POLARITY AND ENDOCYTIC TRAFFIC IN THE MAMMALIAN CELL
Francis Kyei Bugyei
A Thesis
Submitted to the Graduate College of Bowling Green
State University in partial fulfillment of
the requirements for the degree of
MASTER OF SCIENCE
August 2014
Committee:
Carol Heckman, Advisor
Hans Wildschutte
John Wade
ii
ABSTRACT
Carol Heckman, Advisor
Endocytosis involves selective packaging of substances in endosomes for traffic into
cells. The cell traffic pathway can be grouped into two functional compartments: Compartment I
which represents the early stages of sorting and Compartment II representing the final stage of
trafficking and degradation. The sorting events and fate of molecules following uptake is
dependent on a variety of cell signaling pathways. The tumor promoter, phorbol 12-myristate 13acetate (PMA) is known to cause transient loss of filopodia and increased pinocytosis in cells.
Filopodia are finger-like plasma membrane protrusions of a number of animal cells containing
receptors involved in cell sensing. In neurons where filopodia have been extensively studied,
there is some evidence of increased endocytosis during filopodia retraction following encounters
with repulsive cues. We hypothesized that, mammalian cells increase endocytic uptake during
tumor promotion (with loss of filopodia), and polarity may be connected to vesicle trafficking.
PMA effects its action through activation of the enzyme protein kinase C (PKC). To classify
the traffic events in mammalian cells in tumor promotion, we selected conditions that enabled us
to visualize the contents of Compartment II and compare them to the contents of Compartment I.
To investigate the effects of PMA on trafficking three fluorescent markers were applied to rat
liver cells of the IAR20 line. Using pixel-by-pixel analysis and image processing, we found an
increased accumulation of fluorescent marker in compartments I and II in PMA-treated over the
untreated cells. We also found that functional compartments I and II are trafficked in different
directions inside cells. In another study on epithelial cells from the rat trachea, 1000W, we found
iii
an insignificant difference in fluorescent marker accumulated by endocytosis on the two halves
of cells plated on a haptotactic gradient.
iv
To my late father, Kofi Boateng, whose encouragement made me believe I could accomplish
anything, and my mother, Afua Nyame, for the wonderful support to my continuous education.
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ACKNOWLEDGMENTS
I am very grateful to God for giving me the opportunity and wisdom to explore the
wonders of his creation. I am also profoundly thankful to Dr. Carol Heckman, whose love and
support went beyond mere scientific guidance. By providing me with enough liberties to explore
the subject, I was able to acquire a great deal of knowledge in the field. I would also like to thank
Dr. John Wade and Dr. Daria Filippova from the Mathematics department for helping me greatly
with developing a model with which I could confidently analyze my data. I would like to thank
Dr. Hans Wildschutte for critically reviewing my manuscript and providing sound critique
through my experiment. My thanks also go to Dr. Nancy Boudreau and Mr. Calvin Bosumprah
of the Center for Business Analytics without whose help my data would have been reduced to a
statistical mess. I would like to thank the Biological Sciences department for funding my project
and all the professors for giving me a solid foundation to perform scientific inquiry. I am also
grateful to Dr. Marilyn Cayer for technical advice and help on microscopy and image processing.
I cannot sign off without expressing my heartfelt gratitude to the graduate secretary Mrs. DeeDee
Wentland whose tremendous assistance helped me navigate through the maze of graduate school.
Finally my sincere thanks go to my family, friends, lab team, and all names I cannot provide for
lack of space, for providing the friendship and support through my studies. Thank you all.
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TABLE OF CONTENTS
Page
CHAPTER 1. LITERATURE REVIEW ..............................................................................
1
1.1 INTRODUCTION .............................................................................................
1
1.2 TYPES OF ENDOCYTOSIS ............................................................................
4
1.2.1 Phagocytosis .......................................................................................
6
1.2.2 Clathrin-mediated endocytosis............................................................
7
1.2.3 Clathrin-independent endocytosis .......................................................
12
ENDOCYTOSIS, CELL POLARITY AND TUMOR PROMOTION .........
14
1.3.1 Cell polarity ........................................................................................
14
1.3.2 Mammalian cell polarity regulation ....................................................
17
Table 1.1 ............................................................................................
19
1.3.3 Filopodia as mediators of cell polarity................................................
22
1.3.4 Endocytosis and cell polarity regulation .............................................
24
EXPERIMENTAL MODEL..........................................................................
29
CHAPTER 2. MATERIALS AND METHODS ..................................................................
32
2.1 CELL CULTURE AND CHEMICALS ............................................................
32
2.2 SUBSTRATE PREPARATION ........................................................................
33
2.3 PREPARATION OF IAR20 CELLS FOR IMAGING .....................................
34
2.4 PREPARATION OF 1000W CELLS FOR IMAGING ....................................
35
2.5 IMAGING ..........................................................................................................
35
2.6 IMAGE PROCESSING .....................................................................................
36
2.7 STATISTICAL ANALYSIS OF ENDOCYTIC ACTIVITY ...........................
37
1.3
1.4
vi
CHAPTER 3. RESULTS ......................................................................................................
38
3.1 KINETICS OF COMPARTMENT II MARKER TRAFFIC ............................
38
3.2 MARKER FOR COMPARTMENT I AND TRAFFICKING INTO
COMPARTMENT II .........................................................................................
45
3.3 PMA-DEPENDENT DIFFERENCES IN ENDOCYTIC MARKER INFLUX AND
COMPARTMENTALIZATION .......................................................................
49
3.4 SPATIAL DIFFERENCES IN THE LOCALIZATION OF COMPARTMENT I
COMPARTMENTALIZATION .......................................................................
55
3.5 COMPARISON OF FLUORESCENT MARKER ACCUMULATION IN 1000W
CELLS ON HAPTOTACTIC GRADIENT ......................................................
58
CHAPTER 4. DISCUSSION ................................................................................................
60
REFERENCES ......................................................................................................................
65
APPENDIX A .......................................................................................................................
75
APPENDIX B .......................................................................................................................
77
APPENDIX C .......................................................................................................................
79
APPENDIX D .......................................................................................................................
83
APPENDIX E .......................................................................................................................
85
vii
LIST OF FIGURES
Page
Figure
1.1
Illustration of the fundamental features of the endocytic
pathway in a eukaryotic cell ......................................................................................
2
1.2
Classification of endocytosis based on morphology of portals .................................
5
1.3
Mechanism of clathrin coat assembly and internalization
during the endocytic process ......................................................................................
9
1.4
Illustration of G-protein coupled regulation of cellular activities .............................
16
1.5
Illustration of edge features as obtained using 3D imaging and quantitation ............
19
1.6
Illustration of molecules and their roles in filopodia formation ................................
23
1.7
Experimental model of the functional endocytic compartments ...............................
27
1.8
Experimental model of a motile cell on a haptotactic gradient .................................
29
1.9
Stair step model of the endocytic traffic through functional
compartments I and II ................................................................................................
3.1
Relative amount of fluid-phase marker influx over time in
PMA-treated cells versus controls. ............................................................................
3.2
41
Histogram showing the percentage distribution of FITC gray levels
of pixels for the control and PMA-treated cell populations......................................
3.4
39
Histograms of FITC integrated intensity (sum of gray levels)
for two cell populations. ............................................................................................
3.3
31
44
Histograms of CY3integrated intensity (sum of gray levels)
for two cell populations. ............................................................................................
46
viii
3.5
Histograms showing the distribution of CY3 gray levels of pixels for
treated (A) and control cell populations (B) .............................................................
3.6
Histograms of difference between CY3 and FITC pixel-by-pixel gray
level intensities for treated (A) and control cell populations (B)...............................
3.7
52
Color map of fluorescent marker intensity in FITC (A) and CY3 (B)
in a untreated cell showing distribution of two fluorescent markers ........................
3.9
51
Color map of fluorescent marker intensity in FITC (A) and
CY3 (B) in a PMA-treated cell ..................................................................................
3.8
48
53
Ratio of CY3 as a fraction of the two fluorescent markers (CY3 and FITC) on a pixel-bypixel basis in PMA-treated versus control cells .........................................................
54
3.10 Ratio (FITC/CY3) images of (A-D) PMA-treated and (E-H) control cells ...............
57
3.11 Comparison of FITC integrated intensity and optical density (OD) of the right (high side)
against left (low) side of 1000W cells .......................................................................
59
3.12 Image of two cells showing accumulation of FITC vesicles at the rim of 1000W after an
incubation period of 2 minutes ..................................................................................
59
1
CHAPTER 1. LITERATURE REVIEW
1.1 INTRODUCTION
Endocytosis is the general term employed to describe the internalization of extracellular
fluid or particles by invagination and pinching off of the plasma membrane. Endocytosis plays
a variety of important roles in eukaryotic systems including nutrient uptake, scavenging of
extracellular material, and internalization of receptor-bound ligands such as hormones, growth
factors, lipoproteins and antibodies. These roles are essential for organismal homeostasis,
helping to control a number of activities important in maintaining the integrity of a functional
multicellular system. All eukaryotic cells exhibit one or more forms of endocytosis, with the
diversity resulting from the individual cell functions (1). One important property of endocytosis
was first described by Elie Metchnikoff when he recognized that, materials taken up by
endocytosis were degraded after encountering an acidic internal environment (2). With the
advances in molecular genetics, cell biology and high resolution imaging techniques, the basic
organization of the endocytic pathway has been elucidated (1,3-5). This knowledge has
provided a fundamental agreement on the integral features of the pathway across different
eukaryotic systems as illustrated in the figure below (Figure 1.1).
2
Figure 1.1 Illustration of the fundamental features of the endocytic pathway in a eukaryotic
cell.
This figure shows the connnections that exist endocytic traffic in eukaryotic cells. EE=early
endosome, RE=recycling endosome, LE=late endosome, MP=macropinosome,
PHS=phagosome, and LYS=lysosome.
3
A large number of receptor-ligand complexes start off at the clathrin-coated pits of the
plasma membrane, which eventually pinch off to yield clathrin-coated vesicles. The released
vesicles after losing their coats fusion with early endosomes (EEs), a dynamic array of tubules
and vesicles distributed throughout the peripheral and perinuclear cytoplasm. Ligands are
dissociated from their respective receptors in the EEs due to their slightly acidic pH of about
6.0-6.8 (6). This slight acidity is maintained by an ATP-driven proton pump (7,8). Free
receptors selectively accumulate in the early endosome’s tubular extensions, following which
they bud off to form recycling vesicles (RVs) that transport receptors back to the plasma
membrane to be reused. The dissociated ligands collect in the vesicular portions due to a
difference in volume from the endosome’s tubular extensions collect in the vesicular portions
of the EEs. The contents of these vesicles depending properties such as molecular weight, may
be trafficked to the perinuclear cytoplasm on microtubule tracks, and fuse with late endosomes
(LEs) and lysosomes (1,9). Some vesicles, however, are left behind and share the fate of the
RVs being exported out of the cell. In the LEs and lysosomes, ligands may be subject to
degradation by a lower pH of 5 and the high concentration of lysosomal enzymes. Molecules
such as dextran are accumulated in the lysosome. Although dextran is degraded by α-Dglucoside glucohydrolase, this occurs slowly in comparison to its rate of trafficking to the
organelle. This property has made it a preferred label in studies of endocytosis, where they are
usually tagged various fluorescent molecules (10). Once in the lysosomes, ligand recycling is
relatively slow or nonexistent. Thus, cells are capable of accumulating large amounts of
internalized material once transported through the LEs and lysosomes.
The endocytic pathway therefore can be thought of as having functionally and physically
distinct compartments (Figure 1.1). Early endosomes are responsible for sorting receptors and
ligands, helping distinguish and preserve selected receptors from ligands to be internalized and
4
utilized by cells. Late endosomes are responsible for sorting components, and lysosomes are
mainly responsible for digesting exogenous and endogenous macromolecules. With this
knowledge however, it is quite challenging to distinguish these compartments by morphology
due to the pleomorphic nature of endocytic organelles.
An additional challenge of distinguishing compositional difference between compartments
comes from the large quantities of membrane processed by the endocytic pathway (5).
However with the advent of biochemical, functional, and genetic probes for monitoring and
manipulating the endocytic pathway and its organelles, a lot of information has been generated
on the different types of endocytosis in cells. This body of knowledge has also provided an
understanding of the variations responsible for generating specializations of the endocytic
pathway in eukaryotic cells.
1.2 TYPES OF ENDOCYTOSIS
Endocytosis can be grouped into two classes, phagocytosis and pinocytosis, based on the
nature and size of the particles internalized by cells as well as the morphology of the vesicle
formed during internalization (5,11,12). Phagocytosis describes the internalization of large
particles, usually greater than 0.5 µm in diameter, which bind to specific plasma membrane
receptors. These receptors are capable of causing their own uptake, usually by the formation of
F-actin-driven pseudopods that envelope the bound particle during internalization. In contrast,
pinocytosis involves internalization of fluids and smaller particles usually less than 0.2 µm in
diameter. In pinocytosis, smaller vesicles carrying extracellular fluid and smaller
macromolecules are internalized. The macromolecules may be specifically bound to the plasma
membrane before internalization, but such binding can vary from strong to negligible
interaction depending on the nature of the molecules. These vesicles are usually initiated at
5
clathrin-coated pits though other processes involving calveolae and/ or an actin-based
mechanism may be involved (1,11).
Figure 1.2 Classification of endocytosis based on morphology of portals.
Transmission and scanning electron micrographs, and fluorescent micrographs illustrate
structures known to be involved in endocytic events. Adapted with permission from (12)
1.2.1 Phagocytosis
Phagocytosis, which involves the internalization of large particles ( > 0.5 µm), was thought
to be associated only with specialized cells known as phagocytes until proven otherwise.
6
Phagocytic cells include protozoa such as Dictyostelium acanthamoeba, and leukocytes of the
mammalian immune system such as macrophages and neutrophils. Phagocytosis is contactactivated, with uptake triggered by binding of opsonized particles to cell surface receptors
capable of transducing a phagocytic stimulus to the cytoplasm (1). This stimulus results in
polymerization of actin at the sites of particle attachment and subsequent of an extension,
called a pseudopod, that engulfs the bound particle into a cytoplasmic phagosome (1,13,14).
Phagosomes may rapidly fuse with endosomes and/or lysosomes so that the contents are
degraded by their lysosomal hydrolytic enzymes (15-18).
Phagocytosis serves a principal first line defense mechanism against microorganisms in
mammalian systems. It also provides an essential component of the humoral immune response
by allowing the processing and presentation of bacteria-derived peptides to antigen-specific T
lymphocytes (19,20). In protozoa however, phagocytosis serve a nutritional function, as
demonstrated in Dictyostelium discoideum mutants defective for phagocytosis (21). The
mutants eventually died of starvation due to the inability to feed on suspensions of bacteria.
Studies on phagocytosis have showed that membrane receptors play an important role.
Greenberg, et al. have shown that, in macrophages, Fc receptor-mediated phagocytosis of IgGcoated particles is associated with localized tyrosine phosphorylation of a variety of
cytoplasmic proteins (22,23). This demonstrates the active recruitment of cytosolic src-family
kinases to the Fc receptor cytoplasmic domain through its ITAM immunoreceptor tyrosinebased inhibition motif, a consensus motif (24,25). Though it was previously believed that
phagocytosis was restricted to specialized cells, transfection of Fc receptor cDNAs into
normally non-phagocytic cells resulted in phagocytosis (26). Furthermore, fibroblasts can be
made to ingest IgG-coated particles as efficiently as macrophages by ensuring the appropriate
src-family kinases are co-expressed, either as soluble proteins or as cytoplasmic domain
7
fusions to the expressed Fc receptors (27). Thus in the presence of the appropriate receptor
coupled to its corresponding signaling molecule, phagocytic mechanisms can be elicited and
can be assumed common to many or all cell types. Phagocytosis can also be stimulated by
external factors, such as bacteria surface proteins, that permit bacterial attachment and
stimulation of one or more plasma membrane receptors, which stimulate membrane ruffling
and subsequent engulfment of the bound agent, even by epithelial cells (28-30). This kind of
phagocytosis differs somewhat from that mediated by Fc receptors in leucocytes, in that
pseudopod extension is not directed. Pathogenic bacteria and protozoa have also developed
mechanisms to evade by being destroyed by oxidative mechanisms or lysosomal digestion.
Some pathogens evade destruction by immune phagocytic cells after internalization. They can
escape from newly formed phagosomes by utilizing the acidic internal pH of the phagosome to
activate lytic enzymes that rupture the vacuole membrane (31). Others are capable of
modifying the phagocytic process, resulting in the formation of modified vacuoles incapable of
fusing with host cell endosomes and lysosomes (27). This mechanism helps avoid triggering
the cytotoxic respiratory burst which normally would destroy the pathogen. The respiratory
burst, also known as oxidative burst, describes the rapid release of reactive oxygen species
usually from mammalian immune cells, in response to encounters with bacteria or fungi.
1.2.2
Clathrin-mediated endocytosis
Clathrin-coated vesicles (CCVs) have been found to be associated with the uptake of
receptor-bound ligands and extracellular fluids in most animal cell types.
The primary role of clathrin-coated pits (refer to Figure 1.2) in ligand endocytosis was
demonstrated by the correlation between the selective localization of receptor-ligand at coated
pits and rapid ligand uptake (32). It was shown that mutant receptors defective in endocytosis
8
were also lacking in coated pit localization (32). Additional evidence has shown that, deletion
of the single gene for the 180-kDa clathrin heavy chain in Dictyostelium greatly suppressed
pinocytosis, as well as inhibiting the internalization of at least one potential plasma membrane
receptor (33). CCVs were first identified in brain tissue of pigs, where they were found to be
associated with the endocytosis of presynaptic membrane following synaptic vesicle exocytosis
(34). The clathrin assembly unit is called a triskelion, and consists of three 180-kDa heavy
clathrin heavy chains each complexed with a 30-35 kDa light chain (35). When triskelions
assemble into a coat, the legs interdigitate to create a lattice of open hexagonal and pentagonal
faces (35). Even though isolated triskelions can self-assemble into empty cages in vitro in the
absence of energy sources, formation of invaginated coated pits on the plasma membrane has
been found to require energy (36,37). This suggests that, however favorable the energetics for
clathrin self-assembly, it is not sufficient to mediate the entire process of endocytosis in cells.
Clathrin also recruits coated-vesicle cargo through interactions with intermediary proteins
known as adaptors (refer to Figure 1.3).
9
Figure 1.3 Mechanism of clathrin coat assembly and internalization during the endocytic
process.
A number of adaptors mediate processes such as such as coat assembly, excision, and
membrane curvature dring the transport process. Adpated with permission from (12).
10
The adaptors determine the selective inclusion of membrane anchored proteins into coated
vesicles, and further form an interface between the proximal, cargo-bearing, membrane bilayer
and the outer clathrin coat (38-40). Adaptors are known to play a critical role in the attachment
of clathrin to membranes (41-44). Some adaptors are the first to be recruited to membranes in
order to provide the binding site of clathrin coat assembly. Others have been conceived to
induce membrane curvature thereby facilitating clathrin-mediated formation of invaginated
coated pits (1,45).
Adaptors are also known to be involved in recruiting membrane proteins that selectively
localize to clathrin-coated regions (46). Known adaptors involved in this process include
Adaptor ptotein complex 1 and 2 (AP1/2), Eps15, espin, Fer/Cip4 homology domain only
proteins 1 and 2(FCHo1/2) and, and intersectin (47-51). Three central participants known to be
involved in initiation of CCVs include AP2, Phosphatidyl inositide-4,5,bisphosphate (PI4,5,P2), and clathrin (52). The essential role played by PI-4,5-P2 was demonstrated by
experiments that showed the prevention of new coated pits formation from plasma membranes
after acute treatment of cells with inhibitors of PI-4,5-P2 (52). Other experiments have shown
the absence of coated pits in cells depleted of PI-4,5-P2 (53,54).
While AP2 is mainly localized to plasma membrane coated pits, AP1 complexes are
largely restricted to clathrin-coated buds of the TGN, responsible for interactions with proteins
in the TGN(trans golgi network) that exit the Golgi complex via CCVs. AP-2 recruits clathrin
to membranes by binding weakly but specifically to lipid bilayers that contain PI-4,5-P2 as
clathrin has no direct membrane contacts. A membrane-associated AP-2 following a clathrin
triskelion binding reaction brings with it an additional AP-2, which can associate with a second
PI-4,5,-P2 in the membrane. The three-component cluster (one triskelion and two AP2
complexes) then shows a much longer membrane residence time (52). Coated pit formation
11
proceeds by the sequential addition of clathrin triskellions and adaptors, generating a sharply
curved invagination. Adaptor-mediated interactions with membrane-bound proteins and lipids
deform the underlying membrane. FCHo1/2 are part of the complex that stabilizes the nascent
coated pit and facilitates continued growth (52).
Dynamin mediates scission when the deformation has created a suitably narrow neck, and
auxilin, which arrives immediately following scission, recruits Hsc70 to direct uncoating
(55,56). A number of accessory proteins associate with coated pits at specific stages of
assembly and disassembly but their functions are less certain. Adaptor complexes also work
together with accessory membrane proteins that function in the actual targeting of the CCVs to
endosomes. Some of these proteins have been extensively studied, and include members of the
v- or t-SNARE families involved in vesicle docking and/or fusion (57). Cellubrevin, a vSNARE identified in the endocytic pathway, does not function at the level of CCV fusion with
EEs but rather at a later step in recycling (58,59).
The cytoplasmic domains of several receptors, as well as other membrane proteins that
selectively accumulate at plasma membrane coated pits, contain specific sequence information
that facilitates coated pit localization. Though these signals are somewhat degenerate, certain
motifs are strongly represented in many of these localized proteins. Coated pit localization
signals usually involve tyrosine residues placed in a context of one or more amino acids with
large hydrophobic side chains (60). There is another class of signals characterized by leucine
residues as its most critical feature, with di-leucine based motifs being a common occurrence
(61,62). It can therefore be inferred that both tyrosine and di-leucine motifs clearly specify
coat localization. Coated pit localization signals are not restricted to plasma membrane
receptors alone, but have also been shown to reside on membrane proteins found in
intracellular membranes. Most notable are the major membrane glycoproteins of the LEs and
12
lysosomes, lysosomal glycoproteins (lgps), that have short cytoplasmic tails containing a
conserved glycine-tyrosine sequence followed by a hydrophobic amino acid two residues
toward their COOH-termini (63). In a large number of cells, lgps reach their destination by
transport directly from the TGN to the endocytic pathway via CCVs through recognition by
AP1 and AP2 adaptor complexes (63). The glycine residues play an important role in
specifying interactions with AP-1 and AP-2 as alteration of the conserved glycine residues
reduces sorting of newly synthesized Igp/lamps. These processes indicate that direct and
specific protein-protein interactions drive receptor localization at clathrin-coated pits and by
extension, in other sorting events.
1.2.3
Clathrin-independent endocytosis
Other mechanisms of endocytosis are known to be independent of clathrin (see Figure 1.2)
(64,65). This has been demonstrated by experiments where CCV formation was inhibited by
genetics or specific chemical reactions. It has been observed that, under normal conditions,
some plant or bacteria toxins are internalized in structures devoid of clathrin coats, and some of
these uncoated vesicles deliver their contents to endosomes and lysosomes (66). Also despite
various treatments that effectively blocked the proper assembly of clathrin coats, cells still
showed a certain degree of endocytosis of extracellular fluid (67).
Direct genetic evidence of clathrin-independent endocytosis first came from experiments
in the yeast specie, Saccharomyces cerevisae. Several experiments convincingly demonstrated
that yeast exhibit a process reminiscent of endocytosis in animal cells with the isolation and
characterization of yeast mutants with conditional defects in α-factor uptake (68-72). It was
shown that, as in eukaryotic systems, receptor bound α-factor is internalized, appears in a
population of non-lysosomal vesicles (endosomes), and is finally delivered and degraded in the
13
vacuole (lysosomes). Complete deletion of the gene for clathrin heavy chain was found to
decrease but not to block the uptake of receptor-bound α-factor or the marker of fluid phase
endocytosis, Lucifer yellow (68). Also, the intracellular steps of the pathway involve ras-like
GTPases that are directly homologous to the rab proteins that control fusion activities of early
and LEs in mammalian cells (73).
Experiments involving mammalian cells have also demonstrated that elimination of
clathrin function does not always block endocytosis. Dynamin, a GTPase known from genetic,
morphological, and biochemical evidence is known to provide a critical accessory function in
clathrin-dependent endocytosis (74). It is proposed to provide the mechanochemical force to
accomplish the final step in the budding process by complexing around the necks of
invaginated coated pits (75). Mutation of dynamin in mammalian cells has been shown to
inhibit clathrin-mediated endocytosis (76). This mutation however did not totally stop the
endocytic process, but rather resulted in the rapid reduction of both receptor-mediated and fluid
phase endocytosis with fluid uptake restored to normal within 30-60 minutes. These results
expand on the hypothesis that, animal cells have a clathrin-independent pathway of endocytosis
even though the exact mechanism is unknown (76,77).
There have also been studies on caveolae, another plasma membrane pit involved in
endocytosis independent of clathrin coats. Caveolae were initially studied in endothelial cells
and adipocytes. They were recognizable by their characteristic striations, which were thought
to be formed by an accumulation of the integral membrane protein caveolin (78-80). In the
endothelium, caveolae are associated with endocytic transport of solutes across the cell, not
necessarily via the route of endosomes to lysosomes (81). They are also known to be associated
with folate uptake through budding and immediate calveolae, a process referred to as
potocytosis (82). That, however, does not resolve the role played by caveolae in clathrin-
14
independent endocytosis as concrete evidence is lacking on whether it is an essential
component of the trafficking machinery in many or most mammalian cells.
1.3
ENDOCYTOSIS, CELL POLARITY AND TUMOR PROMOTION
1.3.1 Cell polarity
A large number of cells have mechanisms that enable them to detect extracellular gradients
and move toward or away from higher concentrations. There are different types of stimuli that
cells respond to, which have been discovered through experimental testing. Among those
which have been studied by a number of in vitro cell biology, biochemistry, and genetic
techniques are chemotaxis, haptotaxis and durotaxis. Chemotaxis describes the process by
which cells respond to extracellular chemical gradients, and may alter physiological states
depending on specific chemical signals and gradients. In durotaxis and haptotaxis, cells
respond to rather ill-defined mechanochemical gradients but clearly these effect changes in
physiology based on the physical substrates on which they are plated. Haptotactic gradients
are produced by vapor deposition of inert metal atoms onto solid substrates such as glass
coverslips. It was demonstrated that on a substrate sputtered with a heavy metal, the
deposition of serum proteins was dependent on the type of metal used for coating (83). This
differential deposition produces a more attractive substrate where there is a dense deposition of
metal atoms, and therefore more attractive to cultured cells. Durotaxis is associated directly
with the mechanical softness or hardness, measured by Young’s modulus, of the gradient on
which cells are cultured. Although it is thought that cultured cells respond to mechanical cues,
through mechanosensitive channels that relay signals (84), it can also be argued that protein
deposition on the substrate is responsive to these same mechanical properties. Although the
external signals differ in nature, they all contribute to similar intrinsic chemical changes in cells
that produce specific reactions. These intrinsic chemical changes may contribute to alterations
15
in differentiation, proliferation, migration and other developmental processes. During
embryogenesis, polarity is important for individual and group cell migrations events, organ
formation, and wiring of the nervous system (85). Cell polarity continues to play important
roles in the adult, and is particularly crucial to processes such as immune cell trafficking during
inflammation, regenerative processes such as wound healing, and maintenance of tissue
architecture.
Cell polarity mechanisms, mainly chemotaxis, have been thoroughly studied in
Dictyostelium, neutrophils, and a number of transformed mammalian cells. In these cells, it has
been postulated that chemoattractants activate G-protein coupled receptors (GPCRs)(refer to
Figure 1.4) , resulting in localized accumulation of signaling molecules, such as
phosphatidylinositol 3,4,5 trisphosphate (PIP3), toward the high side of the gradient (85). This
accumulation of PIP3 leads to extension at the leading edge of cells, which is thought to be
driven by localized Rac-mediated actin polymerization. The lipid phosphatase activity of
Phosohatase and tensin homolog protein (PTEN) mediates localization of PIP3 to the specific
sides of the cell(86). PTEN is a cytosolic protein recruited to the membrane via several
membrane anchored proteins including Par3, microtubule-associated serine/threonine kinase
(MAST), membrane associated guanylate kinase (MAGI), syntrophin-associated
serine/threonine kinase (SAST), etc. In primordial germ cells (PGCs) however, even though
polarity is mediated by GPCRs, cells maintain uniform PIP3 levels throughout the membrane
and migrate by extending actin-free protrusions (87,88). These ‘non-actin’ protrusions may be
generated by myosin-based contractions, which mediates migration at the lagging edge of
neutrophils and Dictyostelium.
The combination of forces at the leading and lagging edges, together with the balance of
16
extrinsic attractants, control rapid cellular movement. This can be visualized as a push-pull
mechanism where a ‘push’ or a ‘pull’ is dependent on both intrinsic and extrinsic signals.
Figure 1.4 Illustration of G-protein coupled regulation of cellular activities.
Adapted with permission from (12).
Receptor tyrosine kinases (RTKs) have also been found be involved in polarity through
PIP3 accumulation and Rac-mediated actin polymerization at the leading edge (89-91).
17
Fibroblasts and breast carcinoma cells can be stimulated with growth factors that mediate
polarization by binding to RTKs. Actin-mediated events of fibroblasts and carcinoma cells are
coordinated with myosin-based contractions at the lagging edge, which is regulated by Rho and
calcium signaling. These combined regulatory factors contribute to a migratory response that
occurs more slowly than that of amoeboid cells. In spite of the differences that exist in the
migratory behaviors and signaling components of different cell types, the overall pathways that
regulate chemical attraction are similar. Thus, the maintenance of spatial symmetry is an
evolutionary conserved property from yeast to humans (85).
1.3.2
Mammalian cell polarity regulation
Mammalian cells are all involved in some form of directional sensing and polarity. Motile
cells on a suitable substrate, in the absence of a stimulus, can crawl or they may sit still. If a
chemoattractant is present, they move in a random way called chemokinesis. In chemotaxis,
this motility is biased toward or away from a chemoattractant or chemorepellent respectively.
The attraction or repulsion is controlled by molecular mechanisms that are responsive to the
environmental cues. The molecular mechanisms that read and interpret the gradient are referred
to as directional sensing and form an integral component of the cell’s internal compass. It is
however essential to note that, motility and directional sensing are separable, since molecules
within immobilized cells can move toward an external stimulus and mediate changes within the
cell without necessarily altering cell positions on the substrates.
Cell polarity is normally associated with cell-surface protrusions that are essential for
motility, chemotaxis and haptotaxis. These pseudopodia-like protrusions are classified by
differences in size and shape, together with other physiological characteristics. Different
protrusions have been named differently by different investigators because of the difficulty in
18
developing criteria that can be used as a basis for classifying them. The problem of
classification has however been addressed using a method based on 3D imaging combined with
quantitative data (92). The protrusion classes are summarized in Table 1.1. This method of
sampling shape geometrics has been used to generate values for several variables, including: 1)
measures of contour length and curvature, 2) relationship of contour to derived model figures,
3) dimensions of each protrusion modeled as a triangle, and 4) measures of the areas covered
by protrusions. The raw variables are then used to extract latent factors, which are theoretical
variables corresponding to cell features (refer to Figure 1.5). This is a robust method for
classifying protrusions in cultured cells.
Well-studied protrusions include filopodia, lamellipodia and nascent neurites formed by
cultured cells in different conditions. Lamellipodia are broad protrusions, typically found at
the leading edge of the cell (93). They are typically extended in the direction of travel during
locomotion. During the process of wound healing, retracted cells at the wound edge extend
filopodia and lamellipodia into the open space.
These leading egde cells have been shown to develop a distinct polarized morphology, with
lamellipodia and membrane ruffling localized only at the leading edge and not at the sides or
the rear edge of cells (94). Neurites are often defined as protrusions whose lengths exceed
twice the cell diameter. In developing nervous systems, neurons first sprout budding structures
called neurites, which eventually develop into axons. These nascent neurites arise from sites at
the neuronal perimeter that are intitially marked by filopodia. Filopodia are small membrane
protrusions found in numerous cell types in culture. Several studies have been focused on
filopodia because of their proposed role as sensory appendages in cells.
19
Table 1.1 Relations of factor values for protrusions to morphology of the cell edge
Classification
Value increases with increases in
Factor #4
Prevalence of filopodia
Factor #5
Centripetal mass distribution
Factor #7
Prevalence of nascent neurites
Factor #16
Features more massive than filopodia but
pointed
Table taken from reference (93), used by permission of Elsevier
Figure 1.5 Illustration of edge features as obtained using 3D imaging and quantitation.
Data shows contours from cells with a high representation of one edge feature but low
representation of others. A) High factor #4, B) high factor #5, C) high factor #7, and table
showing factor values for the contours pictured. It should be noted that variables used to
construct factor #4 values are entirely confined to the cell edge, and its values are based on
the outline of the cell edge. Values of factors #5 and #7 are based in part on information
20
from higher portions of the cell, but the edge contour supplies enough information to
estimate these values. Illustration adapted with permission from (93).
Filopodia, like microvilli, consist of parallel actin filaments which generate these
protrusions through interactions with a group of molecules at the plasma membrane. Actin
filaments have intrinsic polarity, with one end favoring subunit addition (‘barbed’ end) and the
opposite (‘pointed’ end) favoring subunit subtraction. The actin filaments are organized in
parallel bundles with their barbed ends facing toward the plasma membrane. The fundamental
process of seeding filament growth on the membrane is mediated by a variety of regulatory
factors including the Rho GTPase, Cdc42. Cdc42 is known to activate Wiskott - Aldrich
syndrome protein (WASP), which mediates filament assembly through interactions with
Arp2/3 (95). Arp 2/3, a heterodimeric complex of Arp2 and Arp 3, when activated, forms a
structure resembling the triplet of actin monomers involved in the seeding of actin monomers.
Also, a formin at the membrane facilitates fast addition of subunits, working with Ena/VASP.
Filament bundling in filopodia is also mediated by a number of cross-linking proteins including
espin, fascin, and plastin, which provide stability (96). Fascin, a 58 kDa monomeric protein, is
known to play an important role in actin bundling during filopodia formation. The
arrangement of actin subunits in filaments may provide a suitable surface area for the active
transport of cargo to the tips of these slender protrusions. Active transport is carried out by
motor proteins which act in concert with other stabilizing proteins. Filopodia tips are known to
contain a number of proteins, including integrin which is an essential component of focal
adhesions (96). Focal adhesions serve as signaling platforms that mediate communication
between cells and their microenvironments. The classical view has been that cells use
filopodia to probe the environment for cues and they function in the leading edge as pioneers
21
(97). This may explain the importance of filopodia in physiological processes such as wound
healing, angiogenesis, chemotaxis, morphogenesis and adhesion (93). There is a lot of
evidence to suggest that the filopodia pointing is a directional signal, and it maintains the
persistence of locomotion in motile cells (93,96-101).
Integrins, which are essential components of focal adhesions, are heterodimeric cell
surface adhesion receptors, which link the cellular cytoskeleton and signaling machinery to
molecules of the extacellular matrix (ECM). Members of the integrin family act as receptors
for ECM proteins such as fibronectin, laminin, or collagen. The integrin family consists of
24 heterodimers formed from non-covalently associated α- and β-subunits. Each subunit has
a large extracellular domain, a single membrane spanning region, and a short cytoplasmic
domain, with the exception of the β4 subunit, which has a long cytoplasmic domain. The
integrin family of vertebrates includes at least 18 distinct α- and 8 β-subunits. Integrins are
known to be expressed at high levels on the surface of all anchorage-dependent cell types
(102). Many signaling molecules activated by integrins are implicated in the regulation of cell
motility and survival. In regular focal adhesions, integrins are associated with a number of
adaptor molecules including vinculin, talin, paxillin, FAK, p130Cas, and others through short
cytoplasmic domains. Both α- and β-subunits of integrins contain conserved amino acid motifs
involved in specific adaptor binding. The β-subunit contains two conserved NPxY-motifs
known to bind a large number of proteins, many of which are integral in focal adhesions and
signaling (102).
One such essential interaction is with myosin-X, a motor protein involved in the regulation
of filopodia. There have been several proposed mechanisms involved in transmission of signals
22
from cell surface to intracellular signaling adaptors to effect changes. It is however unresolved
how signals are transmitted through filopodia-integrin interactions.
1.3.3
Filopodia as mediators of cell polarity
Filopodia have generally been described as ‘sensory protrusions’ or ‘antennae’ used by
cells to probe their microenvironment (93,100). Filopodia mediate guidance at the tips of
developing neurites in growth cones capable of distinguishing different chemical cues in both
invertebrate and vertebrate nervous systems. They have also been found to be concentrated
at the growing tips of endothelium in developing blood vessels of adult animals, and at the
dorsal closure in embryonic development (103). Neuronal growth cones are motile actin-rich
and microtubule-rich structures at the end of neurites that guide axons and dendrites to their
proper targets. The correct reading and integration of the environmental cues is essential for
precisely connecting and assembling the different parts of the peripheral and central nervous
systems. Guidance is not only controlled by external cues, but through a combination of
both intrinsic and extrinsic cues. Growth cone and filopodia guidance to their targets is
mediated through chemorepulsion, chemoattraction, contact-dependent repulsion, and
contact-dependent attraction.
23
Figure 1.6 Illustration of molecules and their roles in filopodia formation.
Adpated with permission from (93)
Filopodia contain a variety of receptors involved in communication with a number of
signaling and extracellular matrix molecules. Integrins and cadherins, which are important cell
adhesion molecules, are often found at the tips of filopodia (104,105). The integrins, have been
found in the activated state, and thus primed to probe the extracellular matrix for signals (104).
Integrins subsequently recruit other focal adhesion complex components, as mentioned above,
leading to the formation of mature focal adhesion plaques. The focal adhesions and associated
F-actin structures finally reorganize resulting in the formation of focal adhesion anchored stress
fibers (105).
24
In a study on fibroblasts on micro patterned surface, it was revealed that inhibition of filopodia
formation by the expression of a dominant-negative form of the small GTPase Cdc42 impaired
cell spreading (106).
Filopodia sensing is associated with pathogen tracking and engulfing by macrophages.
During this process, several filopodia after tracking and identifying a pathogen, bind to it and
then retract toward the macrophage cell body (98). Also inhibition of filopodia by formation
by myosin VII in Dictyostelium discoideum caused a decrease in phagocytosis rates as well as
inhibition of migration (107). Filopodia have additional roles during the formation of adherens
junction between epithelial cells, as well as a role during wound healing, dorsal closure and
epithelial-sheet sealing in Drosophila melanogaster and Caenorhabditis elegans embryos
(108,109). In all these processes, protruding filopodia from opposite cells probe the
microenvironment and help sheets of cells to align and adhere together in a precise manner.
Errors of misalignment of cells and tissues result in a variety of developmental problems in
organisms.
1.3.4
Endocytosis and cell polarity regulation
A number of studies have identified connections between endocytosis and cell polarity
(103). These studies related the balance of vesicle trafficking on opposite sides of cells to
polarity and migration. Neurons during development go through a series of morphological
changes that require alterations in the surface area by means of exocytosis and endocytosis
(110-113). This has been observed to occur through integrin signaling which controls the
cytoskeletal machinery (110). These studies have Tojima and coworkers to the hypothesis that
axon guidance involves asymmetric vesicle trafficking through differentially regulating
endocytosis and exocytosis across the growth cone.
25
Regulated membrane trafficking could eventually mediate processes such as receptormediated signal transduction, and downstream mechanical processes of cell reorientation. The
growth cone can adjust its sensitivity to guidance cues by a process of adaptation, which
depends on endocytosis-mediated desensitization and protein synthesis-dependent
resensitization which is aided by exocytic traffic (114,115). This coordinated vesicle trafficking
mechanism has been proposed by a number of groups as essential for cell polarity and
migration. Evidence of the connections between cell traffic and polarity has not only been
found in neurons but also in eukaryotic cells of other tissues.
A study at California Institute of Technology found a high expression of dynamin, a
protein that mediates endocytosis, in human placenta as well as neurons (116). This study
further showed that, the speed of endocytic traffic is dependent on dynamin expression, and this
may reflect its higher expression in cells where the speed of polarization is an absolutely
essential property for function. Another study on mice fibroblasts also reported that, focal
adhesion disassembly is regulated by microtubules through an unknown mechanism that
involves dynamin (117). This study, by means of fluorescent microscopy, showed that clathrin
rapidly accumulated on focal adhesions during microtubule-stimulated disassembly and
departed from focal adhesions with integrin upon their disassembly. Interfering with clathrin
function prevented microtubule-induced focal adhesion disassembly.
Delivery of new components to the membrane appears to be required for steering the cell.
Silencing of VAMP2, a SNARE protein that mediates vesicle fusion at the plasma membrane,
has been known to cause a significant reduction in cell surface integrin expression, with a
resulting disruption of cell adhesion and chemotactic migration (118).
The tumor promoter, phorbol 12-myristate 13-acetate (PMA), is also known to affect the
pinocytosis in cultured cells (119,120).
26
PMA has been reported to stimulate membrane ruffling activity in cultured cells within a few
hours of exposure (121,122). Ruffling involves the assembly and reorganization of the actin
cytoskeleton along the cell’s periphery. This reaction occurs through activation of the enzyme
protein kinase C (PKC), which adds a charged constituent to amino acid residues of various
protein substrates. This cascade of reactions elicited by PMA treatment further leads to
transient loss of filopodia from the surface of cells accompanied by an increase in endocytosis
(122). It has however been difficult to trace the causes of tumor promotion to any of the
physiological processes regulated by PKC. PKC comprises a family of kinases which, when
activated by diacylglycerol or its surrogate, PMA, phosphorylate a great number of substrates.
The above summary of PMA-mediated effects suggests limitations in the biochemical
approach to such effects, due in part to the difficulty of dissecting dynamic changes that occurs
while altering the balance of endocytic and exocytic activity.
Another way of approaching the problem is to design tests for the physiological functions
affected by PMA treatment. It is possible that identifying physiological gateways or
bottlenecks may lead to exclusion of certain mechanisms, e.g. the endocytic/exocytic balance,
as explanations. To this end, the Heckman group showed an increased endocytic uptake of
horseradish peroxidase (HRP) in PMA-treated cells as compared to the controls (120,122,123).
They applied a model first described by Besterman et al. (124) and Steinman et al. (125) to
classify this uptake. Two functional compartments were described to define the endocytic
trafficking of fluid-phase markers in terms of functional compartments (Figure 1.7).
27
Figure 1.7 Experimental model of the functional endocytic compartments.
Compartment I represents the early endosomal stages where rates ã and represent initial
uptake and recycling (efflux) respectively. Compartment II represents the late
endosomal/lysosomal stages where there is little to no exchange with compartment I. Rate
represents maturation into compartment II while rate
represents turnover of the contents of
compartment II. M represents total volume of the cell. Adapted with permission from (119).
Compartment I describes the early endosomal stage where substances trafficked into cells
by endocytosis are sorted, and either processed for efflux back into the ECF through recycling
endosomes or trafficked further to the late endosomal/lysosomal stage. Compartment II
describes the late endosomal/lysosomal stage, marking the final destination of traffic, where
there is insignificant efflux back to compartment I. Kinetic measurements in PMA-treated cells
showed not only an increased endocytosis but also a constant accumulation of marker in
compartment II consistent with the rate of endocytosis (119). Limitations of the previous work
did not allow for the studies of the dynamics of compartment I during PMA treatment.
Also the variability in accumulated HRP among different cells contributed a problem in the
28
analysis of results. With the evidence presented, using fluorescent microscopy and stringent
image analysis, snapshots in time of both compartments were obtained and classified using
pixel-by-pixel analysis of endocytic marker accumulation. It was hypothesized that, the balance
between endocytosis and exocytosis in mammalian cells is associated with directional sensing.
This hypothesis has been explored by Tojima and group concerning directional sensing in the
nervous system during the development of neurons (103). This is however not well
characterized in other mammalian cells such as epithelial cells. The difficulty in studying
polarity in a number of mammalian cells is compounded by their lack of structural changes
during polarization. Changes in endocytic traffic patterns inside cells, by fluorescent
visualization, could help characterize another method of studying intrinsic polarity.
Furthermore, transient disappearance of filopodia has been observed in cells treated with PMA
(122). Our prediction, in line with the Tojima hypothesis, was that transient disappearenace of
filopodia following PMA treatment is accompanied by increased endocytosis. Another
prediction was that, cells plated on a gradient substrate will increase endocytosis on the nonattractive side with a resulting increased fluorescent marker accumulation (Figure 1.8). Thus
cells the balance between influx and will efflux will favor influx on the non-attractive side and
efflux on the attractive side.
29
Figure 1.8 Experimental model of a motile cell on a haptotactic gradient.
Some mammalian cells tend to increase filopodia formation towards the side of the gradient
substrate with increased metal deposition. The attraction is as a result of the increased
concentration of serum proteins (from growth medium) supplanted on the metal atoms. The
method for preparing the gradient substrate is explained in detail in the materials and methods
section.
1.4 EXPERIMENTAL MODEL
Studies have shown that, retention of molecules following endocytic uptake is dependent
on time as well as molecular weight (126).
High molecular weight substances have a higher retention rate, during sorting in the early
endosome and trans-Golgi network, than their lower molecular weight counterparts. By using
30
different fluorescent markers with varying molecular weights at different exposure times,
snapshots were obtained of the same cells containing different fluorescent markers. With
previous knowledge of the influx and efflux dynamics of IAR20 cells, we aim to develop a
better model of endocytic trafficking in these cells.
(A)
31
(B)
Figure 1.9 Stair step model of the endocytic traffic through functional compartments I and II.
The stair step model shown in (B) describes our hypothesis of endocytic traffic through
compartments I and II of our previously described model (A). Blue (Blue-dextran), red (CY3dextran), and green (FITC-dextran) describe the emission colors of the different fluorescent
endocytic markers used in the experiment. The bottom of the stair step (compartment I) is
characterized by either recycling back to the ECF or traffic through the transitional
compartment to compartment II. At the peak (compartment II) however, molecules tend to
accumulate in the lysosomes where there is little to no recycling to compartment I or the ECF.
These reactions are dependent on time and, to a small extent, molecular weight of the substance
being transported.
32
CHAPTER 2. MATERIALS AND METHODS
2.1 CELL CULTURE AND CHEMICALS
The IAR20 cell line was derived from liver cells of inbred BD-VI rats and was maintained
at 37oC and 5%CO2 in 60-mm tissue culture dishes (BD, Franklin Lakes, NJ) with Williams
Medium E (Life Technologies, Grand Island, NY) containing 10% fetal bovine serum
(Hyclone, Logan, UT). The 1000W cell line was generated by treatment of a heterotopic
tracheal transplant from a Fisher rat with 7, 12-dimethylbenz (a) anthracene. The line was
maintained under routine conditions in 60-mm tissue culture dishes with Waymouth's medium
(Life Technologies, Grand Island, NY) containing 10% fetal bovine serum supplemented with
0.1 μg/ml insulin and 0.1 μg/ml hydrocortisone.
A calcium- and magnesium-free Hanks balanced salt solution (CMF-HBSS) with trypsin
and EDTA was prepared for removing confluent/subconfluent cells from the culture dishes.
For the preparation of CMF-HBSS EDTA solution, 50 ml of 10X CMF-HBSS (Life
Technologies, Grand Island, NY) and 0.875 g Na-EDTA were dissolved in 100 ml of deionized
water. The pH indicator, 10 mg phenol red (Matheson, Cincinnati, OH), and 175 mg NaHCO3
(Matheson, Norwood, OH) were dissolved in deionized water to a final volume of 400 ml and
combined with the CMF-HBSS EDTA solution. To adjust the pH, 1N NaOH was added until
the liquid turned a cherry red color. Deionized water then was added to a final volume of
437.5 ml, and the solution was autoclaved. The trypsin working solution was prepared by
adding 20 ml of 0.5% trypsin-EDTA (Life Technologies, Grand Island, NY) to 80 ml of CMFHBSS EDTA solution. It was tested for sterility by addition of 50% sterile Williams medium
E with 10% fetal bovine serum, followed by incubated at 37oC, 5% CO2 for 48 hours. The dish
was observed microscopically for contaminants.
33
Cytoskeletal buffer was prepared at a pH of 6.2 using 150 mM potassium chloride, 5 nM
magnesium chloride, 5 mM EGTA, and 5 mM 4-morpholineethanesulfonic acid (MES)
sodium salt using deionized water as diluent. CY3-Dextran (70,000 MW) was purchased from
Nanocs, Inc (New York, NY). Lysosensor yellow/blue-Dextran (10,000 MW) was purchased
from Life Technologies (Grand Island, NY). Goat anti-mouse IgG conjugated with fluorescein
isothiocyanate (FITC) was purchased from Sigma-Aldrich (St. Louis, MO).
Phorbol 12-myristate 13 acetate (PMA) was purchased from Sigma-Aldrich (St. Louis, MO).
The stock solution was stored at -70° C until added to cultured cells. N-Ethyl-N′-(3dimethylaminopropyl) carbodiimide hydrochloride (EDAC) was purchased from SigmaAldrich and made up at 4% by dissolving 12 g of powder in 300 ml of cytoskeletal buffer at a
pH of 7.2.
PMA was added directly to the tissue culture medium as a 1:500 aliquot (2 nM final
concentration). The goat anti-mouse FITC marker was delivered for 22 minutes in the absence
of PMA for both experimental and control samples. Subsequent treatment times for PMA were
8 min with CY3-Dextran and 2 minutes with Lysosensor-Dextran in the experimental samples.
The control samples received only the markers during this time.
2.2 SUBSTRATE PREPARATION
No.1.5 uncoated round glass coverslips of 25-mm diameter (Electron Microscopy
Sciences, Hatfield, PA) were cleaned for 1 hour with 1 M HCl followed by rinses in deionized
water and absolute ethanol. The cover glasses were sterilized 1 hour under UV light before
cells were put on them. Uncoated coverslips were used for the IAR20 cell culture experiment.
For attaching 1000W cells, the coverslips were coated with a platinum gradient in a high
vacuum coater (Denton, NJ) to produce a haptotactic gradient before cell culture.
34
Nine coverslips were mounted on a stationary table in the coater and a 2-cm length of platinum
wire (Electron Microscopy Sciences, Hatfield, PA) was evaporated at a resistive current of 12
amps for 5 min. The platinum was secured in a tungsten basket positioned at a distance of 4
cm and a height of 1.5 cm from the coverslips being coated. The wire was found to be
completely evaporated from the source, a tungsten basket, by 5 min. Coating was performed in
a vacuum of 2 x 10-5 torr.
2.3 PREPARATION OF IAR20 CELLS FOR IMAGING
Cells from the IAR20 line were grown to confluency under the same conditions as above
(see Cell Culture and Chemicals). Confluent cells were removed from the surface of the dish
using trypsin-EDTA, and the trypsin reaction was stopped using Williams medium E with 10%
fetal bovine serum. The cells were centrifuged at 2,000 rpm for 2 minutes and transferred into
dishes containing glass coverslips at a density of 200,000 cells per 25-mm dish. The cells were
incubated for 12 hours at 37oC and 5% CO2 to allow the cells to attach and spread on the
substrate before treatment with endocytic markers. The culture dishes were labelled as
experimental and control groups before treatment. All endocytic markers- 1 mg/ml GAMFITC, 1 mg/ml Dextran-Cy3, and 1 mg/ml Lysosensor, were made up in Williams medium E
with 10% fetal bovine serum and preincubated for 30 min at 37oC and 5% CO2, before treating
cultured cells. The GAM-FITC marker was used at a concentration of 1 mg/ml to label the
lysosomal compartment. GAM-FITC was delivered for 20 min in the absence of PMA for
both experimental and control samples. This medium was aspirated, followed by a quick rinse
with pre-incubated Williams medium E at 37oC to remove excess GAM-FITC and then
addition of 1 mg/ml of CY3-Dextran. Subsequent treatment times were 8 min with CY3Dextran and 2 min with Lysosensor-Dextran in the experimental samples.
35
2 nM PMA was added to the pre-incubated solutions of Dextran-CY3 and Lysosensor-Dextran
for the experimental treatments only. The CY3-Dextran solution was replaced with 1 mg/ml
Lysosensor-Dextran solution for a period of 2 min followed immediately by fixation in 3%
EDAC in cytoskeletal buffer. Samples were mounted in Immunofluor mountant (ICN
Pharmaceuticals). The coverglass edges were sealed with clear nail polish to prevent drying,
and the samples were stored at 4oC for 24 hours before imaging.
2.4 PREPARATION OF 1000W CELLS FOR IMAGING
The cells were grown to confluency in a 60-mm tissue culture dish. Confluent cells were
then removed from the surface of the surface of the dish using trypsin-EDTA, centrifuged at
3000 rpm for 2 minutes, and transferred to a 25-mm tissue culture dish containing glass
coverslips. The density was 200,000 cells per dish.
The trypsin reaction was stopped using Williams medium E with 10% fetal bovine serum. The
cells were incubated for 24 hours at 37oC and 5% CO2 to allow for attachement of cells to the
substrate before treatment with endocytic markers. The culture dishes were randomly separated
into experimental and control groups, and labelled as such before treatment. The endocytic
labelling markers- 1mg/ml GAM-FITC, was made up in Williams medium E with 10% fetal
bovine serum, and preincubated for 30 minutes at 37C and 5% Co2 before treating cultured
cells. The cultured cells were treated with endocytic marker for approximately 2 minutes, and
fixed immediately using 3% EDAC in cytoskeletal buffer for 15 minutes. Excess fluorescent
label was rinsed three times with PBS and the slides mounted on a glass slide using Immuno
fluor (ICN Pharmaceuticals) as mountant. The sample edges were sealed with clear nail polish
to prevent drying, and stored at 4 oC for 24 hours before imaging.
36
2.5 IMAGING
Images were acquired with a Zeiss Axiophot epifluorescence light microscope (Carl Zeiss,
Inc) using the Plan-Neofluar 100x/1.30 oil objective lens (Carl Zeiss, Inc). Digital images (12
bit) were obtained using a Micromax 1300 YHS CCD camera (Roper Scientific, Trenton, NJ)
with 1300 x 1030 imaging array, and spatial resolution of 6.7 x 6.7um pixels. Images were
acquired with exposure times of 3 seconds using a FluoArc mercury lamp (Carl Zeiss,Inc). A
Zeiss Axioline slider (Carl Zeiss, Inc) with three filter sets (Chroma, VT) was used to excite
FITC, Cy3, and Yellow/Blue at 495nm, 550nm, and 345nm respectively (Life
Technologies,NY) respectively. Fluorescent signals were collected at emission wavelengths of
519 nm, 570 nm, and 455 nm for FITC, Cy3, and Lysosensor Yellow/Blue respectively.
2.6 IMAGE PROCESSING
For the IAR20 cell analysis, the 3 channels (corresponding to GAM-FITC, Dextran-Cy3,
and Dextran-Lysosensor) of the fluorescent images were first corrected for microscope
misalignment and uneven illumination. Image alignment was performed by using Cell Profiler
(Broad Institute, Boston, MA) to create a mask on the misaligned areas, followed by manual
cropping in Metamorph (Molecular devices, Sunnyvale, CA). The uneven illumination was
corrected in Metamorph by performing an inclusive threshold to determine the upper and lower
threshold limit of the background.
The upper threshold limit of the background of each image was then subtracted from the
image. Since negative intensity values were not allowed, this procedure resulted in zero
background intensities. The images were then cropped to single cells for each channel, and
pixel-by-pixel intensity values were obtained from each cell using MatLab R2014a
(MathWorks, Natick, MA). These values were stored in Microsoft Excel worksheets.
37
For the 1000W cells, following background subtraction as above, each cell was divided into
two equal halves using the region selection tool. This was done to enable the separate analysis
of the left and right halves, which corresponded to the attractive and repulsive directions on the
haptotactic gradient.
The integrated intensity and integrated optical density (OD) were determined for each half
using the integrated morphometric analysis menu, and the data were stored in Microsoft Excel
spreadsheets for further analysis.
2.7 STATISTICAL ANALYSIS OF ENDOCYTIC ACTIVITY
For the IAR20 cells, the pixel-by pixel intensity data for each cell and endocytic marker
were compiled and computed using SAS/STAT (SAS Institute, Cary, NC). Because of their
low intensity values, the Lysosensor Yellow/Blue signal images were not used in the analysis.
Then, the corresponding pixels from the cell that contained a value of zero in both channels
(CY3/FITC) were eliminated from the analysis. Graphical representations of the data
computed in SAS/STAT were produced in Minitab 17 (Minitab, Inc, State College, PA). The
sum of the intensities (integrated intensity) for each cell and the corresponding histogram for
each channel was computed and displayed with MatLab R2014a. The image color maps for
each channel per cell were also obtained with MatLab R2014a. The ratio images were obtained
with Metamorph.
For the 1000W cells, the integrated intensity and integrated OD data for each half of the
cell were computed and plotted in Microsoft Excel software.
38
CHAPTER 3. RESULTS
3.1 KINETICS OF COMPARTMENT II MARKER TRAFFIC
Previous studies on endocytosis in several cell types have demonstrated that, internalized
materials that are sorted at the early endosomal stage are either recycled back to the membrane
and ECF, or trafficked into the late endosomal and lysosomal compartment. Trafficking is
time-dependent with an exponentially increased accumulation of endocytic marker in the
lysosomes with time. Previous studies by many other laboratories showed that uptake and
accumulation were enhanced in cells treated with PMA (Figure 1.7). Fluid flow rates ã and
represent influx and efflux of compartment I respectively, whereas rates
and
represent
influx and efflux of compartment II respectively. The amount of substances carried by rates ã,
,
,
are a, b, c, and d respectively. Filling of compartment II is dependent on rate
, and
the pool of endocytosed contents drawn on for compartment II depends on the balance between
rates ã and
. This balance between recycling and maturation into compartment II ultimately
decides the accumulation.
Attempts to determine the rates
and
in the treated cells, compared to controls, were
unsuccessful to date. As compartment I is loaded at a higher rate, the concentration of marker
in the cell is expected to be greater at all experimental times. The expectation would be
violated only if the volume of compartment I expanded to exactly compensate for the increased
uptake rate. Otherwise, the amount of marker trafficked to the extracellular fluid and to
compartment II is necessarily greater. Thus, the determination requires distinguishing whether
these enhanced rates of marker trafficking also depend on a change in either the rates
or
,
or both. Also, the variability in the cell populations assayed by biochemical means compounds
the difficulty in making this experimental determination (123).
39
Plots of the average values obtained from four instantaneous uptake experiments by
Christopher Runyeon suggested that the influx was remarkably linear over short periods of time
(119,120). PMA-treated samples already showed greater uptake at the shortest time that was
feasible to assay, however, and the standard deviations of the data values were large (Figure
3.1).
Figure 3.1 Relative amount of fluid-phase marker influx over time in PMA-treated cells
versus controls.
The marker, HRP, was added at a final concentration of 1 mg/ml to cells in replicate dishes,
which were then incubated for the indicated times. In control dishes, the solvent vehicle was
added simultaneously with HRP. In experimental dishes, PMA was added to a final
concentration of 2 nM. Each point on the curve represents the average amount of HRP in the
cells as determined by assaying the HRP enzymatic activity. Details of the experimental
methods can be found in reference (120). Error bars represent the standard deviation.
40
The linear time course shown is superimposed on the actual time course of accumulation in
Appendix A, in order to visualize the effect of trafficking into Compartment II.
In previous work with the HRP marker, we measured the efflux from the physiological
compartment I. The data, shown in appendix A (120) suggests that efflux is essentially
complete by 12 minutes. Similar conditions were chosen here, so that the predictions from the
original work could be tested experimentally. The time permitted for efflux in the current work
is designated by an arrow on the experiment with HRP flux (shown in Appendix A).
Quantification of accumulation by fluorescence microscopy and image analysis showed the
amount of FITC retained was greater after sequential influx and efflux in a few of the PMAtreated cells than controls. Whereas most of the cells showed integrated intensity between 5 x
106 and 1.75 x 107, a fraction of ~10% of the PMA-treated cells showed very high intensity
values. This increase was demonstrated in histograms of integrated intensity (summed gray
levels) per cell (Figure 3.2). It is important to note that integrated intensities of FITC reflect the
marker in compartment II, which was loaded in the absence of PMA. However, values of >2.6
x107 intensity were only found when PMA was present, as shown in Figure 3.2. This was an
unexpected result. Not only were both cell populations loaded with FITC marker under the
same conditions for 20 minutes, but the amounts effluxed in the presence of PMA were known
to be greater than in control untreated cells (see Appendix A). Although the number of cells
showing an increased integrated intensity was small, they did represent 4 out of 45 cells or
around 9% of the total PMA-treated cells. Trivial explanations for the difference were ruled
out, as explained in Figure 3.2.
41
(A)
Number of cells
Integrated intensity
(B)
Number of cells
Integrated intensity
Figure 3.2 Histograms of FITC integrated intensity (sum of gray levels) for two cell
populations. (A) Distribution of intensity values for 45 PMA-treated cells, (B) Distribution of
intensity values for 32 untreated cells. The average background intensity subtracted from the
42
images was 302 and 318 for the 45 PMA-treated and 32 untreated cells, respectively. This
represented a small increment but did allow higher integrated gray levels to be observed in the
PMA-treated cells. This increase was compensated for by the difference in cell size (pixels)
between the PMA-treated and control cells. The 45 PMA-treated and 32 untreated cells had an
average size of 46722 and 47993 pixels respectively.
Comparison of the gray level values on a pixel-by-pixel basis was performed for both cell
populations in the experiment to determine the range of pixel intensities across cell populations.
Control cells showed a greater number of dim pixels, i.e. those with values from zero to around
200, when compared to PMA-treated cells (Figure 3.3). This result could have arisen from
several effects. Although PMA is known to cause some cultured cells to round up (121,122),
this effect was ruled out with the results of average size of cells in each population using pixel
count. Information on the size of the respective cell populations was performed by counting
the pixels contained in each whole cell. After cropping, the PMA-treated cells were enclosed in
a smaller frame, but the difference was very slight (see Figure 3.2 legend). The data on the cell
sizes is shown in Appendix B.
43
(A)
Histogram of FITC
1.4
1.2
Percent
1.0
0.8
0.6
0.4
0.2
0.0
0
255
510
(B)
765
1020
FITC
1275
1530
1785
1275
1530
1785
Histogram of FITC
1.4
1.2
Percent
1.0
0.8
0.6
0.4
0.2
0.0
0
255
510
765
1020
FITC
44
(C)
Figure 3.3 Histogram showing the percentage distribution of FITC gray levels of pixels for
the control and PMA-treated cell populations. (A) and (B) show the percentage distribution
of FITC in the PMA-treated and control cells respectively. (C) shows the overlay of the
histograms and demonstrates the comparative distributions for treated (black) and control
(grey) cell populations.
PMA-treated cells, overlaid in gray, include many pixels with values above 1070.
Moreover, the pixels with ranges 535 to 1070 are somewhat overrepresented in cells from the
PMA-treated population. These data clearly show that the difference in marker retention is a
real finding and not a result of a trivial difference in the conditions used for acquiring or
processing the images. This however represents a slight difference between the treated and
45
control cells which was an expected finding, since they were both treated without PMA
during FITC incubation. The main aim of treating both cell populations at this stage without
PMA was to establish a standard for comparison with FITC. Though there was a slight
difference between them, it was not quite as pronounced as in the subsequent fluorescent dye
treatment.
3.2 MARKER FOR COMPARTMENT I AND TRAFFICKING INTO COMPARTMENT II
CY3 marker was delivered in order to represent the dynamic loading of compartment I and
transition of the marker to compartment II in experimental and control cells. These processes
can be visualized as going up the stair steps (as modeled in Figure 1.9). Quantification of
accumulation by fluorescence microscopy and image analysis showed an increase in CY3
accumulation in the PMA-treated cells as compared to the controls after an incubation period of
10 minutes (Figure 3.4). In previous experiments, Christopher Runyeon found that PMAtreated cells took up more HRP than controls. By 10 minutes, the treated and control cells
contained 0.577 and 0.297 ng/105 cells, respectively (120). The treated cells had 1.9x the
amount of HRP. This difference in CY3-dextran accumulation was reflected in the comparison
of histograms of integrated intensity (summed gray levels) per cell (Figure 3.4).
The histograms show that, above the level of 7.8 x 107, there were only 3 control cells as
compared to 7 PMA-treated cells. These represent 9.4% and 16% of the cells sampled in the
experimental population. Moreover, values >9.2 x107 intensity were only found in the treated
cells and the cells with the highest accumulation showed values of 1.5 x 108, as shown in
Figure 3.4 (A). The enhanced accumulation in treated cells certainly reflects an increase in rate
ã, as mentioned above. It is unknown whether there is an increase in rate
treatment.
with PMA
46
(A)
Number of cells
Integrated intensity
(B)
Number of cells
Integrated intensity
Figure 3.4 Histograms of CY3integrated intensity (sum of gray levels) for two cell
populations. A) Distribution of intensity values for 45 PMA-treated cells, B) Distribution of
intensity values for 32 untreated cells. The latter distribution shows only 3 cells in the
categories above the level 7.8 x 107.
Comparison of the pixel-by-pixel gray levels for both cell populations in the experiment
was undertaken for the CY3marker. The control cells showed a greater number of dim pixels
when compared to the PMA-treated cells (Figure 3.5).
47
A fraction of the pixels represented at values ~400 and below were missing in the experimental
cell population, which showed instead an increase in values of ~900 and greater. Moreover, at
all gray-level values above 1500, the percentage of pixels represented in the collective PMAtreated population exceeds that in the control cells. A comparison of the histograms shows that
PMA-treated cells have many pixels with values above 2800 (see Figure 3.5 (C)). Conversely,
values between 1000 and 1350 are more represented in untreated cells (shown in white),
suggesting that this intensity range represents compartments with less marker. From the
results above it can be concluded that, the treated cells contain a greater number of areas
contining concentrated CY3 vesicles which represent the transition between compartment I
and II. These results suggest that, the increase in pinocytic accumulation caused by PMA
treatment may occur through increased influx of materials into both compartment I and II.
(A)
48
(B)
(C)
Figure 3.5 Histograms showing the distribution of CY3 gray levels of pixels for treated (A)
and control cell populations (B). (C) Overlay of the histograms showing comparative
distributions for treated (black) and control (white) cell populations.
49
3.3 PMA-DEPENDENT DIFFERENCES IN ENDOCYTIC MARKER INFLUX AND
COMPARTMENTALIZATION
Quantification of the FITC and CY3markers accumulated in the cells showed greater
amounts of CY3 than FITC in both PMA-treated and control cells. This was consistent with
expectations (see Introduction). The relationship was reflected in the histogram of the
difference (CY3-FITC) between gray level intensities of markers in each pixel. The data
mainly represent areas inside the cells, because nearly all of the pixels in the background were
converted to zero by the background subtraction procedure. The difference in CY3 over FITC
is represented by the areas greater than zero on the horizontal axis in Figure 3.6.
The vertical axis shows the percentage of pixels by each difference value. As predicted, the
intensity of CY3 is greater in PMA-treated cells. The difference in CY3 over FITC in these
cells is represented by a range 0 to 2850 whereas the difference in the control cells ranges from
0 to 2350. In a few pixels the FITC intensity is greater than CY3 (less than zero on the
horizontal axis), and there is still a greater range (0 to -480) in the PMA-treated cells as
compared to the controls (0 to -300). Comparing this distribution with the integrated intensity
for the whole cells (Figure 3.3), it appears that the intensity in certain PMA-treated cells is
higher than the control. In some locations, it exceeds the intensity of the CY3 marker. It can be
concluded from the above results that, an number of areas of the PMA-treatment causes an
increased traffic into both functional compartment I and II.
50
(A)
(B)
51
(C)
Figure 3.6 Histograms of difference between CY3 and FITC pixel-by-pixel gray level
intensities for treated (A) and control cell populations (B). (C) Overlay of the histograms
showing comparative distributions for treated (black) and control (white) cell populations.
The possibility that areas with extremely high and low intensity of markers represent
structural compartments was further investigated by imaging techniques. Since markers are
carried inside small vesicles that resemble bubbles in the cytoplasm, their localization might
reveal features related to those described in the experimental model (see Figure 1.5). The
intensity of each marker was represented in color map images, called heat maps, to illustrate
the compartmentalization of the markers. FITC is a label of functional compartment II. In
contrast, CY3 marker represents influx into portions of Compartment I and transition between
compartment I and II. As expected based on the fact that CY3 was a more intense marker, the
intensity color maps showed a difference in the areas occupied by CY3 and FITC markers
inside the cells. The comparison of the FITC and CY3 distribution for a treated cell is imaged
in Figure 3.7.
52
Whereas the FITC marker was typically localized on one side of the cell, the CY3 marker
appeared to be more distributed through the majority of the cell area. Of the PMA-treated
cells, 93% showed an eccentric localization of the FITC marker while this localization pattern
was represented in 97% of the control cell population. The eccentric localization describes the
distribution of marker in the poles of the cell (away from the center). The typical appearance of
the markers in a control cell is shown in Figure 3.8. In some cases, the concentration of the
two markers occurred on the opposite sides of the cell. The following results demonstrate the
functional compartmentalization exhibited by cells. Though the traffic pattern cannot be
explained, it can be concluded that, markers incubated with cells for longer periods tends to
accumulate in smaller areas than those incubated for shorter periods. The smaller areas
representing compartment II could be explained by the accumulation in lysosomes following
influx and sorting in compartment II. For additional color map images, refer to Appendix C.
(B)
(A)
Figure 3.7 Color map of fluorescent marker intensity in FITC (A) and CY3 (B) in a PMAtreated cell. (A) and (B) show the different distribution of fluorescent markers in the same
cell. The FITC image color map of the cell (A) shows an intensity range between 0 (deep blue)
and 600 (dark red), with 250-300 (green) representing the middle range of fluorescent
intensities.
53
The CY3 image color map (B) shows an intensity range between 0 (deep blue) and 1600 (dark
red), with 750-800 (green) representing the middle range of fluorescent intensities
(C)
(D)
Figure 3.8 Color map of fluorescent marker intensity in FITC (A) and CY3 (B) in a
untreated cell showing distribution of two fluorescent markers. The FITC image color map of
the cell (A) shows an intensity range between 0 (deep blue) and 500 (dark red), with 250-300
(green) representing the middle range of fluorescent intensities. The CY3 image color map (B)
shows an intensity range between 0 (deep blue) and 1400 (dark red), with 600-800 (green)
representing the middle range of fluorescent intensities.
The ratio of CY3 as a fraction of the two markers (FITC and CY3) was plotted as a
histogram (Figure 3.9). This ratio represented showed the distribution of the amount of CY3
over FITC within the same cell in overlaid images. In the histogram, a value of 1.0 would be
observed only where CY3 accounted for all of the fluorescence intensity. The control cells’
plot is shown in black while the PMA-treated cells’ plot is shown in grey. Values greater 0.5
on the horizontal axis represent areas in cells where the amount of CY3 is greater than FITC,
and vice versa for values less than 0.5.
For values between 0.60 and 0.91, the relative amounts of CY3 compared to FITC are greater
54
in the PMA-treated cells than in control cells. The area between 0.39 and 0.52 on the
horizontal axis show the areas in PMA-treated cells (grey) where the intensity of the FITC
marker is greater than CY3.
In these data, the pixel-by-pixel comparison reveals that there are many areas where CY3 is
more intense than FITC in the PMA-treated cell populations. Whereas there are still many such
areas in the control cells, the excess number in the treated cells is clearly indicated by the bins
in the histogram where the gray display (PMA-treated) exceeds the black (control). Thus, these
results expand on those recorded earlier in Figures 3.3 and 3.5.
Figure 3.9 Ratio of CY3 as a fraction of the two fluorescent markers (CY3 and FITC) on a
pixel-by-pixel basis in PMA-treated versus control cells. The control cells’ plot is shown in
black while the PMA-treated cells’ plot is shown in grey.
Values greater 0.5 on the horizontal axis represent areas in cells where the amount of CY3 is
55
greater FITC, and vice versa for values less than 0.5. The area between 0.39 and 0.52 on the
horizontal axis show the areas in PMA-treated cells (grey) where the amount is FITC is greater
than CY3.
3.4 SPATIAL DIFFERENCES IN THE LOCALIZATION OF COMPARTMENT I AND II
Metamorph software was used to make a ratio image and display the ratios of FITC and
CY3 relative to one another on a pixel-by-pixel basis. Therefore, ratio data similar to that
displayed in Figure 3.10 could be visualized in a color map image.
Although the exact conditions for each display had to be matched to the range of intensities in
the two channels, the ratio images of FITC over CY3 generally displayed the difference in
distribution of the two fluorescent markers inside cells. These images reinforced the
observation above (Figure 3.9), that the FITC distribution was eccentric in most cells. This
could not be an artifact of image shifts during the image processing steps, because, when two
cells were observed in the same image, the appearance of Compartment II was generally
localized in different quarters of the cells. One qualitative difference was observed for PMAtreated cells (A), compared to controls (B), namely that some treated cells showed an increased
concentration of FITC on the periphery of cells in a ring-like pattern (Figure 3.10). This pattern
could reinforce our prediction of different traffic patterns in PMA treated cells over control
cells, which could have consequences on polarity. However, as this pattern was observed in a
total of 4 of 45 PMA-treated cells, it is difficult to make assertive conclusions to this effect.
Nevertheless, none of the control cells showed this pattern in the ratio images. Both treated and
untreated cell populations, however, could show a different directional orientation of
compartment I and II within a single cell, as shown in Figure 3.10.
56
Whereas FITC marker showed a typical striated or spotty pattern on one side of the cell or
mixed with CY3, CY3 shows a more uniform and concentrated localization on one side of the
cell. For additional ratio images, refer to Appendix D.
(A)
(C)
(B)
(D)
57
(F)
(E)
(G)
(H)
Figure 3.10 Ratio (FITC/CY3) images of (A-D) PMA-treated and (E-H) control cells. Each
image shows color map of ratio intensity of cells obtained by overlaying FITC on CY3
intensities in the same cells. Green represents the mid ratio showing areas where FITC and
CY3 intensities are the same. Colors above green in the scale (yellow-red) represent areas
where FITC is greater than CY3, and vice versa for the range below green (deep green to
dark blue).
58
3.5 COMPARISON OF FLUORESCENT MARKER ACCUMULATION IN 1000W CELLS
ON HAPTOTACTIC GRADIENT
One objective of my work was to test the Tojima hypothesis by measuring the amount of
total fluorescent marker (FITC) accumulated on opposite halves of the cells on a haptotactic
gradient. Cells accumulated FITC marker over an incubation period of 2 min and the intensity
on the high side (facing the attractive end of the gradient) against the low side (facing the
repulsive end of the gradient) for each cell was determined. From Tojima’s hypothesis, we
expected the low side of the cell to do more endocytosis, and therefore accumulate a greater
concentration of FITC than the high side over the short exposure period. There was no
significant difference between the markers accumulated FITC on the low and high side of the
cell. In Figure 3.12, the high side is represented on the left side of each image. A comparison
of the normalized integrated gray level with other measures, however, however, showed a
consistent difference between the high side and low side. The same difference was reflected in
the other measures of intensity, i.e. optical density (OD) and integrated OD. This suggested
that the balance of endocytosis was slightly skewed in favor of the low side of the cell, although
it was not significant. The raw data obtained in Metamorph used in for making histograms in
Figure 3.11 is shown in appendix E. Sample of cell images used in this analysis is shown in
Figure 3.12.
59
Figure 3.11 Comparison of FITC integrated intensity and optical density (OD) of the right
(high side) against left (low) side of 1000W cells.
It was also observed in the 1000W cells that, the FITC marker contained in vesicles only
accumulated along the edges (rim) of the cells after a short incubation period of 2 minutes
(Figure 3.12). Additional images of this phenomenon can be found in appendix E.
Figure 3.12 Image of two cells showing accumulation of FITC vesicles at the rim of 1000W
after an incubation period of 2 minutes.
60
CHAPTER 4. DISCUSSION
Since the kinetics of marker trafficking are well known for the IAR20 rat liver cell
line(119,120), this enabled the selection of proper conditions for visualizing accumulation in
Compartment II in relation to accumulation in Compartment I. The images, along with the
analysis of the intensity levels, which are equivalent to marker concentration, allowed the
characterization of the spatial compartments corresponding to previous models for pinocytic
traffic. Particular attention was paid to the relationship of marker accumulation to signaling
pathways activated after PMA treatment. PMA activates a regulatory molecule known as
protein kinase C (PKC), and is a chemical surrogate for diacylglycerol, the endogenous
activator of PKC. After activation by either of these molecules, PKC adds a charged
constituent to amino acid residues of a number of protein substrates. The PKC family of
proteins comprises a number of serine/threonine kinases, some of which bind to phorbol esters
to modulate signal transduction pathways and cellular functions. This family is composed of at
least 12 different isoforms that play roles in cellular functions including cell cycle control,
proliferation, differentiation, metastasis, and apoptosis (127). The difficulty of dissecting the
roles of the PKC family derives from the different combinations and roles of individual
isoenzymes within cells and across tumor types, along with the complexity of regulatory
mechanisms invoked in the cell.
A number of studies have shown a strong connection between protein kinase and Rho
GTPases in the regulation of actin cytoskeletal organization (128-130), and indeed, PKC binds
Rho directly (131). Although the GTPase most closely linked with filopodia dynamics was
Cdc42, there is evidence of its binding affinity for only one isoform, PKC-alpha (131). As
mentioned in the Introduction (section 1.3.4), the complexity of effects downstream of
PKCactivation has made it difficult to identify the physiological processes essential for tumor
promotion.
61
PMA has been reported to cause an enhancement of membrane ruffling activity in many cell
types within a few hours of exposure, its effect on ruffling in IAR20 cells has not been tested.
The cell ruffling activity in 1000W cells, stimulated by PMA treatment, is accompanied by an
increase in fluid traffic into cells by nonspecific pinocytosis as previously shown (122).
Despite the fact that ruffling is a rearrangement of actin-based cytoskeleton and is often
abolished by the actin-depolymerizing cytochalasins, there is little evidence of Rho GTPasemediated regulation of ruffling.
Macropinocytosis, like phagocytosis, is an actin-dependent process regulated by one or
more members of the Ras-related Rho GTPase family (132,133). Rho GTPases comprise a
number of signaling proteins that work together with various effector molecules to transmit
signals within cells and mediate changes in the actin cytoskeleton at specific sites. In humans,
about 22 Rho GTPases exist of which isoforms of Rho and Rac and Cdc42 have been best
studied (134). About 1% of the genome consists of Rho-family GTPase or its regulatory
proteins, guanine nucleotide exchange factors (GEFs) or GTPase-activating proteins (GAPs),
however. Upon activation Rho GTPases bind to a number of effectors, including protein
kinases and some actin binding proteins (135). The rate of macropinocytosis in mammalian
cells can either be increased or decreased through a number of reactions under the control of the
Rho family of GTPases. While immature dendritic cells actively carry out macropinocytosis
and phagocytosis, mature cells cease endocytic activity, becoming more specialized for antigen
presentation to T cells (136). Studies have shown that endocytic downregulation in mature
dendritic cells is controlled by Rho family of GTPases, especially Cdc42 (136). A number of
pathogens, including Salmonella typhimirium and Shigella flexnerri, drive their own
internalization by activating Rho GTPases, by employing a type III secretion mechanism to
inject specific GEFs into the cytoplasm of their intended host cell (133,137).
62
Inactivation of Rho GTPases with Toxin B caused a dramatic decrease in macropinocytosis in
immature dendritic cells (136). Toxin B is a bacteria pathogenesis effector toxin produced by
Clostridium difficile, which acts to inactivate many Rho GTPases by blocking their capacity for
nucleotide exchange. Runyeon showed an increased rate of nonspecific pinocytosis of HRP in
PMA-treated over the control rat liver cells (120). The results of my work strongly support the
increased pinoctyic rate shown by the increased accumulation of both FITC-GAM and CY3dextran in PMA-treated cells over the controls (see Figures 3.3 and 3.9). It also suggests that
the model previously used to interpret the flow of traffic in cells is incomplete. Despite the fact
that, in both cell populations in my experiment, FITC was accumulated without PMA
treatment, there was an increased FITC accumulation in the treated cells over the controls. This
suggests that FITC marker may continue to leave from the control cells but accumulate
specifically in treated cells during the period of PMA exposure, which was 10 minutes. Since a
2-minute rinse period was inserted between the loading of FITC marker and the efflux period,
the result suggests differences in continuous traffic of remnant FITC molecules. This was
unexpected and indicates that a reduction in rate and/or an enhancement of rate
with PMA
treatment. Further work must be done in order to test the possibility that marker concentration
in localized organelles is responsible for the finding, rather than the gross amount of FITC
marker retained.
Typically, in experimental analysis, we look for the simplest explanation that can account
for the observations. According to the previous model of Heckman, et al., PMA, through PKC
activation, could stimulate macropinocytosis through activation of certain Rho GTPases, and
the effect may be confined to rate ã (119). A graphical model for physiological trafficking is
shown in the Introduction (Section 1.4). The rapid influx of substances begins with an
increased rate of traffic to compartment I, followed by continuous traffic to compartment II.
63
Since there is more CY3 marker loaded in treated cells during the 10-min interval allowed for
partially filling compartment I, my results are consistent with this interpretation. It cannot be
determined from my results, whether the treated and control cells differ in rates of efflux and
trafficking between Compartment I and Compartment II. The results suggest the possibility of
developing a method to measure the rates
and
. Data relevant to the volume of the
functional compartment I in the presence and absence of PMA treatment were also subjected to
preliminary evaluation by estimating the relative size of the CY3-containing compartment.
Even though the specific isoform of PKC or the Rho GTPase involved was not determined, it
opens the door for future studies by applying this model.
Evidence from the ratio images suggested a compartmentalization of the endocytic
markers. Materials taken up by cells are first sorted in the compartment I which represents a
larger area inside the cells. Following sorting, materials that are not recycled are sent into
compartment II. The results (Figures 3.7 and 3.8) show that compartment I occupies larger
areas inside the cells than compartment II. Furthermore the distribution of fluorescent marker
is general for compartment I, as the CY3 marker rarely shows a strong directional localization
in the cell. In contrast, the marker for Compartment II is strongly localized within a crescentshaped area of the cell. This localization shows that uptake is generally nondirectional, because
marker distribution is not polarized. This supports, with spatial evidence, the functional
compartmentalization model of endocytosis, which had already been developed by others based
on physiological data. The displacement is a little weaker for PMA-treated cells, as mentioned
above (see Results).
The ratio images suggest traffic in different directions of compartment I and II substances.
In non-migratory mammalian cells that lack visible cell surface protrusions, it is difficult to
develop a cue to their polarization. The ruffling activity of IAR20 cells was studied in earlier
work (122), and ruffles appeared in more than one quadrant on the perimeter of the cell.
64
Whereas ruffling was relatively delocalized, the above results show that lysosomes
(compartment II) are stored at one side of the cell. Using the endocytic marker for the lysosome
now provides valuable information on the position of the Compartment II organelles.
Localization of the FITC marker corresponds to a known phenomenon inside the cells, in
which the lysosomes surround the area of the microtubule-organizing center or centrosome
(138). Thus a cue was discovered that will be useful in assessing polarity, which is otherwise
difficult to assess because, in epithelial cells, the leading and trailing edges do not show a
visible difference in shape.
In the experiment designed specifically to measure marker uptake relative to polarity,
using the 1000W cell line, derived from rat tracheal epithelium, there was no significant
difference in integrated intensity between the high and low side of cells on a gradient. There
was however a higher optical density (OD) on the high side than the low side of the cells. The
optical OD, which represents a measure of the opacity of the image, is a similar measure when
applied to a bright object on a black background. Even though there was no significant
difference in integrated intensity between the two cell halves, the consistent direction of the
difference may still suggest differences which may have not been statistically significant. The
results (Figures 3.11 and 3.12) suggest that the balance between endocytosis and endocytosis
proposed as a model for the neuronal development may be similar in association with polarity
and migration in epithelial cells.
Future research will be directed at determining the corresponding fluorescent marker
concentration for the fluorescent intensity measurements for each marker. With the aid of these
measurements and the model developed by Runyeon et al (119) , we aim to determine the
volume changes that occur in compartment I during endocytic traffic. Also it would be prudent
to target and deactivate specific Rho GTPases in subsequent experiments to determine whether
endocytic traffic will be affected by PMA treatment.
65
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APPENDIX A
Experimental determination of influx and efflux kinetics for HRP (Runyeon, unpublished data).
Influx describes the uptake of materials into a cellular comparments while efflux describes
emptying of the cell by exocytosis.Accumulation curves in red (diamond markers) and black
(square markers) represent PMA-treated and control cells respectively. The time used for FITC
accumulation in the current work, i.e., 20 min and efflux for a period of 12 min, is marked with
an arrow. The amount of HRP taken up by untreated cells was 0.694 ng/100,000 cells, and the
amount of HRP remaining in the cells was 0.278 ng. In theory, these amounts would be less
than those determined from the intensity of FITC marker as endocytic marker rentention is
enhanced with increased molecular weight. Since the marker is sorted in a fashion that depends
on molecular weight, more of the FITC molecules are passed to compartment II and fewer are
effluxed. The FITC marker is expected to be equally bright in the treated and untreated cells,
however. Theoretical curves were calculated to represent the accumulation time course that
would have been observed if initial uptake rates had continued for 120 min. These values are
plotted in thin red lines (PMA) and thin black lines (control).
76
77
APPENDIX B
The table depicted below shows the cell sizes of both PMA-treated and control cells. Pixel
count of the total area covered by cells was used as this provides a more sensitive index of cell
measurement in pre-processed images for fluorescent analysis. Pixel count does not change
with image processing proceeses, especially in cases where images have to be enlarged to
complete the analysis.
Sample 2 (45 PMA-treated
sample 4 (32 control cells)
Cell numbers cells) total pixel count
total pixel count
1
24490
40330
2
32130
43456
3
82636
61244
4
41600
46512
5
53520
41808
6
105608
49000
7
37989
71680
8
32384
64152
9
32955
64728
10
36330
29750
11
38522
23707
12
37370
18720
13
55575
26895
14
29050
23103
15
32886
21170
16
36288
116800
17
50456
44485
18
69778
44485
19
69250
39601
20
46690
31122
21
32399
56196
22
57178
41370
23
26235
33389
24
23840
58149
25
36848
59829
26
22040
39312
27
41860
44197
28
32436
40533
29
24568
38415
30
36091
61596
31
23405
78925
32
24327
81125
78
33
34
35
36
37
38
39
40
41
42
43
44
45
Mean
Maximum
42188
28710
22496
24335
54264
46434
33950
77520
140160
65949
113085
69126
57536
46721.93 47993.25
140160
116800
79
APPENDIX C
Additional color map (heat map images are shown below). Group A shows PMA-treated and
group B shows control cells. Each image pair shows the same cell containing FITC on the left
and CY3 on the right.
Group A
(1)
(2)
(3)
80
(4)
(5)
(6)
81
Group B
(1)
(2)
(3)
82
(4)
(5)
(6)
83
APPENDIX D
Additional ratio images (FITC/CY3) are shown below. Group A shows PMA-treated and group
B shows control cells.
Group A (PMA treated cells)
84
Group B (control cells)
85
APPENDIX E
The FITC accumulation data obtained from the two cell halves of 1000W cells plated on a
haptotactic gradient is shown below in table A. The right side represents the cell half on the
attractive side of the gradient (high side), and the left represents the other half on the repulsive
side of the gradient (low side)
Table A: Data of FITC accumulation on the two halves of 1000W cells on platinum
haptotactic gradient
ima
ge
08-513_FB_FIT
C_T2_08
08-513_FB_FIT
C_T2_09B
08-513_FB_FIT
C_T2_10
7-30-13_
FB_FITC_T
1_01
7-3013_FB_FIT
C_T1_02
7-3013_FB_FIT
C_T1_03
7-3013_FB_FIT
C_T1_04
7-3013_FB_FIT
C_T1_05
7-3013_FB_FIT
C_T1_06
7-3013_FB_FIT
C_T1_07
normali normaliz normali normaliz normalized
normalized
zed gray ed gray
zed OD ed OD
integrated
integrated
left
right
left
right
OD left
OD right
0.342195 0.657804 0.50420 0.495798 0.678686949
1.304646384
1
9
1681
319
0.469965
93
0.530034
07
0.50137
5894
0.498624
106
0.937352464
1.057159062
0.294663
928
0.705336
072
0.5032
0.4968
0.585580142
1.401701257
0.381334
093
0.618665
907
0.50142
3825
0.498576
175
0.760502541
1.23381833
0.492960
689
0.507039
311
0.5
0.5
0.985921378
0.657459999
0.506564
902
0.493435
098
0.49922
3602
0.500776
398
1.014705433
0.988404987
0.299341
755
0.092206
377
0.31756
3291
0.365189
873
0.942620773
0.290355904
0.518225
677
0.608451
868
0.481774
323
0.50325
0975
0.317246
835
0.496749
025
0.440412
693
0.559587
307
0.50081
15
0.596448
294
0.403551
706
0.49774
7214
1.917913121
1.029755933
0.969854592
0.499188
5
0.879398123
1.120993987
0.502252
786
1.198295595
0.803483261
86
7-3013_FB_FIT
C_T1_08
7-3013_FB_FIT
C_T1_09
7-3013_FB_FIT
C_T1_10
0.453357
891
0.546642
109
0.50248
2622
0.497517
378
0.902648833
1.098739727
0.398693
744
0.601306
256
0.50133
1203
0.498668
797
0.801366467
1.205822904
0.469241
699
0.200451
681
0.06284
0789
0.063533
665
0.940988694
3.155046735
7-3013_FB_FIT
C_T1_11
7-3013_FB_FIT
C_T1_12
7-3013_FB_FIT
C_T1_14
7-3013_FB_FIT
C_T1_15
7-3013_FB_FIT
C_T1_16
7-3013_FB_FIT
C_T1_17
7-3013_FB_FIT
C_T1_18
7-3013_FB_FIT
C_T1_19
7-3013_FB_FIT
C_T1_20
7-3013_FB_FIT
C_T1_21
7-3013_FB_FIT
C_T1_22
7-3013_FB_FIT
C_T1_24
0.535696
859
0.072300
348
0.464303
141
0.50025
2016
0.063834
915
0.499747
984
0.471782
511
0.528217
489
0.50131
2336
0.574609
833
0.425390
167
0.511885
34
7-30-
1.137984848
1.070853973
0.928138471
0.498687
664
0.941094956
1.053669442
0.49863
8277
0.501361
723
1.152358051
0.853103716
0.488114
66
0.50012
4039
0.499875
961
1.023516768
0.9759872
0.547968
439
0.452031
561
0.49926
1884
0.500738
116
1.097557128
0.905399702
0.523214
364
0.476785
636
0.50049
4071
0.499505
929
1.045395729
0.952629938
0.584845
599
0.415154
401
0.49888
0875
0.501119
125
1.17231513
0.83217141
0.524938
206
0.475061
794
0.50086
1857
0.499138
143
1.048069839
0.948488666
0.482532
498
0.517467
502
0.50405
7428
0.495942
572
0.957296671
1.026604258
0.573912
323
0.426087
677
0.49831
4877
0.501685
123
1.151706179
0.855057106
0.535842
525
0.464157
475
0.49826
3027
0.501736
973
1.075421004
0.931551108
0.177015
64
0.633335
151
0.03536
2578
0.034983
817
5.00573341
18.10366087
0.377755
0.03494
9384
0.49866
0.189649
21
0.622244
5.362991571
0.501331
1.247811339
0.753504897
87
13_FB_FIT
C_T1_25
7-3013_FB_FIT
C_T1_28
7-3013_FB_FIT
C_T1_29
7-3013_FB_FIT
C_T1_30
08-513_FB_FIT
C_T2_04
02-0813_FITC_F
B_T2_03
02-0813_FITC_F
B_T2_01
02-0813_FITC_F
B_T2_06
08-513_FB_FIT
C_T2_08
08-513_FB_FIT
C_T2_02
Mean
Stdev
338
662
8603
397
0.434100
612
0.565899
388
0.50157
3977
0.498426
023
0.865476743
1.135372878
0.449380
909
0.550619
091
0.50368
6753
0.496313
247
0.892183299
1.109418486
0.404311
879
0.595688
121
0.50013
9082
0.499860
918
0.808398891
1.191707732
0.262283
824
0.221850
228
0.01250
8229
0.012691
098
20.96890151
17.48077505
0.517967
721
0.515865
948
0.482032
279
0.49805
6365
0.014563
675
17.00680
272
0.623137
842
0.376862
158
0.49711
4376
0.078473
547
0.458870
267
0.01323
8498
0.462656
185
0.328054
792
0.671945
208
0.49006
9471
0.50434
0521
0.495659
479
0.650462889
1.355658948
0.546746
416
0.453253
584
0.49897
3306
0.501026
694
1.095742816
0.904649572
0.449369
13
0.128461
346
0.476719
366
0.141297
559
0.43201
6066
0.16333
0191
0.851885
844
2.696987
237
1.877198357
3.753416797
3.458209505
8.509352644
35.42141321
1.039978117
0.028343498
0.502885
624
1.253510003
0.749399345
0.012821
277
5.927677415
35.78974768
0.94406245
88
Additional images of the data shown in the table are shown below.
89