-12.sgm
LaTeX2e(2002/01/18)
P1: GCE
DRUBIN
to the D. melanogaster shibire mutant abolished podosomes, which suggests that
dynamin is required for formation and/or maintenance of podosomes.
How is dynamin physically connected to the actin cytoskeleton? In the past
couple of years, several proteins that could act at the interface between dynamin
and actin have been identified (Table 3). Syndapin, Abp1, cortactin, and intersectin
all contain SH3 domains that can bind to the proline-rich domain of dynamin
(Figure 4). In addition, profilin binds to dynamin (Witke et al. 1998). Each of these
dynamin-binding proteins is in some way connected to the machinery involved in
actin polymerization. Syndapin and intersectin-l bind to N-WASP (Hussain et al.
2001, Qualmann et al. 1999), a potent activator of the Arp2/3 complex, whereas
cortactin and yeast Abp1 can directly activate the Arp2/3 complex (Goode et al.
2001, Uruno et al. 2001, Weaver et al. 2001). This activation activity has not been
demonstrated for mammalian Abp1, although this protein closely colocalizes with
the Arp2/3 complex in growth-factor-treated cells (Kessels et al. 2000). Profilin
binds to G-actin, promotes nucleotide exhange, and permits assembly at barbed
ends of actin filaments (Pantaloni et al. 2001). One might therefore speculate that
some or all of these dynamin-binding proteins might promote actin polymerization
in the vicinity of dynamin during different endocytic events. This may occur, for
example, during the budding of CCVs and caveolae-derived vesicles. A burst of
actin polymerization at the constricted neck may facilitate detachment of vesicles
from the membrane, may move vesicles away from the cortex, or both.
SYNDAPIN Syndapin (synaptic, dynamin-associated protein), also referred to as
PACSIN, has been implicated in both clathrin-mediated endocytosis and actin
cytoskeletal functions (Table 3, Figure 4f ). There are three closely related syndapin isoforms in mammalian cells. Syndapin I is expressed primarily in the brain
(Qualmann et al. 1999), syndapin II is expressed ubiquitiously (Qualmann & Kelly
2000), and syndapin III is expressed primarily in lung and muscle (Modregger et al.
2000). Members of the syndapin protein family share a similar domain structure.
Each family member contains a conserved N-terminal region, followed by two
predicted coiled-coil regions, several arginine-proline-phenylalanine (NPF) sequences, and a highly conserved SH3 domain at the C terminus. Syndapin interacts
via its SH3 domain with several proteins involved in membrane trafficking including dynamin, synaptojanin, and synapsin (Qualmann & Kelly 2000, Qualmann
et al. 1999). Additionally, syndapin interacts with N-WASP and Son-of sevenless
(Qualmann et al. 1999, Wasiak et al. 2001). In support of a role for syndapin in endocytosis, overexpression of its SH3 domain, which presumably sequesters dynamin,
inhibits RME (Qualmann & Kelly 2000). This inhibition occurs at a step after
constriction of the coated pit, as demonstrated using a permeabilized cell assay for
endocytic internalization (Simpson et al. 1999). In support of the possibility that
syndapin has both endocytic and actin functions, overexpression of the full-length
protein induces the formation of filopodia (Qualmann & Kelly 2000). This phenotype may occur through activation of the Arp2/3 complex by N-WASP because this
phenotype was suppressed by coexpressing the region of N-WASP that interacts
Dynamin, F-actin
Arp2/3 complex,
CortBP1,
Dynamin,
F-actin, ZO-1
Cdc42, Dynamin,
Eps15, Epsin,
SOS,
Synaptojanin,
N-WASP
Dynamin, SOS,
Synaptojanin,
Synapsin,
N-WASP
Abp1
Cortactin
Intersectin
Syndapin
Overexpression induces filopodia formation;
SH3 domain overexpression inhibits
endocytosis (transferrin uptake)
Qualmann et al. 1999, Simpson et al. 1999,
Qualmann & Kelly 2000, Kessels &
Qualmann 2002
Yamabhai et al. 1988, Roos & Kelly 1998,
Sengar et al. 1999, Tong et al. 2000,
Hussain et al. 2001, McPherson 2002
GEF for Cdc42b; overexpression induces
filopodia; overexpression of SH3 domains
inhibits endocytosis (transferrin uptake)
Only intersectin-l contains this feature.
b
AR197-CB19-12.sgm
Mammalian Abp1 does not contain the acidic motif found in the yeast protein; however, mammalian Abp1 colocalizes with the Arp2/3 complex in cells.
Localizes to tips of filopodia;
partially colocalizes with dynamin
Wu et al. 1991, Schuuring et al. 1993,
Wu & Parsons 1993, Huang et al. 1998,
Bowden et al. 1999, Kaksonen et al.
2000, Uruno et al. 2001, Cao et al. 2003
Lock et al. 1998; Larbolette et al. 1999;
Kessels et al. 2000, 2001; Goode et al.
2001; Mise-Omata et al. 2003
References
Activator of the Arp2/3 complex; inhibits
debranching of actin filaments; substrate
for Src
Overexpression of SH3 domain or knock
down of Abp1 inhibits endocytosis
(transferrin uptake); subtrate for Src;
yeast Abp1 activates the Arp2/3 complexa
Function/activities
AR197-CB19-12.tex
Present but not enriched in CCVs;
colocalizes with clathrin
Lammelipodia, podosomes;
invadopodia; endosomes; CCPs;
partially colocalizes with dynamin
Lammelipodia, partially colocalizes
with dynamin and clathrin coat
components
Subcellular localization
AR
a
Binding partners
Protein
20:13
TABLE 3 Putative links between dynamin and the actin cytoskeleton
Annu. Rev. Cell Dev. Biol. 2003.19:287-332. Downloaded from arjournals.annualreviews.org
by University of Pittsburgh on 08/17/07. For personal use only.
26 Aug 2003
LaTeX2e(2002/01/18)
ACTIN AND ENDOCYTOSIS
P1: GCE
317
26 Aug 2003
20:13
Annu. Rev. Cell Dev. Biol. 2003.19:287-332. Downloaded from arjournals.annualreviews.org
by University of Pittsburgh on 08/17/07. For personal use only.
318
AR
AR197-CB19-12.tex
ENGQVIST-GOLDSTEIN
¥
AR197-CB19-12.sgm
LaTeX2e(2002/01/18)
P1: GCE
DRUBIN
with the Arp2/3 complex. Furthermore, targeting of syndapin-N-WASP complexes
to the mitochondria causes N-WASP-dependent polymerization of F-actin at these
sites (Kessels & Qualmann 2002). Syndapin is therefore an excellent candidate for
recruiting N-WASP to the endocytic machinery to promote assembly of F-actin
during late stages of CCV formation.
Further evidence for the possibility that syndapin-N-WASP drives actin assembly proximal to dynamin comes from a recent study showing that N-WASP is
directly involved in RME and that this function is coupled to its ability to bind to
syndapin (Kessels & Qualmann 2002). Overexpression of the PRD of N-WASP,
or microinjection of anti-WASP antibodies, inhibited transferrin uptake (Kessels
& Qualmann 2002). These findings are consistent with results of studies in lymphocytes showing that WASP knock-out mice exhibit defects in T cell–receptor
endocytosis in addition to defects in actin organization (Zhang et al. 1999). Syndapin binds to N-WASP via the PRD, and overexpression of syndapin rescues the
endodocytic defects asssociated with overexpression of the PRD domain of NWASP. This finding suggests that the syndapin-N-WASP interaction is important
for endocytosis. It is not clear, however, whether syndapin can interact with dynamin and N-WASP simultaneously. To fulfill such a role, syndapin would have to
use the same SH3 domain to interact with both proteins. This may be accomplished
through higher-order protein interactions and/or oligomerization (Qualmann &
Kelly 2000).
INTERSECTIN Intersectin is a modular scaffolding protein implicated in clathrinmediated endocytosis, actin cytoskeletal organization, and cell signaling (Table 3,
Figure 4e). There are two intersectin genes and several spliced variants of each
(Guipponi et al. 1998, Sengar et al. 1999). We discuss one short form (intersectin-s)
and two long forms (intersectin-l and intersectin 2-l). Intersectin-s is ubiquitously
expressed and contains two N-terminal Eps15-homology domains, followed by a
central coiled-coil domain, then by five SH3 domains (Hussain et al. 1999). The
Eps15-homology domains of intersectin bind to epsin; the coiled-coil domain interacts with Eps15; and a subset of the SH3 domains bind to dynamin, synaptojanin,
and Son-of sevenless (Hussain et al. 1999, Roos & Kelly 1999, Sengar et al. 1999,
Tong et al. 2000, Yamabhai et al. 1998). Most of the intersectin-interacting proteins are involved in clathrin-mediated endocytosis, suggesting that intersectin has
an endocytic function. This possibility is supported by the observation that overexpression of intersectin in Cos-7 cells inhibits transferrin uptake (Sengar et al.
1999), although this is likely caused by the sequestering of its binding partners. In
addition, expression of individual intersectin SH3 domains inhibits endocytosis at
different stages, which suggests that intersectin may function both in early events
leading to the formation of constricted coated pits and in late events leading to
CCV fission (Simpson et al. 1999).
Intersectin-l is predominately expressed in the brain, and it contains, in addition
to the domains found in intersectin-s, a C-terminal extension containing a Dblhomology, pleckstrin-homology, and C2 domain (Hussain et al. 1999). Tandem
26 Aug 2003
20:13
AR
AR197-CB19-12.tex
AR197-CB19-12.sgm
LaTeX2e(2002/01/18)
Annu. Rev. Cell Dev. Biol. 2003.19:287-332. Downloaded from arjournals.annualreviews.org
by University of Pittsburgh on 08/17/07. For personal use only.
ACTIN AND ENDOCYTOSIS
P1: GCE
319
Dbl-homology/pleckstrin-homology domains are found in GEFs that activate
the Rho family of proteins. Recently, Hussain et al. (2001) demonstrated that
intersectin-l functions as a GEF for Cdc42. Overexpression of intersectin-l in culture cells caused induction of filopodia, which is a phenotype associated with
Cdc42 activation. Furthermore, intersectin-l stimulated actin nucleation through
N-WASP and the Arp2/3 complex in a permeabilized cell assay using human
neutrophils. Intersectin-l also binds directly to N-WASP, and this interaction upregulates the GEF activity. Thus intersectin-l can not only recruit machinery required for actin polymerization to the plasma membrane, it also can stimulate actin
assembly via Cdc42 and N-WASP. Because intersectins also interact with epsin,
dynamin, and Eps15 (three main players in endocytosis), intersectin is an excellent
candidate for facilitating polymerization of actin at endocytic sites.
Additional evidence that intersectin may function to integrate Cdc42 signaling with WASP-mediated actin polymerization comes from a study of T cells
(McGavin et al. 2001). Intersectin 2-l interacts with WASP and activates Cdc42.
Furthermore, while overexpression of full-length intersectin 2-l increased ligandinduced endocytosis of the T cell antigen receptor (TCR), an intersectin construct
lacking the Dbl-homology domain efficiently inhibited internalization of the TCR
complex. This complex is internalized via the clathrin-mediated pathway, and internalization is dependent on a functional actin cytoskeleton (McGavin et al. 2001).
Ligand-induced TCR internalization may therefore be an excellent pathway with
which to dissect the specific functions of intersectin and other proteins thought to
act at the interface between actin and the endocytic machinery
CORTACTIN AND ABP1 Cortactin was originally identified as a substrate for Srcrelated kinases (Wu et al. 1991), but it has recently been implicated in membrane
dynamics and actin cytoskeletal functions (Table 3, Figure 4d). Cortactin is widely
expressed in tissues and is often overexpressed in transformed cell lines, including
certain cancers (Schuuring et al. 1993, Wu & Parsons 1993). In invasive breast
cancer cells, cortactin localizes to invadopodia, which are invasive structures, possibly related to podosomes (Schuuring et al. 1993). Invadopodia are rich in F-actin,
and they degrade the extracellular matrix (Bowden et al. 1999). Cortactin also localizes to both the leading edge of the cell in lamellipodia (Wu et al. 1991) and the
endocytic compartments. Kaksonen et al. (2000) showed that cortactin is present
on endosomal vesicles, where it colocalizes with the Arp2/3 complex in actin
comet tails. Furthermore, Cao et al. (2003) showed that cortactin is a component
of CCPs. Microinjection of anticortactin antibodies inhibited transferrin uptake,
which suggests that cortactin participates in RME (Cao et al. 2003).
Cortactin is a multidomain protein consisting of an acidic motif, followed by
6.5 tandemly repeated 37–amino acid segments, an α-helical region, a prolinerich segment, and an SH3 domain located at the C terminus (Wu et al. 1991). It
was recently shown that cortactin directly activates the Arp2/3 complex and inhibits
debranching of the resulting filament networks in vitro (Uruno et al. 2001, Weaver
et al. 2001). The acidic motif of cortactin is sufficient for it to bind to the Arp2/3
26 Aug 2003
20:13
Annu. Rev. Cell Dev. Biol. 2003.19:287-332. Downloaded from arjournals.annualreviews.org
by University of Pittsburgh on 08/17/07. For personal use only.
320
AR
AR197-CB19-12.tex
ENGQVIST-GOLDSTEIN
¥
AR197-CB19-12.sgm
LaTeX2e(2002/01/18)
P1: GCE
DRUBIN
complex; however, Arp2/3 activation also requires its F-actin binding domain (the
tandem repeats) (Uruno et al. 2001, Weaver et al. 2001). Because cortactin localizes
to actin-rich membrane structures such as podosomes, invadopodia, rocketing endosomes, and lamellipodia, one might speculate that cortactin functions to promote
F-actin assembly on membranes. Alternatively, cortactin may affect actin filament
turnover because it inhibits filament debranching (Weaver et al. 2001). In support
of these various possibilities, Schafer et al. (2002) recently showed that cortactin
caused association of F-actin bundles with PtdIns(4,5)P2-containing liposomes in
vitro. Dynamin, which binds directly to cortactin (McNiven et al. 2000), was important for this activity, and this activity was dependent on the ability of cortactin
to bind dynamin via cortactin’s SH3 domain (Schafer et al. 2002). These results
suggest that dynamin may recruit cortactin to PtdIns(4,5)P2-rich clusters in the
plasma membrane (e.g., endocytic sites, podosomes) and may regulate cortactin’s
ability to activate the Arp2/3 complex and/or its ability to inhibit debranching of
filament networks.
Abp1 shares many features with cortactin (Table 3, Figure 4c): It is a Src
substrate (Larbolette et al. 1999, Lock et al. 1998), it binds to F-actin, it colocalizes
with the Arp2/3 complex in lamellipodia upon growth factor stimulation, and it
binds to dynamin through an SH3 domain (Kessels et al. 2000, 2001). Recently,
Goode et al. (2001) found that yeast Abp1 binds and activates the Arp2/3 complex.
The mechanism of activation appears similar to that of cortactin; both proteins
appear to increase the affinity of the Arp2/3 complex for actin filaments (Olazabal
& Machesky 2001). Although mammalian Abp1 lacks the acidic motif involved
in Arp2/3 activation, it may form a complex with other proteins that bind to the
Arp2/3 complex. Upon growth-factor treatment, Abp1 colocalizes with the Arp2/3
complex at lamellipodia (Kessels et al. 2000) and with dynamin in puncta that also
contain clathrin coat components such as AP-2 and Hip1R, which suggests that the
latter structures are sites of endocytosis (Kessels et al. 2001). In support of a role in
endocytosis, overexpression of the Abp1 SH3 domain inhibits transferrin uptake
(Kessels et al. 2001). The endocytosis block was rescued by coexpressing either
dynamin or an Abp1 construct containing the actin-binding modules together with
the SH3 domain. Thus Abp1 may support endocytosis by combining cytoskeletal
functions with its ability to interact with dynamin. Further support for a role for
Abp1 in clathrin-mediated endocytosis comes from a recent study showing that
knock down of Abp1 expression using RNA interference inhibits transferrin uptake
(Mise-Omata et al. 2003).
CONCLUDING REMARKS
Recent work has provided evidence that the actin cytoskeleton directly participates
in endocytic internalization. Microscopy studies in mammalian cells show that Factin is recruited to endocytic sites during internalization in caveolae-mediated
endocytosis, clathrin-mediated endocytosis, and macropinocytosis. In addition, in
26 Aug 2003
20:13
AR
AR197-CB19-12.tex
AR197-CB19-12.sgm
LaTeX2e(2002/01/18)
Annu. Rev. Cell Dev. Biol. 2003.19:287-332. Downloaded from arjournals.annualreviews.org
by University of Pittsburgh on 08/17/07. For personal use only.
ACTIN AND ENDOCYTOSIS
P1: GCE
321
the past couple of years a number of proteins that could function at the interface
between actin and the endocytic machinery have been identified. Studies in both
yeast and mammalian cells have also begun to unravel some of the activities of
these proteins. The stage is now set for addressing specific questions regarding
the role of actin in endocytosis. At which step(s) during internalization does actin
function? How is the assembly of F-actin at endocytic sites regulated? To address
these questions, the relationship of endocytic sites to the actin assembly machinery
must be defined. Time-lapse video microscopy of actin and different proteins
critical for internalization should be fruitful. Functional studies on the putative
linker proteins will be needed to determine that the actin-associated activities of
these proteins (e.g., actin binding, activation of the Arp2/3 complex) are important
for their endocytic functions. RNA interference to reduce the expression of these
proteins, in combination with total internal reflection microscopy to visualize the
recruitment of F-actin to endocytic sites, should be informative. Finally, it will be
important to develop in vitro assays for endocytic internalization that faithfully
recapitulate the requirement for F-actin.
ACKNOWLEDGMENTS
We thank the members of the Drubin laboratory for helpful discussions. We also
thank Adam Martin, Walter Gall, Marko Kaksonen, Mariko Sekiya-Kawasaki,
Sebastien Carreno, and Claire Zhang for helpful comments on the manuscript.
We are grateful to Beverly Wendland, Peter McPherson, and Mark McNiven for
sharing unpublished data. We regret that space limitations prevented us from citing
all of the literature relevant to this review.
The Annual Review of Cell and Developmental Biology is online at
http://cellbio.annualreviews.org
LITERATURE CITED
Adams AE, Pringle JR. 1984. Relationship of
actin and tubulin distribution to bud growth
in wild-type and morphogenetic-mutant Saccharomyces cerevisiae. J. Cell Biol. 98:934–
45
Aguilar RC, Watson HA, Wendland B. 2003.
The yeast epsin Ent1 is recruited to membranes through multiple independent interactions. J. Biol. Chem. 278:10737–43
Apodaca G. 2001. Endocytic traffic in polarized
epithelial cells: role of the actin and microtubule cytoskeleton. Traffic 2:149–59
Ayscough KR. 2000. Endocytosis and the development of cell polarity in yeast require
a dynamic F-actin cytoskeleton. Curr. Biol.
10:1587–90
Ayscough KR, Eby JJ, Lila T, Dewar H,
Kozminski KG, Drubin DG. 1999. Sla1p is a
functionally modular component of the yeast
cortical actin cytoskeleton required for correct localization of both Rho1p-GTPase and
Sla2p, a protein with talin homology. Mol.
Biol. Cell 10:1061–75
Ayscough KR, Stryker J, Pokala N, Sanders M,
Crews P, Drubin DG. 1997. High rates of
actin filament turnover in budding yeast and
roles for actin in establishment and maintenance of cell polarity revealed using the
26 Aug 2003
20:13
Annu. Rev. Cell Dev. Biol. 2003.19:287-332. Downloaded from arjournals.annualreviews.org
by University of Pittsburgh on 08/17/07. For personal use only.
322
AR
AR197-CB19-12.tex
ENGQVIST-GOLDSTEIN
¥
AR197-CB19-12.sgm
LaTeX2e(2002/01/18)
P1: GCE
DRUBIN
actin inhibitor latrunculin-A. J. Cell Biol.
137:399–416
Baggett JJ, Wendland B. 2001. Clathrin function in yeast endocytosis. Traffic 2:297–302
Bauer F, Urdaci M, Aigle M, Crouzet M.
1993. Alteration of a yeast SH3 protein leads
to conditional viability with defects in cytoskeletal and budding patterns. Mol. Cell.
Biol. 13:5070–84
Belmont LD, Drubin DG. 1998. The yeast
V159N actin mutant reveals roles for actin
dynamics in vivo. J. Cell Biol. 142:1289–
99
Benmerah A, Gagnon J, Begue B, Megarbane
B, Dautry-Varsat A, Cerf-Bensussan N.
1995. The tyrosine kinase substrate eps15
is constitutively associated with the plasma
membrane adaptor AP-2. J. Cell Biol. 131:
1831–38
Benmerah A, Lamaze C, Begue B, Schmid SL,
Dautry-Varsat A, Cerf-Bensussan N. 1998.
AP-2/Eps15 interaction is required for receptor-mediated endocytosis. J. Cell Biol. 140:
1055–62
Bennett EM, Chen CY, Engqvist-Goldstein ÅE,
Drubin DG, Brodsky FM. 2001. Clathrin hub
expression dissociates the actin-binding protein Hip1R from coated pits and disrupts their
alignment with the actin cytoskeleton. Traffic
2:851–58
Bowden ET, Barth M, Thomas D, Glazer RI,
Mueller SC. 1999. An invasion-related complex of cortactin, paxillin and PKCmu associates with invadopodia at sites of extracellular matrix degradation. Oncogene 18:4440–
49
Brodin L, Low P, Shupliakov O. 2000. Sequential steps in clathrin-mediated synaptic
vesicle endocytosis. Curr. Opin. Neurobiol.
10:312–20
Brodsky FM, Chen CY, Knuehl C, Towler
MC, Wakeham DE. 2001. Biological basket
weaving: formation and function of clathrincoated vesicles. Annu. Rev. Cell Dev. Biol.
17:517–68
Buss F, Arden SD, Lindsay M, Luzio JP, Kendrick-Jones J. 2001. Myosin VI isoform localized to clathrin-coated vesicles with a role
in clathrin-mediated endocytosis. EMBO J.
20:3676–84
Buss F, Luzio JP, Kendrick-Jones J. 2002.
Myosin VI, an actin motor for membrane
traffic and cell migration. Traffic 3:851–58
Cao H, Orth JD, Chen J, Weller SG, Heuser JE,
McNiven MA. 2003. Cortactin is a component of clathrin-coated pits and participates
in receptor-mediated endocytosis. Mol. Cell
Biol. 23:2162–70
Carbone R, Fre S, Iannolo G, Belleudi F,
Mancini P, et al. 1997. eps15 and eps15R are
essential components of the endocytic pathway. Cancer Res. 57:5498–504
Cardelli J. 2001. Phagocytosis and macropinocytosis in Dictyostelium: phosphoinositidebased processes, biochemically distinct.
Traffic 2:311–20
Chen H, Fre S, Slepnev VI, Capua MR, Takei K,
et al. 1998. Epsin is an EH-domain-binding
protein implicated in clathrin-mediated endocytosis. Nature 394:793–97
Chopra VS, Metzler M, Rasper DM, EngqvistGoldstein AE, Singaraja R, et al. 2000.
HIP12 is a non-proapoptotic member of a
gene family including HIP1, an interacting protein with huntingtin. Mamm. Genome
11:1006–15
Cope MJ, Yang S, Shang C, Drubin DG. 1999.
Novel protein kinases Ark1p and Prk1p associate with and regulate the cortical actin
cytoskeleton in budding yeast. J. Cell Biol.
144:1203–18
Cremona O, De Camilli P. 2001. Phosphoinositides in membrane traffic at the synapse. J.
Cell Sci. 114:1041–52
Damke H, Baba T, Warnock DE, Schmid SL.
1994. Induction of mutant dynamin specifically blocks endocytic coated vesicle formation. J. Cell Biol. 127:915–34
Desrivieres S, Cooke FT, Morales-Johansson H,
Parker PJ, Hall MN. 2002. Calmodulin controls organization of the actin cytoskeleton
via regulation of phosphatidylinositol (4,5)bisphosphate synthesis in Saccharomyces
cerevisiae. Biochem. J. 366:945–51
Dharmawardhane S, Schurmann A, Sells MA,
Chernoff J, Schmid SL, Bokoch GM. 2000.
26 Aug 2003
20:13
AR
AR197-CB19-12.tex
AR197-CB19-12.sgm
LaTeX2e(2002/01/18)
Annu. Rev. Cell Dev. Biol. 2003.19:287-332. Downloaded from arjournals.annualreviews.org
by University of Pittsburgh on 08/17/07. For personal use only.
ACTIN AND ENDOCYTOSIS
Regulation of macropinocytosis by p21–
activated kinase-1. Mol. Biol. Cell 11:3341–
52
D’Hondt K, Heese-Peck A, Riezman H. 2000.
Protein and lipid requirements for endocytosis. Annu. Rev. Genet. 34:255–95
Donnelly SF, Pocklington MJ, Pallotta D, Orr
E. 1993. A proline-rich protein, verprolin, involved in cytoskeletal organization and cellular growth in the yeast Saccharomyces cerevisiae. Mol. Microbiol. 10:585–96
Doyle T, Botstein D. 1996. Movement of yeast
cortical actin cytoskeleton visualized in vivo.
Proc. Natl. Acad. Sci. USA 93:3886–91
Dramsi S, Cossart P. 1998. Intracellular
pathogens and the actin cytoskeleton. Annu.
Rev. Cell Dev. Biol. 14:137–66
Drees BL, Sundin B, Brazeau E, Caviston JP,
Chen GC, et al. 2001. A protein interaction
map for cell polarity development. J. Cell
Biol. 154:549–71
Dulic V, Egerton M, Elguindi I, Raths S, Singer
B, Riezman H. 1991. Yeast endocytosis assays. Methods Enzymol. 194:697–710
Dunaevsky A, Connor EA. 2000. F-actin is concentrated in nonrelease domains at frog neuromuscular junctions. J. Neurosci. 20:6007–
12
Duncan MC, Cope MJ, Goode BL, Wendland
B, Drubin DG. 2001. Yeast Eps15-like endocytic protein, Pan1p, activates the Arp2/3
complex. Nat. Cell Biol. 3:687–90
Dunn R, Hicke L. 2001. Multiple roles for
Rsp5p-dependent ubiquitination at the internalization step of endocytosis. J. Biol. Chem.
276:25974–81
Engqvist-Goldstein ÅE, Kessels MM, Chopra
VS, Hayden MR, Drubin DG. 1999. An
actin-binding protein of the Sla2/Huntingtin
interacting protein 1 family is a novel component of clathrin-coated pits and vesicles. J.
Cell Biol. 147:1503–18
Engqvist-Goldstein ÅE, Warren RA, Kessels
MM, Keen JH, Heuser J, Drubin DG. 2001.
The actin-binding protein Hip1R associates
with clathrin during early stages of endocytosis and promotes clathrin assembly in vitro.
J. Cell Biol. 154:1209–23
P1: GCE
323
Evangelista M, Klebl BM, Tong AH, Webb BA,
Leeuw T, et al. 2000. A role for myosin-I
in actin assembly through interactions with
Vrp1p, Bee1p, and the Arp2/3 complex. J.
Cell Biol. 148:353–62
Feng Y, Davis NG. 2000. Akr1p and the type I
casein kinases act prior to the ubiquitination
step of yeast endocytosis: Akr1p is required
for kinase localization to the plasma membrane. Mol. Cell. Biol. 20:5350–59
Ford MG, Mills IG, Peter BJ, Vallis Y, Praefcke
GJ, et al. 2002. Curvature of clathrin-coated
pits driven by epsin. Nature 419:361–66
Ford MG, Pearse BM, Higgins MK, Vallis Y,
Owen DJ, et al. 2001. Simultaneous binding
of PtdIns(4,5)P2 and clathrin by AP180 in the
nucleation of clathrin lattices on membranes.
Science 291:1051–55
Freeman NL, Chen Z, Horenstein J, Weber A,
Field J. 1995. An actin monomer binding activity localizes to the carboxyl-terminal half
of the Saccharomyces cerevisiae cyclaseassociated protein. J. Biol. Chem. 270:5680–
85
Frischknecht F, Moreau V, Rottger S, Gonfloni S, Reckmann I, et al. 1999. Actin-based
motility of vaccinia virus mimics receptor
tyrosine kinase signalling. Nature 401:926–
29
Fujimoto LM, Roth R, Heuser JE, Schmid SL.
2000. Actin assembly plays a variable, but
not obligatory role in receptor-mediated endocytosis in mammalian cells. Traffic 1:161–
71
Gad H, Ringstad N, Low P, Kjaerulff O,
Gustafsson J, et al. 2000. Fission and uncoating of synaptic clathrin-coated vesicles
are perturbed by disruption of interactions
with the SH3 domain of endophilin. Neuron
27:301–12
Gaidarov I, Keen JH. 1999. PhosphoinositideAP-2 interactions required for targeting to
plasma membrane clathrin-coated pits. J.
Cell Biol. 146:755–64
Gaidarov I, Santini F, Warren RA, Keen JH.
1999. Spatial control of coated-pit dynamics
in living cells. Nat. Cell Biol. 1:1–7
Galán JE. 2001. Salmonella interactions with
26 Aug 2003
20:13
Annu. Rev. Cell Dev. Biol. 2003.19:287-332. Downloaded from arjournals.annualreviews.org
by University of Pittsburgh on 08/17/07. For personal use only.
324
AR
AR197-CB19-12.tex
ENGQVIST-GOLDSTEIN
¥
AR197-CB19-12.sgm
LaTeX2e(2002/01/18)
P1: GCE
DRUBIN
host cells: type III secretion at work. Annu.
Rev. Cell Dev. Biol. 17:53–86
Garrett WS, Chen LM, Kroschewski R, Ebersold M, Turley S, et al. 2000. Developmental
control of endocytosis in dendritic cells by
Cdc42. Cell 102:325–34
Geli MI, Lombardi R, Schmelzl B, Riezman
H. 2000. An intact SH3 domain is required
for myosin I-induced actin polymerization.
EMBO J. 19:4281–91
Geli MI, Riezman H. 1996. Role of type I
myosins in receptor-mediated endocytosis in
yeast. Science 272:533–35
Gervais FG, Singaraja R, Xanthoudakis S,
Gutekunst CA, Leavitt BR, et al. 2002. Recruitment and activation of caspase-8 by the
Huntingtin-interacting proteins Hip-1 and a
novel partner Hippi. Nat. Cell Biol. 4:95–105
Gold ES, Underhill DM, Morrissette NS, Guo J,
McNiven MA, Aderem A. 1999. Dynamin 2
is required for phagocytosis in macrophages.
J. Exp. Med. 190:1849–56
Goode BL, Rodal AA. 2001. Modular complexes that regulate actin assembly in budding yeast. Curr. Opin. Microbiol. 4:703–12
Goode BL, Rodal AA, Barnes G, Drubin DG.
2001. Activation of the Arp2/3 complex by
the actin filament binding protein Abp1p. J.
Cell Biol. 153:627–34
Goodman OB Jr, Krupnick JG, Gurevich VV,
Benovic JL, Keen JH. 1997. Arrestin/clathrin
interaction. Localization of the arrestin binding locus to the clathrin terminal domain. J.
Biol. Chem. 272:15017–22
Goodson HV, Anderson BL, Warrick HM, Pon
LA, Spudich JA. 1996. Synthetic lethality
screen identifies a novel yeast myosin I gene
(MYO5): Myosin I proteins are required for
polarization of the actin cytoskeleton. J. Cell
Biol. 133:1277–91
Gottlieb TA, Ivanov IE, Adesnik M, Sabatini
DD. 1993. Actin microfilaments play a critical role in endocytosis at the apical but not
the basolateral surface of polarized epithelial
cells. J. Cell Biol. 120:695–710
Guipponi M, Scott HS, Chen H, Schebesta A,
Rossier C, Antonarakis SE. 1998. Two isoforms of a human intersectin (ITSN) protein
are produced by brain-specific alternative
splicing in a stop codon. Genomics 53:369–
76
Hacker U, Albrecht R, Maniak M. 1997. Fluidphase uptake by macropinocytosis in Dictyostelium. J. Cell Sci. 110(Pt 2):105–12
Haffner C, Takei K, Chen H, Ringstad N, Hudson A, et al. 1997. Synaptojanin 1: localization on coated endocytic intermediates in
nerve terminals and interaction of its 170 kDa
isoform with Eps15. FEBS Lett. 419:175–80
Heese-Peck A, Pichler H, Zanolari B, Watanabe R, Daum G, Riezman H. 2002. Multiple
functions of sterols in yeast endocytosis. Mol.
Biol. Cell 13:2664–80
Henley JR, Krueger EW, Oswald BJ, McNiven
MA. 1998. Dynamin-mediated internalization of caveolae. J. Cell Biol. 141:85–99
Henry KR, D’Hondt K, Chang J, Newpher T,
Huang K, et al. 2002. Scd5p and clathrin
function are important for cortical actin organization, endocytosis, and localization of
Sla2p in yeast. Mol. Biol. Cell 13:2607–25
Heuser JE, Morisaki JH. 1992. Time-lapse
video microscopy of endosomal “rocketing”
of La/Zn-treated cells. Mol. Biol. Cell 3:172
(Abstr.)
Hicke L. 2001. Protein regulation by monoubiquitin. Nat. Rev. Mol. Cell Biol. 2:195–201
Hicke L, Riezman H. 1996. Ubiquitination of a
yeast plasma membrane receptor signals its
ligand-stimulated endocytosis. Cell 84:277–
87
Hicke L, Zanolari B, Riezman H. 1998. Cytoplasmic tail phosphorylation of the α-factor
receptor is required for its ubiquitination and
internalization. J. Cell Biol. 141:349–58
Higgins MK, McMahon HT. 2002. Snap-shots
of clathrin-mediated endocytosis. Trends
Biochem. Sci. 27:257–63
Hinshaw JE. 2000. Dynamin and its role in
membrane fission. Annu. Rev. Cell Dev. Biol.
16:483–519
Holtzman DA, Yang S, Drubin DG. 1993.
Synthetic-lethal interactions identify two
novel genes, SLA1 and SLA2, that control
membrane cytoskeleton assembly in Saccharomyces cerevisiae. J. Cell Biol. 122:635–44
26 Aug 2003
20:13
AR
AR197-CB19-12.tex
AR197-CB19-12.sgm
LaTeX2e(2002/01/18)
Annu. Rev. Cell Dev. Biol. 2003.19:287-332. Downloaded from arjournals.annualreviews.org
by University of Pittsburgh on 08/17/07. For personal use only.
ACTIN AND ENDOCYTOSIS
Howard JP, Hutton JL, Olson JM, Payne GS.
2002. Sla1p serves as the targeting signal
recognition factor for NPFX(1,2)D-mediated
endocytosis. J. Cell Biol. 157:315–26
Huang C, Liu J, Haudenchild CC, Zhan X.
1998. The role of tyrosine phosphorylation
of cortactin in the locomotion of endothelial
cells. J. Biol. Chem. 273:25770–6
Huang KM, D’Hondt K, Riezman H, Lemmon
SK. 1999. Clathrin functions in the absence
of heterotetrameric adaptors and AP180related proteins in yeast. EMBO J. 18:3897–
908
Hussain NK, Jenna S, Glogauer M, Quinn
CC, Wasiak S, et al. 2001. Endocytic protein intersectin-l regulates actin assembly via
Cdc42 and N-WASP. Nat. Cell Biol. 3:927–
32
Hussain NK, Yamabhai M, Ramjaun AR, Guy
AM, Baranes D, et al. 1999. Splice variants
of intersectin are components of the endocytic machinery in neurons and nonneuronal
cells. J. Biol. Chem. 274:15671–77
Idrissi FZ, Wolf BL, Geli MI. 2002. Cofilin, but
not profilin, is required for Myosin-I-induced
actin polymerization and the endocytic uptake in yeast. Mol. Biol. Cell 13:4074–
87
Inaba K, Inaba M, Naito M, Steinman RM.
1993. Dendritic cell progenitors phagocytose
particulates, including bacillus CalmetteGuerin organisms, and sensitize mice to mycobacterial antigens in vivo. J. Exp. Med.
178:479–88
Insall R, Muller-Taubenberger A, Machesky L,
Kohler J, Simmeth E, et al. 2001. Dynamics
of the Dictyostelium Arp2/3 complex in endocytosis, cytokinesis, and chemotaxis. Cell
Motil. Cytoskelet. 50:115–28
Itoh T, Koshiba S, Kigawa T, Kikuchi A,
Yokoyama S, Takenawa T. 2001. Role of the
ENTH domain in phosphatidylinositol-4,5bisphosphate binding and endocytosis. Science 291:1047–51
Jarousse N, Kelly RB. 2001. Endocytotic mechanisms in synapses. Curr. Opin. Cell Biol.
13:461–69
Jenness DD, Spatrick P. 1986. Down regula-
P1: GCE
325
tion of the α-factor pheromone receptor in
S. cerevisiae. Cell 46:345–53
Kaksonen M, Peng HB, Rauvala H. 2000. Association of cortactin with dynamic actin in
lamellipodia and on endosomal vesicles. J.
Cell Sci. 113(Pt 24):4421–26
Kalchman MA, Koide HB, McCutcheon K,
Graham RK, Nichol K, et al. 1997. HIP1, a
human homologue of S. cerevisiae Sla2p, interacts with membrane-associated huntingtin
in the brain. Nat. Genet. 16:44–53
Kaminska J, Gajewska B, Hopper AK, Zoladek
T. 2002. Rsp5p, a new link between the actin
cytoskeleton and endocytosis in the yeast
Saccharomyces cerevisiae. Mol. Cell. Biol.
22:6946–48
Kanzaki M, Watson RT, Khan AH, Pessin JE.
2001. Insulin stimulates actin comet tails
on intracellular GLUT4-containing compartments in differentiated 3T3L1 adipocytes. J.
Biol. Chem. 276:49331–36
Kay BK, Yamabhai M, Wendland B, Emr SD.
1999. Identification of a novel domain shared
by putative components of the endocytic and
cytoskeletal machinery. Protein Sci. 8:435–
38
Kessels MM, Engqvist-Goldstein ÅE, Drubin
DG. 2000. Association of mouse actinbinding protein 1 (mAbp1/SH3P7), an Src kinase target, with dynamic regions of the cortical actin cytoskeleton in response to Rac1
activation. Mol. Biol. Cell 11:393–412
Kessels MM, Engqvist-Goldstein ÅE, Drubin
DG, Qualmann B. 2001. Mammalian Abp1,
a signal-responsive F-actin-binding protein,
links the actin cytoskeleton to endocytosis via the GTPase dynamin. J. Cell Biol.
153:351–66
Kessels MM, Qualmann B. 2002. Syndapins integrate N-WASP in receptor-mediated endocytosis. EMBO J. 21:6083–94
Koenig JH, Ikeda K. 1989. Disappearance and
reformation of synaptic vesicle membrane
upon transmitter release observed under reversible blockage of membrane retrieval. J.
Neurosci. 9:3844–60
Kosaka T, Ikeda K. 1983. Reversible blockage
of membrane retrieval and endocytosis in the
26 Aug 2003
20:13
Annu. Rev. Cell Dev. Biol. 2003.19:287-332. Downloaded from arjournals.annualreviews.org
by University of Pittsburgh on 08/17/07. For personal use only.
326
AR
AR197-CB19-12.tex
ENGQVIST-GOLDSTEIN
¥
AR197-CB19-12.sgm
LaTeX2e(2002/01/18)
P1: GCE
DRUBIN
garland cell of the temperature-sensitive mutant of Drosophila melanogaster, shibirets1.
J. Cell Biol. 97:499–507
Kubler E, Riezman H. 1993. Actin and fimbrin
are required for the internalization step of endocytosis in yeast. EMBO J. 12:2855–62
Lamaze C, Chuang TH, Terlecky LJ, Bokoch
GM, Schmid SL. 1996. Regulation of
receptor-mediated endocytosis by Rho and
Rac. Nature 382:177–79
Lamaze C, Fujimoto LM, Yin HL, Schmid
SL. 1997. The actin cytoskeleton is required
for receptor-mediated endocytosis in mammalian cells. J. Biol. Chem. 272:20332–35
Lappalainen P, Drubin DG. 1997. Cofilin promotes rapid actin filament turnover in vivo.
Nature 388:78–82
Larbolette O, Wollscheid B, Schweikert J,
Nielsen PJ, Wienands J. 1999. SH3P7 is a
cytoskeleton adapter protein and is coupled
to signal transduction from lymphocyte antigen receptors. Mol. Cell. Biol. 19:1539–46
Lechler T, Shevchenko A, Li R. 2000. Direct involvement of yeast type I myosins in Cdc42dependent actin polymerization. J. Cell Biol.
148:363–73
Lee E, De Camilli P. 2002. Dynamin at actin
tails. Proc. Natl. Acad. Sci. USA 99:161–66
Lee WL, Bezanilla M, Pollard TD. 2000. Fission yeast myosin-I, Myo1p, stimulates actin
assembly by Arp2/3 complex and shares
functions with WASp. J. Cell Biol. 151:789–
800
Legendre-Guillemin V, Metzler M, Charbonneau M, Gan L, Chopra V, et al. 2002. HIP1
and HIP12 display differential binding to Factin, AP2, and clathrin. Identification of a
novel interaction with clathrin light chain. J.
Biol. Chem. 277:19897–904
Lemmon SK. 2001. Clathrin uncoating: auxilin
comes to life. Curr. Biol. 11:R49–52
Li H, Page N, Bussey H. 2002. Actin patch
assembly proteins Las17p and Sla1p restrict
cell wall growth to daughter cells and interact
with cis-Golgi protein Kre6p. Yeast 19:1097–
112
Li R. 1997. Bee1, a yeast protein with homology to Wiscott-Aldrich syndrome protein, is
critical for the assembly of cortical actin cytoskeleton. J. Cell Biol. 136:649–58
Li R, Zheng Y, Drubin DG. 1995. Regulation
of cortical actin cytoskeleton assembly during polarized cell growth in budding yeast. J.
Cell Biol. 128:599–615
Lin Q, Lo CG, Cerione RA, Yang W. 2002.
The Cdc42 target ACK2 interacts with sorting nexin 9 (SH3PX1) to regulate epidermal
growth factor receptor degradation. J. Biol.
Chem. 277:10134–38
Lock P, Abram CL, Gibson T, Courtneidge SA.
1998. A new method for isolating tyrosine kinase substrates used to identify fish, an SH3
and PX domain-containing protein, and Src
substrate. EMBO J. 17:4346–57
Lorenzi R, Brickell PM, Katz DR, Kinnon
C, Thrasher AJ. 2000. Wiskott-Aldrich syndrome protein is necessary for efficient IgGmediated phagocytosis. Blood 95:2943–46
Madania A, Dumoulin P, Grava S, Kitamoto H,
Scharer-Brodbeck C, et al. 1999. The Saccharomyces cerevisiae homologue of human
Wiskott-Aldrich syndrome protein Las17p
interacts with the Arp2/3 complex. Mol. Biol.
Cell 10:3521–38
Maniak M. 2002. Conserved features of endocytosis in Dictyostelium. Int. Rev. Cytol.
221:257–87
May RC, Machesky LM. 2001. Phagocytosis
and the actin cytoskeleton. J. Cell Sci. 114:
1061–77
McCann RO, Craig SW. 1997. The I/LWEQ
module: a conserved sequence that signifies
F-actin binding in functionally diverse proteins from yeast to mammals. Proc. Natl.
Acad. Sci. USA 94:5679–84
McCann RO, Craig SW. 1999. Functional genomic analysis reveals the utility of the
I/LWEQ module as a predictor of protein:
actin interaction. Biochem. Biophys. Res.
Commun. 266:135–40
McGavin MK, Badour K, Hardy LA, Kubiseski TJ, Zhang J, Siminovitch KA. 2001. The
intersectin 2 adaptor links Wiskott Aldrich
Syndrome protein (WASp)-mediated actin
polymerization to T cell antigen receptor endocytosis. J. Exp. Med. 194:1777–87
26 Aug 2003
20:13
AR
AR197-CB19-12.tex
AR197-CB19-12.sgm
LaTeX2e(2002/01/18)
Annu. Rev. Cell Dev. Biol. 2003.19:287-332. Downloaded from arjournals.annualreviews.org
by University of Pittsburgh on 08/17/07. For personal use only.
ACTIN AND ENDOCYTOSIS
McMahon HT. 1999. Endocytosis: an assembly protein for clathrin cages. Curr. Biol.
9:R332–35
McNiven MA, Kim L, Krueger EW, Orth JD,
Cao H, Wong TW. 2000. Regulated interactions between dynamin and the actin-binding
protein cortactin modulate cell shape. J. Cell
Biol. 151:187–98
McPherson PS. 2002. The endocytic machinery
at an interface with the actin cytoskeleton: a
dynamic, hip intersection. Trends Cell Biol.
12:312–15
Merrifield CJ, Feldman ME, Wan L, Almers
W. 2002. Imaging actin and dynamin recruitment during invagination of single clathrincoated pits. Nat. Cell Biol. 4:691–98
Merrifield CJ, Moss SE, Ballestrem C, Imhof
BA, Giese G, et al. 1999. Endocytic vesicles
move at the tips of actin tails in cultured mast
cells. Nat. Cell Biol. 1:72–74
Metzler M, Legendre-Guillemin V, Gan L,
Chopra V, Kwok A, et al. 2001. HIP1
functions in clathrin-mediated endocytosis
through binding to clathrin and adaptor protein 2. J. Biol. Chem. 276:39271–76
Michaely P, Kamal A, Anderson RG, Bennett
V. 1999. A requirement for ankyrin binding
to clathrin during coated pit budding. J. Biol.
Chem. 274:35908–13
Mise-Omata S, Montagne B, Deckert M, Wienands J, Acuto O. 2003. Mammalian actin
binding protein 1 is essential for endocytosis but not lamellipodia formation: functional
analysis by RNA interference. Biochem. Biophys. Res. Commun. 301:704–10
Mishra SK, Agostinelli NR, Brett TJ, Mizukami
I, Ross TS, Traub LM. 2001. Clathrin- and
AP-2–binding sites in HIP1 uncover a general assembly role for endocytic accessory
proteins. J. Biol. Chem. 276:46230–36
Modregger J, Ritter B, Witter B, Paulsson M,
Plomann M. 2000. All three PACSIN isoforms bind to endocytic proteins and inhibit
endocytosis. J. Cell Sci. 113(Pt 24):4511–
21
Moreau V, Galán JM, Devilliers G, Haguenauer-Tsapis R, Winsor B. 1997. The yeast
actin-related protein Arp2p is required for the
P1: GCE
327
internalization step of endocytosis. Mol. Biol.
Cell 8:1361–75
Moreau V, Madania A, Martin RP, Winson B.
1996. The Saccharomyces cerevisiae actinrelated protein Arp2 is involved in the actin
cytoskeleton. J. Cell Biol. 134:117–32
Morris SM, Arden SD, Roberts RC, KendrickJones J, Cooper JA, et al. 2002. Myosin VI
binds to and localises with Dab2, potentially
linking receptor-mediated endocytosis and
the actin cytoskeleton. Traffic 3:331–41
Morris SM, Cooper JA. 2001. Disabled-2 colocalizes with the LDLR in clathrin-coated pits
and interacts with AP-2. Traffic 2:111–23
Mulholland J, Konopka J, Singer-Kruger B, Zerial M, Botstein D. 1999. Visualization of
receptor-mediated endocytosis in yeast. Mol.
Biol. Cell 10:799–817
Mulholland J, Preuss D, Moon A, Wong A, Drubin D, Botstein D. 1994. Ultrastructure of
the yeast actin cytoskeleton and its association with the plasma membrane. J. Cell Biol.
125:381–91
Mulholland J, Wesp A, Riezman H, Botstein D.
1997. Yeast actin cytoskeleton mutants accumulate a new class of Golgi-derived secretary
vesicle. Mol. Biol. Cell 8:1481–99
Munn AL. 2001. Molecular requirements for
the internalisation step of endocytosis: insights from yeast. Biochim. Biophys. Acta
1535:236–57
Munn AL, Heese-Peck A, Stevenson BJ, Pichler H, Riezman H. 1999. Specific sterols required for the internalization step of endocytosis in yeast. Mol. Biol. Cell 10:3943–57
Munn AL, Riezman H. 1994. Endocytosis is
required for the growth of vacuolar H(+)ATPase-defective yeast: identification of six
new END genes. J. Cell Biol. 127:373–86
Munn AL, Stevenson BJ, Geli MI, Riezman H.
1995. end5, end6, and end7: mutations that
cause actin delocalization and block the internalization step of endocytosis in Saccharomyces cerevisiae. Mol. Biol. Cell 6:1721–
42
Naqvi SN, Zahn R, Mitchell DA, Stevenson
BJ, Munn AL. 1998. The WASp homologue
Las17p functions with the WIP homologue
26 Aug 2003
20:13
Annu. Rev. Cell Dev. Biol. 2003.19:287-332. Downloaded from arjournals.annualreviews.org
by University of Pittsburgh on 08/17/07. For personal use only.
328
AR
AR197-CB19-12.tex
ENGQVIST-GOLDSTEIN
¥
AR197-CB19-12.sgm
LaTeX2e(2002/01/18)
P1: GCE
DRUBIN
End5p/verprolin and is essential for endocytosis in yeast. Curr. Biol. 8:959–62
Nobes C, Marsh M. 2000. Dendritic cells: new
roles for Cdc42 and Rac in antigen uptake?
Curr. Biol. 10:R739–41
Ochoa GC, Slepnev VI, Neff L, Ringstad N,
Takei K, et al. 2000. A functional link between dynamin and the actin cytoskeleton at
podosomes. J. Cell Biol. 150:377–89
Oh P, McIntosh DP, Schnitzer JE. 1998. Dynamin at the neck of caveolae mediates their
budding to form transport vesicles by GTPdriven fission from the plasma membrane of
endothelium. J. Cell Biol. 141:101–14
Olazabal IM, Machesky LM. 2001. Abp1p and
cortactin, new “hand-holds” for actin. J. Cell
Biol. 154:679–82
Orth JD, Krueger EW, Cao H, McNiven MA.
2002. The large GTPase dynamin regulates
actin comet formation and movement in living cells. Proc. Natl. Acad. Sci. USA 99:167–
72
Orth JD, McNiven MA. 2003. Dynamin at the
actin-membrane interface. Curr. Opin. Cell
Biol. 15:31–9
Pantaloni D, Le Clainche C, Carlier MF. 2001.
Mechanism of actin-based motility. Science
292:1502–6
Payne GS, Baker D, van Tuinen E, Schekman R. 1988. Protein transport to the vacuole and receptor-mediated endocytosis by
clathrin heavy chain-deficient yeast. J. Cell
Biol. 106:1453–61
Pelkmans L, Helenius A. 2002. Endocytosis via
caveolae. Traffic 3:311–20
Pelkmans L, Puntener D, Helenius A. 2002.
Local actin polymerization and dynamin recruitment in SV40-induced internalization of
caveolae. Science 296:535–39
Pruyne D, Bretscher A. 2000a. Polarization of
cell growth in yeast. J. Cell Sci. 113(Pt 4):
571–85
Pruyne D, Bretscher A. 2000b. Polarization of
cell growth in yeast. I. Establishment and
maintenance of polarity states. J. Cell Sci.
113(Pt 3):365–75
Qualmann B, Kelly RB. 2000. Syndapin isoforms participate in receptor-mediated endo-
cytosis and actin organization. J. Cell Biol.
148:1047–62
Qualmann B, Kessels MM. 2002. Endocytosis
and the cytoskeleton. Int. Rev. Cytol. 220:93–
144
Qualmann B, Roos J, DiGregorio PJ, Kelly
RB. 1999. Syndapin I, a synaptic dynaminbinding protein that associates with the neural Wiskott-Aldrich syndrome protein. Mol.
Biol. Cell 10:501–13
Raths S, Rohrer J, Crausaz F, Riezman H. 1993.
end3 and end4: two mutants defective in
receptor-mediated and fluid-phase endocytosis in Saccharomyces cerevisiae. J. Cell Biol.
120:55–65
Razani B, Lisanti MP. 2001. Caveolins and
caveolae: molecular and functional relationships. Exp. Cell Res. 271:36–44
Reneke JE, Blumer KJ, Courchesne WE,
Thorner J. 1988. The carboxy-terminal segment of the yeast α-factor receptor is a regulatory domain. Cell 55:221–34
Riezman H. 1985. Endocytosis in yeast: several
of the yeast secretory mutants are defective
in endocytosis. Cell 40:1001–9
Ringstad N, Gad H, Low P, Di Paolo G, Brodin
L, et al. 1999. Endophilin/SH3p4 is required
for the transition from early to late stages in
clathrin-mediated synaptic vesicle endocytosis. Neuron 24:143–54
Rohatgi R, Ma L, Miki H, Lopez M, Kirchhausen T, et al. 1999. The interaction between N-WASP and the Arp2/3 complex
links Cdc42-dependent signals to actin assembly. Cell 97:221–31
Rohrer J, Benedetti H, Zanolari B, Riezman
H. 1993. Identification of a novel sequence
mediating regulated endocytosis of the G
protein-coupled alpha-pheromone receptor
in yeast. Mol. Biol. Cell 4:511–21
Roos J, Kelly RB. 1998. Dap160, a neuralspecific Eps15 homology and multiple SH3
domain-containing protein that interacts with
Drosophila dynamin. J. Biol. Chem. 273:
19108–19
Roos J, Kelly RB. 1999. The endocytic machinery in nerve terminals surrounds sites of exocytosis. Curr. Biol. 9:1411–14
26 Aug 2003
20:13
AR
AR197-CB19-12.tex
AR197-CB19-12.sgm
LaTeX2e(2002/01/18)
Annu. Rev. Cell Dev. Biol. 2003.19:287-332. Downloaded from arjournals.annualreviews.org
by University of Pittsburgh on 08/17/07. For personal use only.
ACTIN AND ENDOCYTOSIS
Rothberg KG, Heuser JE, Donzell WC, Ying
YS, Glenney JR, Anderson RG. 1992. Caveolin, a protein component of caveolae membrane coats. Cell 68:673–82
Rozelle AL, Machesky LM, Yamamoto M,
Driessens MH, Insall RH, et al. 2000.
Phosphatidylinositol 4,5-bisphosphate induces actin-based movement of raft-enriched
vesicles through WASP-Arp2/3. Curr. Biol.
10:311–20
Salisbury JL, Condeelis JS, Satir P. 1980.
Role of coated vesicles, microfilaments, and
calmodulin in receptor-mediated endocytosis
by cultured B lymphoblastoid cells. J. Cell
Biol. 87:132–41
Sallusto F, Cella M, Danieli C, Lanzavecchia
A. 1995. Dendritic cells use macropinocytosis and the mannose receptor to concentrate
macromolecules in the major histocompatibility complex class II compartment: downregulation by cytokines and bacterial products. J. Exp. Med. 182:389–400
Sandvig K, van Deurs B. 1990. Selective modulation of the endocytic uptake of ricin
and fluid phase markers without alteration
in transferrin endocytosis. J. Biol. Chem.
265:6382–88
Santini F, Keen JH. 2002. A glimpse of coated
vesicle creation? Well almost! Nat. Cell Biol.
4:E230–32
Schafer DA. 2002. Coupling actin dynamics
and membrane dynamics during endocytosis. Curr. Opin. Cell Biol. 14:76–81
Schafer DA, D’Souza-Schorey C, Cooper JA.
2000. Actin assembly at membranes controlled by ARF6. Traffic 1:896–907
Schafer DA, Weed SA, Binns D, Karginov AV,
Parsons JT, Cooper JA. 2002. Dynamin2 and
cortactin regulate actin assembly and filament organization. Curr. Biol. 12:1852–57
Schmid SL, Smythe E. 1991. Stage-specific assays for coated pit formation and coated vesicle budding in vitro. J. Cell Biol. 114:869–
80
Schmidt A, Wolde M, Thiele C, Fest W, Kratzin
H, et al. 1999. Endophilin I mediates synaptic
vesicle formation by transfer of arachidonate
to lysophosphatidic acid. Nature 401:133–41
P1: GCE
329
Schuuring E, Verhoeven E, Litvinov S,
Michalides RJ. 1993. The product of the
EMS1 gene, amplified and overexpressed in
human carcinomas, is homologous to a vsrc substrate and is located in cell-substratum
contact sites. Mol. Cell. Biol. 13:2891–98
Seastone DJ, Harris E, Temesvari LA, Bear
JE, Saxe CL, Cardelli J. 2001. The WASplike protein scar regulates macropinocytosis,
phagocytosis and endosomal membrane flow
in Dictyostelium. J. Cell Sci. 114:2673–83
Sellers JR. 2000. Myosins: a diverse superfamily. Biochim. Biophys. Acta 1496:3–22
Sengar AS, Wang W, Bishay J, Cohen S, Egan
SE. 1999. The EH and SH3 domain Ese proteins regulate endocytosis by linking to dynamin and Eps15. EMBO J. 18:1159–71
Sever S, Damke H, Schmid SL. 2000. Garrotes,
springs, ratchets, and whips: putting dynamin
models to the test. Traffic 1:385–92
Shaw JD, Cummings KB, Huyer G, Michaelis
S, Wendland B. 2001. Yeast as a model system for studying endocytosis. Exp. Cell Res.
271:1–9
Shih SC, Katzmann DJ, Schnell JD, Sutanto
M, Emr SD, Hicke L. 2002. Epsins and
Vps27p/Hrs contain ubiquitin-binding domains that function in receptor endocytosis.
Nat. Cell Biol. 4:389–93
Shih SC, Sloper-Mould KE, Hicke L. 2000.
Monoubiquitin carries a novel internalization
signal that is appended to activated receptors.
EMBO J. 19:187–98
Shupliakov O, Bloom O, Gustafsson JS,
Kjaerulff O, Low P, et al. 2002. Impaired recycling of synaptic vesicles after acute perturbation of the presynaptic actin cytoskeleton. Proc. Natl. Acad. Sci. USA 99:14476–81
Simons K, Toomre D. 2000. Lipid rafts and signal transduction. Nat. Rev. Mol. Cell Biol.
1:31–39
Simpson F, Hussain NK, Qualmann B, Kelly
RB, Kay BK, et al. 1999. SH3-domaincontaining proteins function at distinct steps
in clathrin-coated vesicle formation. Nat.
Cell Biol. 1:119–24
Singer-Kruger B, Nemoto Y, Daniell L, FerroNovick S, De Camilli P. 1998. Synaptojanin
26 Aug 2003
20:13
Annu. Rev. Cell Dev. Biol. 2003.19:287-332. Downloaded from arjournals.annualreviews.org
by University of Pittsburgh on 08/17/07. For personal use only.
330
AR
AR197-CB19-12.tex
ENGQVIST-GOLDSTEIN
¥
AR197-CB19-12.sgm
LaTeX2e(2002/01/18)
P1: GCE
DRUBIN
family members are implicated in endocytic
membrane traffic in yeast. J. Cell Sci. 111(Pt
22):3347–56
Smith MG, Swamy SR, Pon LA. 2001. The life
cycle of actin patches in mating yeast. J. Cell
Sci. 114:1505–13
Srinivasan S, Seaman M, Nemoto Y, Daniell
L, Suchy SF, et al. 1997. Disruption of
three phosphatidylinositol-polyphosphate 5phosphatase genes from Saccharomyces
cerevisiae results in pleiotropic abnormalities of vacuole morphology, cell shape, and
osmohomeostasis. Eur. J. Cell Biol. 74:350–
60
Tan PK, Davis NG, Sprague GF, Payne GS.
1993. Clathrin facilitates the internalization
of seven transmembrane segment receptors
for mating pheromones in yeast. J. Cell Biol.
123:1707–16
Tan PK, Howard JP, Payne GS. 1996. The sequence NPFXD defines a new class of endocytosis signal in Saccharomyces cerevisiae.
J. Cell Biol. 135:1789–800
Tang HY, Cai M. 1996. The EH-domaincontaining protein Pan1 is required for normal organization of the actin cytoskeleton in
Saccharomyces cerevisiae. Mol. Cell. Biol.
16:4897–914
Tang HY, Munn A, Cai M. 1997. EH domain
proteins Pan1p and End3p are components of
a complex that plays a dual role in organization of the cortical actin cytoskeleton and endocytosis in Saccharomyces cerevisiae. Mol.
Cell. Biol. 17:4294–304
Tang HY, Xu J, Cai M. 2000. Pan1p, End3p, and
S1a1p, three yeast proteins required for normal cortical actin cytoskeleton organization,
associate with each other and play essential
roles in cell wall morphogenesis. Mol. Cell.
Biol. 20:12–25
Taunton J, Rowning BA, Coughlin ML, Wu
M, Moon RT, et al. 2000. Actin-dependent
propulsion of endosomes and lysosomes by
recruitment of N-WASP. J. Cell Biol. 148:
519–30
Teo M, Tan L, Lim L, Manser E. 2001. The tyrosine kinase ACK1 associates with clathrincoated vesicles through a binding motif
shared by arrestin and other adaptors. J. Biol.
Chem. 276:18392–98
Terrell J, Shih S, Dunn R, Hicke L. 1998. A
function for monoubiquitination in the internalization of a G protein-coupled receptor.
Mol. Cell 1:193–202
Thomsen P, Roepstorff K, Stahlhut M, van
Deurs B. 2002. Caveolae are highly immobile plasma membrane microdomains, which
are not involved in constitutive endocytic
trafficking. Mol. Biol. Cell 13:238–50
Tong XK, Hussain NK, Adams AG, O’Bryan
JP, McPherson PS. 2000. Intersectin can regulate the Ras/MAP kinase pathway independent of its role in endocytosis. J. Biol. Chem.
275:29894–99
Uruno T, Liu J, Zhang P, Fan Y, Egile C,
et al. 2001. Activation of Arp2/3 complexmediated actin polymerization by cortactin.
Nat. Cell. Biol. 3:259–66
Vaduva G, Martin NC, Hopper AK. 1997.
Actin-binding verprolin is a polarity development protein required for the morphogenesis
and function of the yeast actin cytoskeleton.
J. Cell Biol. 139:1821–33
Waddle JA, Karpova TS, Waterston RH, Cooper
JA. 1996. Movement of cortical actin patches
in yeast. J. Cell Biol. 132:861–70
Waelter S, Scherzinger E, Hasenbank R, Nordhoff E, Lurz R, et al. 2001. The huntingtin
interacting protein HIP1 is a clathrin and
alpha-adaptin-binding protein involved in
receptor-mediated endocytosis. Hum. Mol.
Genet. 10:1807–17
Wanker EE, Rovira C, Scherzinger E, Hasenbank R, Walter S, et al. 1997. HIP-1: a huntingtin interacting protein isolated by the yeast
two-hybrid system. Hum. Mol. Genet. 6:487–
95
Warren DT, Andrews PD, Gourlay CW,
Ayscough KR. 2002. Sla1p couples the yeast
endocytic machinery to proteins regulating
actin dynamics. J. Cell Sci. 115:1703–15
Wasiak S, Quinn CC, Ritter B, de Heuvel
E, Baranes D, et al. 2001. The Ras/Rac
guanine nucleotide exchange factor mammalian Son-of-sevenless interacts with PACSIN 1/syndapin I, a regulator of endocytosis
26 Aug 2003
20:13
AR
AR197-CB19-12.tex
AR197-CB19-12.sgm
LaTeX2e(2002/01/18)
Annu. Rev. Cell Dev. Biol. 2003.19:287-332. Downloaded from arjournals.annualreviews.org
by University of Pittsburgh on 08/17/07. For personal use only.
ACTIN AND ENDOCYTOSIS
and the actin cytoskeleton. J. Biol. Chem.
276:26622–28
Watson HA, Cope MJ, Groen AC, Drubin
DG, Wendland B. 2001. In vivo role for
actin-regulating kinases in endocytosis and
yeast epsin phosphorylation. Mol. Biol. Cell
12:3668–79
Weaver AM, Karginov AV, Kinley AW, Weed
SA, Li Y, et al. 2001. Cortactin promotes and
stabilizes Arp2/3-induced actin filament network formation. Curr. Biol. 11:370–74
Welch MD, Mullins RD. 2002. Cellular control
of actin nucleation. Annu. Rev. Cell Dev. Biol.
18:247–88
Wells AL, Lin AW, Chen LQ, Safer D, Cain SM,
et al. 1999. Myosin VI is an actin-based motor that moves backwards. Nature 401:505–
8
Wendland B. 2002. Epsins: adaptors in endocytosis? Nat. Rev. Mol. Cell Biol. 3:971–77
Wendland B, Emr SD. 1998. Pan1p, yeast
eps15, functions as a multivalent adaptor that
coordinates protein-protein interactions essential for endocytosis. J. Cell Biol. 141:71–
84
Wendland B, McCaffery JM, Xiao Q, Emr SD.
1996. A novel fluorescence-activated cell
sorter-based screen for yeast endocytosis mutants identifies a yeast homologue of mammalian eps15. J. Cell Biol. 135:1485–500
Wendland B, Steece KE, Emr SD. 1999. Yeast
epsins contain an essential N-terminal ENTH
domain, bind clathrin and are required for endocytosis. EMBO J. 18:4383–93
Wesp A, Hicke L, Palecek J, Lombardi R, Aust
T, et al. 1997. End4p/Sla2p interacts with
actin-associated proteins for endocytosis in
Saccharomyces cerevisiae. Mol. Biol. Cell
8:2291–306
Wienke DC, Knetsch ML, Neuhaus EM, Reedy
MC, Manstein DJ. 1999. Disruption of
a dynamin homologue affects endocytosis,
organelle morphology, and cytokinesis in
Dictyostelium discoideum. Mol. Biol. Cell
10:225–43
Winter D, Lechler T, Li R. 1999. Activation
of the yeast Arp2/3 complex by Bee1p, a
WASP-family protein. Curr. Biol. 9:501–4
P1: GCE
331
Witke W, Podtelejnikov AV, Di Nardo A,
Sutherland JD, Gurniak CB, et al. 1998. In
mouse brain profilin I and profilin II associate
with regulators of the endocytic pathway and
actin assembly. EMBO J. 17:967–76
Wolkoff AW, Klausner RD, Ashwell G, Harford
J. 1984. Intracellular segregation of asialoglycoproteins and their receptor: a prelysosomal event subsequent to dissociation of
the ligand-receptor complex. J. Cell Biol. 98:
375–81
Wu H, Parsons JT. 1993. Cortactin, an 80/85–
kilodalton pp60src substrate, is a filamentous
actin-binding protein enriched in the cell cortex. J. Cell Biol. 120:1417–26
Wu H, Reynolds AB, Kanner SB, Vines RR,
Parsons JT. 1991. Identification and characterization of a novel cytoskeleton-associated
pp60src substrate. Mol. Cell. Biol. 11:5113–
24
Yamabhai M, Hoffman NG, Hardison NL,
McPherson PS, Castagnoli L, et al. 1998. Intersectin, a novel adaptor protein with two
Eps15 homology and five Src homology 3
domains. J. Biol. Chem. 273:31401–7
Yang S, Cope MJ, Drubin DG. 1999. Sla2p is
associated with the yeast cortical actin cytoskeleton via redundant localization signals.
Mol. Biol. Cell 10:2265–83
Yang W, Lin Q, Zhao J, Guan JL, Cerione
RA. 2001a. The nonreceptor tyrosine kinase
ACK2, a specific target for Cdc42 and a negative regulator of cell growth and focal adhesion complexes. J. Biol. Chem. 276:43987–
93
Yang W, Lo CG, Dispenza T, Cerione
RA. 2001b. The Cdc42 target ACK2 directly interacts with clathrin and influences
clathrin assembly. J. Biol. Chem. 276:17468–
73
Yeung BG, Phan HL, Payne GS. 1999. Adaptor complex-independent clathrin function in
yeast. Mol. Biol. Cell 10:3643–59
Zeng G, Cai M. 1999. Regulation of the actin
cytoskeleton organization in yeast by a novel
serine/threonine kinase Prk1p. J. Cell Biol.
144:71–82
Zeng G, Yu X, Cai M. 2001. Regulation
26 Aug 2003
20:13
332
AR
AR197-CB19-12.tex
ENGQVIST-GOLDSTEIN
¥
AR197-CB19-12.sgm
P1: GCE
DRUBIN
of yeast actin cytoskeleton-regulatory complex Pan1p/Sla1p/End3p by serine/threonine
kinase Prk1p. Mol. Biol. Cell 12:3759–
72
Zhang J, Shehabeldin A, da Cruz LA, Butler J, Somani AK, et al. 1999. Antigen
receptor-induced activation and cytoskeletal rearrangement are impaired in WiskottAnnu. Rev. Cell Dev. Biol. 2003.19:287-332. Downloaded from arjournals.annualreviews.org
by University of Pittsburgh on 08/17/07. For personal use only.
LaTeX2e(2002/01/18)
Aldrich syndrome protein-deficient lymphocytes. J. Exp. Med. 190:1329–42
Zoladek T, Tobiasz A, Vaduva G, Boguta M,
Martin NC, Hopper AK. 1997. MDP1, a Saccharomyces cerevisiae gene involved in mitochondrial/cytoplasmic protein distribution,
is identical to the ubiquitin-protein ligase
gene RSP5. Genetics 145:595–603
Annu. Rev. Cell Dev. Biol. 2003.19:287-332. Downloaded from arjournals.annualreviews.org
by University of Pittsburgh on 08/17/07. For personal use only.
12-HI-RESOLUTION-VERSION
9/15/2003
See legend on next page.
7:56 PM
Page 1
ACTIN AND ENDOCYTOSIS
C-1
12-HI-RESOLUTION-VERSION
Annu. Rev. Cell Dev. Biol. 2003.19:287-332. Downloaded from arjournals.annualreviews.org
by University of Pittsburgh on 08/17/07. For personal use only.
C-2
9/15/2003
ENGQVIST-GOLDSTEIN
7:56 PM
■
Page 2
DRUBIN
Figure 1 Protein complexes in the yeast endocytic machinery. Proteins important for
endocytic internalization in yeast appear to form complexes that can promote F-actin
assembly at endocytic sites. Endocytosis in yeast can be classified into two main pathways
based on cargo: (a, b) receptor-mediated endocytosis (RME) and (c) fluid-phase endocytosis. RME can further be divided into (a) ubiquitin-dependent internalization and
(b) NPFX(1,2)D-dependent internalization; this division depends on the signal for internalization. Sla1p is an adaptor for NPFX(1,2)D-dependent internalization, whereas Ent1/2p
appears to be an adaptor for ubiquitin-mediated internalization. The dashed circles identify three putative functional modules based on the Arp2/3 activators Las17p, Pan1p, and
Abp1p (Goode & Rodal 2001). Physical interactions between proteins (coimmunoprecipitation, yeast two-hybrid, and/or direct interactions) are indicated with double-headed
arrows. Proteins important for internalization are colored and outlined in black. Known
identities or activities of these proteins are indicated in the key.
12-HI-RESOLUTION-VERSION
9/15/2003
7:56 PM
Page 3
Annu. Rev. Cell Dev. Biol. 2003.19:287-332. Downloaded from arjournals.annualreviews.org
by University of Pittsburgh on 08/17/07. For personal use only.
ACTIN AND ENDOCYTOSIS
C-3
Figure 3 Putative actin functions in different endocytic pathways (clathrin- and caveolaemediated endocytosis and macropinocytosis). Actin is likely to function at different stages
during endocytic internalization. First, actin may provide a scaffold for the endocytic
machinery during formation of CCVs and caveolae (see 1a and 2a). Second, actin may provide a force driving invagination and/or fission of CCVs and caveolae (see 1b, 1c, and 2b).
Third, actin may provide a force to move vesicles formed by each of these pathways away
from the plasma membrane (see 1c9, 2c, and 3c). Additionally, the actin cytoskeleton plays
a role during the formation of membrane protrusions required for the formation of
macropinocytic vesicles (see 3a).
12-HI-RESOLUTION-VERSION
ENGQVIST-GOLDSTEIN
7:56 PM
■
Page 4
DRUBIN
Annu. Rev. Cell Dev. Biol. 2003.19:287-332. Downloaded from arjournals.annualreviews.org
by University of Pittsburgh on 08/17/07. For personal use only.
C-4
9/15/2003
Figure 4 Domain structures of proteins that may function at the interface between
actin and the endocytic machinery in clathrin-mediated endocytosis.
(a) Hip1 and Hip1R, (b) myosin VI, (c) Abp1, (d) cortactin, (e) intersectin-s and
intersectin-l, and (f) syndapin. The arrows point to interacting proteins.
Abbreviations: ANTH, AP180 N-terminal homology domain; CL HC, clathrin heavy
chain; CL LC, clathrin light chain; SH3, src-homology 3 domain; A, acidic motif;
EH, Eps15-homology domain; DH, Dbl-homology domain; PH, pleckstrin-homology domain.
P1: FRK
September 4, 2003
21:45
Annual Reviews
AR197-FM
Annual Review of Cell and Developmental Biology
Volume 19, 2003
Annu. Rev. Cell Dev. Biol. 2003.19:287-332. Downloaded from arjournals.annualreviews.org
by University of Pittsburgh on 08/17/07. For personal use only.
CONTENTS
ADULT STEM CELL PLASTICITY: FACT OR ARTIFACT, Martin Raff
CYCLIC NUCLEOTIDE-GATED ION CHANNELS, Kimberly Matulef and
William N. Zagotta
23
ANTHRAX TOXIN, R. John Collier and John A.T. Young
45
GENES, SIGNALS, AND LINEAGES IN PANCREAS DEVELOPMENT,
L. Charles Murtaugh and Douglas A. Melton
REGULATION OF MAP KINASE SIGNALING MODULES BY SCAFFOLD PROTEINS
IN MAMMALS, Deborah Morrison and Roger J. Davis
FLOWER DEVELOPMENT: INITIATION, DIFFERENTIATION, AND
DIVERSIFICATION, Moriyah Zik and Vivian F. Irish
REGULATION OF MEMBRANE PROTEIN TRANSPORT BY UBIQUITIN AND
UBIQUITIN-BINDING PROTEINS, Linda Hicke and Rebecca Dunn
POSITIONAL CONTROL OF CELL FATE THROUGH JOINT INTEGRIN/RECEPTOR
PROTEIN KINASE SIGNALING, Filippo G. Giancotti and Guido Tarone
CADHERINS AS MODULATORS OF CELLULAR PHENOTYPE,
Margaret J. Wheelock and Keith R. Johnson
GENOMIC IMPRINTING: INTRICACIES OF EPIGENETIC REGULATION IN
CLUSTERS, Raluca I. Verona, Mellissa R.W. Mann, and
Marisa S. Bartolomei
THE COP9 SIGNALOSOME, Ning Wei and Xing Wang Deng
ACTIN ASSEMBLY AND ENDOCYTOSIS: FROM YEAST TO MAMMALS,
Åsa E.Y. Engqvist-Goldstein and David G. Drubin
TRANSPORT PROTEIN TRAFFICKING IN POLARIZED CELLS, Theodore R. Muth
and Michael J. Caplan
MODULATION OF NOTCH SIGNALING DURING SOMITOGENESIS,
Gerry Weinmaster and Chris Kintner
TETRASPANIN PROTEINS MEDIATE CELLULAR PENETRATION, INVASION,
AND FUSION EVENTS AND DEFINE A NOVEL TYPE OF MEMBRANE
MICRODOMAIN, Martin E. Hemler
INTRAFLAGELLAR TRANSPORT, Jonathan M. Scholey
1
71
91
119
141
173
207
237
261
287
333
367
397
423
vii
P1: FRK
September 4, 2003
viii
21:45
Annual Reviews
AR197-FM
CONTENTS
Annu. Rev. Cell Dev. Biol. 2003.19:287-332. Downloaded from arjournals.annualreviews.org
by University of Pittsburgh on 08/17/07. For personal use only.
THE DYNAMIC AND MOTILE PROPERTIES OF INTERMEDIATE FILAMENTS,
Brian T. Helfand, Lynne Chang, and Robert D. Goldman
PIGMENT CELLS: A MODEL FOR THE STUDY OF ORGANELLE TRANSPORT,
Alexandra A. Nascimento, Joseph T. Roland, and Vladimir I. Gelfand
SNARE PROTEIN STRUCTURE AND FUNCTION, Daniel Ungar and
Frederick M. Hughson
445
469
493
STRUCTURE, FUNCTION, AND REGULATION OF BUDDING YEAST
KINETOCHORES, Andrew D. McAinsh, Jessica D. Tytell, and Peter K. Sorger
ENA/VASP PROTEINS: REGULATORS OF THE ACTIN CYTOSKELETON AND
CELL MIGRATION, Matthias Krause, Erik W. Dent, James E. Bear,
Joseph J. Loureiro, and Frank B. Gertler
541
PROTEOLYSIS IN BACTERIAL REGULATORY CIRCUITS, Susan Gottesman
565
NODAL SIGNALING IN VERTEBRATE DEVELOPMENT, Alexander F. Schier
BRANCHING MORPHOGENESIS OF THE DROSOPHILA TRACHEAL SYSTEM,
Amin Ghabrial, Stefan Luschnig, Mark M. Metzstein, and
Mark A. Krasnow
589
QUALITY CONTROL AND PROTEIN FOLDING IN THE SECRETORY PATHWAY,
E. Sergio Trombetta and Armando J. Parodi
ADHESION-DEPENDENT CELL MECHANOSENSITIVITY,
Alexander D. Bershadsky, Nathalie Q. Balaban, and Benjamin Geiger
PLASMA MEMBRANE DISRUPTION: REPAIR, PREVENTION, ADAPTATION,
Paul L. McNeil and Richard A. Steinhardt
INDEXES
Subject Index
Cumulative Index of Contributing Authors, Volumes 15–19
Cumulative Index of Chapter Titles, Volumes 15–19
ERRATA
An online log of corrections to Annual Review of Cell and Developmental
Biology chapters (if any, 1997 to the present) may be found at
http://cellbio.annualreviews.org
519
623
649
677
697
733
765
768