University of Pennsylvania ScholarlyCommons
Publicly Accessible Penn Dissertations
Summer 2010
The Role of the Exocyst in Exocytosis and Cell Migration
Jianglan Liu University of Pennsylvania, [email protected]
Follow this and additional works at: https://repository.upenn.edu/edissertations
Part of the Cell Biology Commons
Recommended Citation Liu, Jianglan, "The Role of the Exocyst in Exocytosis and Cell Migration" (2010). Publicly Accessible Penn Dissertations. 201. https://repository.upenn.edu/edissertations/201
This paper is posted at ScholarlyCommons. https://repository.upenn.edu/edissertations/201 For more information, please contact [email protected]. The Role of the Exocyst in Exocytosis and Cell Migration
Abstract The exocyst, an evolutionarily conserved octameric protein complex, plays a crucial role in the tethering of post-Golgi secretory vesicles to the plasma membrane for exocytosis and cell migration. How the exocyst is targeted to sites of exocytosis and how this complex regulates cell migration are poorly understood. I have carried out experiments to characterize Exo70, a component of the exocyst complex. Firstly, I found that Exo70 directly interacts with phosphatidylinositol 4,5-bisphosphate through positively charged residues at its C-terminus, and this interaction is critical for the plasma membrane targeting of Exo70. Using the ts045 vesicular stomatitis virus glycoprotein trafficking assay, I found that the Exo70-lipid interaction is critical for the docking and fusion of post-Golgi secretory vesicles with the plasma membrane. Secondly, I demonstrate that the exocyst plays a pivotal role in tumor cell invasion. Invadopodia are actin-rich membrane protrusions formed by tumor cells that degrade the extracellular matrix for invasion. Depletion of the exocyst component Exo70 or Sec8 led to failure in invadopodia formation in MDA-MB-231 cells expressing constitutively active c-Src, whereas the overexpression of Exo70 promoted invadopodia formation. Disrupting the exocyst function by RNA interference of EXO70 or SEC8, or by expression of a dominant negative fragment of Exo70 blocked the secretion of matrix metalloproteinases. I further found that the exocyst interacts with the Arp2/3 complex in cells with high invasion potential; blocking the exocyst-Arp2/3 interaction inhibited Arp2/3-mediated actin polymerization and invadopodia formation. Finally, the exocyst also functions in directly regulating Arp2/ 3-mediated actin polymerization. Using pyrene actin assay and total internal reflection fluorescence microscopy, I found that Exo70 synergizes with WAVE2 to promote Arp2/3-mediated actin polymerization and branching. In addition, I have examined how the Exo70-Arp2/3 interaction is regulated in the cell downstream of growth factor signaling. Overall, these studies provide mechanistic insights to the function of the exocyst in exocytosis and cell migration.
Degree Type Dissertation
Degree Name Doctor of Philosophy (PhD)
Graduate Group Biology
First Advisor Wei Guo
Keywords Exocytosis, Exocyst, Invadopodia, The Arp2/3 complex, cell migration, actin polymerization
Subject Categories Cell Biology
This dissertation is available at ScholarlyCommons: https://repository.upenn.edu/edissertations/201
THE ROLE OF THE EXOCYST IN EXOCYTOSIS AND CELL MIGRATION
JIANGLAN LIU
A DISSERTATION in BIOLOGY
Presented to the Faculties of the University of Pennsylvania in Partial Fulfillment of the Requirements for the Degree of Doctor of Philosophy 2010
Supervisor of Dissertation
______Wei Guo, Associate Professor of Biology
Graduate Group Chairperson
______Paul Sniegowski, Associate Professor of Biology
Dissertation Committee Tatyana Svitkina, Associate Professor of Biology Michael Lampson, Assistant Professor of Biology Kimberly Gallagher, Assistant Professor of Biology Margaret Chou, Associate Professor of Pathology and Laboratory Medicine Mickey Marks, Associate Professor of Pathology and Laboratory Medicine ACKNOWLEDGEMENTS
First of all, I would like to give my deepest gratitude to my mentor Dr. Wei
Guo. I regard Wei as my scientific father as he guided me into the wonderful scientific field and directed me all the way till now. I am so impressed with his enthusiasms and curiosity on science, his persistence on research and his extensive knowledge. And I really appreciate that he gives me inspiring advices and warm encouragements in all the time of research. I would benefit from his mentoring throughout my entire scientific career.
Secondly, I really appreciate all the members in my thesis committee: Prof.
Tatyana Svitkina, Prof. Margaret M. Chou, Prof. Mickey Marks, Prof. Kimberly
Gallagher and Prof. Michael A.Lampson. They have committed much time and efforts to follow my research and have always been a great source of constructive suggestions and encouragements.
I enjoy the wonderful time that I have spent with all the previous and current members of Guo lab. They make the lab such a pleasant place for everyday work.
They are not only my scientific partners, but also my incredible friends. In particular,
I would like to thank Dr. Bing He who has offered me so much help and valuable advices. I appreciate Dr. Xiaofeng Zuo for his contribution to the first part of this study. I am grateful to Dr Xiaoyu Zhang and Jian Zhang for their nice help since I
ii entered the lab. I also want to thank Dr. Kelly Orlando for her help on my scientific
English writing. Finally, I want to thank all other lab folks for their constant discussions and encouragements.
I would also like to extend thanks to the people who have been instrumental in completion of this work. I am grateful to Prof. Susette C. Mueller, Dr. Vira V. Artym and Mr. Peter Johnson for their collaboration on the invadopodia research; Prof. Yale
Goldman and Dr. Yujie Sun for their collaboration on the TIRFM experiments; Dr.
Jian Jing for giving me advices for zymography experiments; Prof. Roberto
Dominguez, Dr. Grzegorz Rebowski and Dr. Boczkowska Malgorzata for their help on the pyrene actin assay.
Finally, I would like to give my special thanks to my family. I want to thank my parents with my heart for their love, support and encouragement throughout my life.
Most of all, I would give my thanks to my dear husband, Peng Yue. He is my labmate, my soulmate and my lifemate. His solid help for my research and his endless love for my life make me strong enough to go this far. I want to give my last but most special thanks to my little angel Shannon Xianning Yue. You make mommy’s life complete.
Love you forever.
iii ABSTRACT
THE ROLE OF THE EXOCYST
IN EXOCYTOSIS AND CELL MIGRATION
Jianglan Liu
Supervisor: Dr. Wei Guo
The exocyst, an evolutionarily conserved octameric protein complex, plays a crucial role in the tethering of post-Golgi secretory vesicles to the plasma membrane for exocytosis and cell migration. How the exocyst is targeted to sites of exocytosis and how this complex regulates cell migration are poorly understood. I have carried out experiments to characterize Exo70, a component of the exocyst complex. Firstly, I found that Exo70 directly interacts with phosphatidylinositol 4,5-bisphosphate through positively charged residues at its C-terminus, and this interaction is critical for the plasma membrane targeting of Exo70. Using the ts045 vesicular stomatitis virus glycoprotein trafficking assay, I found that the Exo70-lipid interaction is critical for the docking and fusion of post-Golgi secretory vesicles with the plasma membrane. Secondly, I demonstrate that the exocyst plays a pivotal role in tumor cell invasion. Invadopodia are actin-rich membrane protrusions formed by tumor cells that degrade the extracellular matrix for invasion. Depletion of the exocyst component
Exo70 or Sec8 led to failure in invadopodia formation in MDA-MB-231 cells
iv expressing constitutively active c-Src, whereas the overexpression of Exo70 promoted invadopodia formation. Disrupting the exocyst function by RNA interference of EXO70 or SEC8, or by expression of a dominant negative fragment of
Exo70 blocked the secretion of matrix metalloproteinases. I further found that the exocyst interacts with the Arp2/3 complex in cells with high invasion potential; blocking the exocyst-Arp2/3 interaction inhibited Arp2/3-mediated actin polymerization and invadopodia formation. Finally, the exocyst also functions in directly regulating Arp2/3-mediated actin polymerization. Using pyrene actin assay and total internal reflection fluorescence microscopy, I found that Exo70 synergizes with WAVE2 to promote Arp2/3-mediated actin polymerization and branching. In addition, I have examined how the Exo70-Arp2/3 interaction is regulated in the cell downstream of growth factor signaling. Overall, these studies provide mechanistic insights to the function of the exocyst in exocytosis and cell migration.
v Table of Contents
Chapter 1. Introduction································································································· 1
1.1 Overview··············································································································· 1
1.2 The exocyst complex···························································································· 3
1.3 Phosphoinositides in cell regulation and membrane dynamics·························· 56
1.4 Cell invasion and invadopodia structures··························································· 60
1.5 Thesis overview·································································································· 69
Chapter 2. Phosphatidylinositol 4,5-bisphosphate mediates the targeting of the exocyst to the plasma membrane for exocytosis in mammalian cells························ 70
2.1. Introduction········································································································ 71
2.2. Results················································································································ 73
2.3. Discussion········································································································ 106
2.4. Materials and Methods····················································································· 110
Chapter 3. The role of the exocyst in matrix metalloproteinase secretion and actin dynamics during tumor cell invadopodia formation················································· 116
3.1. Introduction······································································································ 117
3.2. Results·············································································································· 120
3.3. Discussion········································································································ 152
3.4. Materials and Methods····················································································· 156
vi Chapter 4: The role of Exo70 in Arp2/3-mediated actin assembly during cell migration··················································································································· 163
4.1. Introduction······································································································ 164
4.2. Results·············································································································· 170
4.3. Discussion and future perspectives·································································· 184
4.4. Materials and Methods····················································································· 194
Chapter 5: Discussion and future perspectives························································· 199
5.1. Impact of my research in the field··································································· 199
5.2. The molecular mechanism of exocyst targeting to the plasma membrane······ 200
5.3. A crosstalk between actin cytoskeleton and exocytosis machineries during cell
migration········································································································· 201
5.4. The functions of the exocyst in many cellular processes································· 202
5.5. Future perspectives·························································································· 202
References················································································································· 203
vii List of Tables
Table 1. Comparison of the affinities of Exo70 and exo70-1 for phospholipids. ········ 80
Table 2. Exo70 C-terminus mutants. ············································································ 85
viii List of Figures
Figure 1.1 An outline of membrane trafficking events in the cell.······························· 2
Figure 1.2 Four major steps in vesicular trafficking. ··················································· 4
Figure 1.3 Protein-protein interactions among the exocyst subunits. ·························· 9
Figure 1.4 Structural studies of the exocyst subunits. ··············································· 11
Figure 1.5 Subcellular localization of the exocyst in various cell types. ··················· 16
Figure 1.6 The exocyst is the downstream effector of a variety of Ras family of small
GTPases. ···················································································································· 44
Figure 2.1 Association of Exo70 with the plasma membrane (PM) in HeLa cells. ··· 75
Figure 2.2 The interaction between Exo70 and phospholipids in vitro. ···················· 79
Figure 2.3 Mutations at Exo70 C-terminus abolish the association between Exo70 and the PM. ······················································································································· 84
Figure 2.4 Mutations at the C-terminus of Exo70 affect its interaction with phospholipids. ············································································································ 87
Figure 2.5 Mutations in the exo70-1 mutant did not affect its interaction with TC10
(Q75L) or Sec8. ········································································································· 89
Figure 2.6 Exo70 is required for the PM localization of Sec8. ································· 92
Figure 2.7 Exocytosis of VSV-G ts045 is blocked in EXO70 siRNA knockdown cells. ··························································································································· 95
Figure 2.8 VSV-G ts045 exocytosis defect in EXO70 siRNA knockdown cells expressing the exo70-1 mutant. ··············································································· 102
ix Figure 3.1 Knockdown of the exocyst inhibits invadopodial activity. ···················· 122
Figure 3.2 Overexpression of Exo70 stimulates invadopodial activity. ·················· 127
Figure 3.3 Localization of Exo70 at the focal degradation sites. ···························· 131
Figure 3.4 The exocyst is required for the secretion of MMPs in MDA-MB-231
(Y527F c-Src) cells. ································································································· 135
Figure 3.5 Zymography analyses of MMP-2 and MMP-9 secreted from
MDA-MB-231 (Y527F c-Src) cells overexpressing Exo70∆C (amino acids 1–408) or full-length Exo70. ···································································································· 138
Figure 3.6 The interaction between Exo70 and the Arp2/3 complex is required for invadopodia formation. ···························································································· 141
Figure 3.7 Exo70 is involved in Arp2/3-mediated actin polymerization in the cell. ··························································································································· 147
Figure 4.1 rExo70 enhances actin polymerization mediated by the Arp2/3 complex and WAVE2. ············································································································ 172
Figure 4.2 Elongation and branching of actin filaments visualized by
TIRFM. ···················································································································· 175
Figure 4.3 GST-WAVE2 pulled down GFP/myc-tagged rExo70 from HeLa cells. ························································································································· 179
Figure 4.4 Examine the in vitro interaction of GST-tagged rExo70 and His(6)-tagged
Arpc1 wt or Arpc1 (21E) mutant. ············································································ 182
x Abbreviations
AMPAR, a-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid receptor. aPKC, atypical protein kinase C.
ARE, apical recycling endosomes.
Arf, ADP ribosylation factor.
BFA, Brefeldin A.
COG, conserved oligomeric Golgi.
ECM, extracellular matrix
EGF, epidermal growth factor
ER, endoplasmic reticulum.
EST, expressed sequence tag
FRAP, fluorescence recovery after photobleaching.
GARP, Golgi-associated retrograde protein.
GEF, guanine nucleotide exchange factor.
GLUT4, glucose transporter 4
HDM, high-density microsomal membranes
HSS, high speed supernatant.
LDLR, LDL receptor.
LDM, low-density microsomal membranes
LUV, large unilamellar vesicle.
xi MAGUK, membrane-associated guanylate kinase.
MDCK, Madin-Darby canine kidney.
MMP, matrix metalloprotease
NGF, nerve growth factor
NMDAR, N-methyl D-aspartate receptor.
NPF, nucleation-promoting factor.
NRK, normal rat kidney
PBS, phosphate-buffered saline.
PC, phosphotidylcholine.
PH, pleckstrin homology domain.
PI(4,5)P2, phosphatidylinositol 4,5-bisphosphate.
PKD, protein kinase D
PM, plasma membrane.
PS, phosphatidylserine.
PSD, postsynaptic density.
PI, phosphatidylinositol.
RBD, Ral binding domain.
RNAi, RNA interference. shRNA, short hairpin RNA.
SNARE, soluble JV-ethylmaleimide-sensitive fusion attachment protein receptor.
xii TIRFM, total internal reflection fluorescence microscopy.
TGN, trans-Golgi network. tSNARE, target membrane-associated SNARE.
VSV-G, vesicular stomatitis virus G.
xiii Jianglan Liu THE ROLE OF THE EXOCYST IN EXOCYTOSIS AND CELL MIGRATION Chapter 1 Chapter 1. Introduction
1.1. Overview
Membrane trafficking, or the transport of membrane-bound materials between cellular endomembrane compartments and the plasma membrane, is essential for delivery of proteins and other macromolecules to various sites inside and outside of the cell (Figure 1.1). The membrane trafficking system involves distinct organelles, including the endoplasmic reticulum (ER), Golgi apparatus, endosomal compartments, and plasma membrane. In the biosynthetic pathway, secretory cargos are synthesized and modified in the ER and then transported to the Golgi apparatus for further modifications. Upon arrival at the trans-Golgi network (TGN), the cargos are sorted and packaged into post-Golgi vesicles that take different routes towards the plasma membrane or endosomal compartments, respectively. In the endocytic pathway, membrane and proteins are internalized at the plasma membrane and are either delivered through early and late endosomes to the lysosomes for degradation or recycled back to the plasma membrane via recycling endosomes.
Between different membrane compartments, proteins and lipids are mostly transported by small membrane-enclosed vesicles. The basic scheme of membrane trafficking consists of several major steps: budding of vesicles from the donor
1 Jianglan Liu THE ROLE OF THE EXOCYST IN EXOCYTOSIS AND CELL MIGRATION Chapter 1
Figure 1.1
Figure 1.1 An outline of membrane trafficking events in the cell. In this diagram, the exocytic and endocytic routes are illustrated with solid and dashed arrows, respectively. In the biosynthetic-secretory pathway, cargo leaves the TGN or recycling endosomes in vesicular carriers to the plasma membrane. In the endocytic pathway, proteins are internalized and transported to early endosomes, and then either travel through late endosomes to the lysosome to be degraded or return to the plasma membrane through the recycling endosomes. Early endosomes may serve as sorting stations for the next stages of cargo transport. Figure modified from Orlando and Guo,
2009.
2 Jianglan Liu THE ROLE OF THE EXOCYST IN EXOCYTOSIS AND CELL MIGRATION Chapter 1 compartment, transport of vesicles along cytoskeleton, tethering of the vesicles to specific domains and fusion of vesicles with the target compartment. Coat proteins and cytoskeleton elements play major roles during the budding and transport steps, respectively. The tethering step, defined as the initial recognition and attachment of secretory vesicles with the target membrane, is mediated by tethering proteins. In the subsequent fusion step, the SNARE (soluble NSF attachment protein receptors) proteins drive the final fusion of the two membranes (Cai et al., 2007, Figure 1.2).
Exocytosis is a fundamental membrane trafficking event in eukaryotic cells that mediates the incorporation of membrane proteins and lipids into specific plasma membrane domains as well as the secretion of vesicle contents to the exterior of the cell. It is essential for a number of biological processes including cell growth, morphogenesis and cell migration. Exocytosis is achieved by the fusion of secretory vesicles with the plasma membrane, which is catalyzed by the assembly of the
SNARE complex. Before membrane fusion, the tethering of secretory vesicles to the plasma membrane is thought to be mediated by the exocyst, an evolutionarily conserved octameric protein complex.
1.2. The exocyst complex
Up to date, a number of proteins and protein complexes have been identified
3 Jianglan Liu THE ROLE OF THE EXOCYST IN EXOCYTOSIS AND CELL MIGRATION Chapter 1 Figure 1.2
Figure 1.2 Four major steps in vesicular trafficking. The diagram reveals the four essential steps in vesicular transport. (1) Budding: coat proteins are recruited onto the donor membrane to promote the formation of a vesicle. Cargo and SNAREs are incorporated into the budding vesicle through interaction with coat subunits. (2)
Transport: the vesicle moves toward the acceptor compartment through a cytoskeletal track. (3) Tethering: tethering factors work in conjunction with upstream regulators to tether the vesicle to their acceptor membrane. (4) Fusion: the vesicle-associated
SNARE and the SNARE on the acceptor membrane assemble into a four-helix bundled SNARE complex, which drives membrane fusion and the delivery of cargo.
Figure adapted from Cai et al., 2007.
4 Jianglan Liu THE ROLE OF THE EXOCYST IN EXOCYTOSIS AND CELL MIGRATION Chapter 1 and characterized that function at the tethering steps at various stages of membrane trafficking (Pfeffer, 1999; Guo et al., 2000; Waters and Hughson, 2000; Whyte and
Munro, 2002). In particular, the tethering of post-Golgi secretory vesicles to the plasma membrane is mediated by the evolutionarily conserved exocyst complex, which is composed of Sec3, Sec5, Sec6, Sec8, Sec10, Sec15, Exo70 and Exo84.
1.2.1. The identification and molecular organization of the exocyst complex
Most exocyst components were originally identified in late 1970’s in a genetic screen for temperature-sensitive secretory (sec) mutants of the yeast Saccharomyces cerevisiae (Novick and Schekman, 1979; Novick et al., 1980). The screen identified
23 complementation groups of sec mutants defective in secretion, which could be divided into two basic categories, 13 genes important for ER-to-Golgi and/or intra-Golgi membrane trafficking and 10 genes required for Golgi-to-plasma membrane (PM) transport (Novick et al., 1980 & 1981). These 10 genes are known as the ‘‘late-acting’’ or “late” secretory genes and six out of them were later shown to encode exocyst components: Sec3, Sec5, Sec6, Sec8, Sec10 and Sec15. Two other exocyst components, Exo70 and Exo84, were biochemically identified from the purified yeast complex or the homologous mammalian complex.
The observation that Sec15 and Sec8 associated with a 19.5 svedberg (19.5S)
5 Jianglan Liu THE ROLE OF THE EXOCYST IN EXOCYTOSIS AND CELL MIGRATION Chapter 1 particle in a linear density gradient (Bowser and Novick, 1991; Bowser et al., 1992) leads to the hypothesis that some late SEC genes might be present in a high molecular weight protein complex. Subsequently, Novick’s group purified this protein complex from yeast lysates (TerBush et al., 1996; TerBush et al., 2001). This complex is composed of the products of six late SEC genes Sec3, Sec5, Sec6, Sec8, Sec10 and
Sec15, and a novel gene product Exo70 (TerBush and Novick, 1995; TerBush et al.,
1996). All these components are present as single copy in the complex (TerBush et al., 1996). The last yeast exocyst component, Exo84, was identified based on sequence similarity to the mammalian homologue (Guo et al., 1999b). Genetic analysis indicates that yeast Exo84, like the other exocyst subunits, is essential for exocytosis (Zhang et al., 2005).
The identification of mammalian exocyst complex was largely promoted by the identification of yeast exocyst complex as well as the timely realization of the conservation of the secretory pathway from yeast to mammals. The mammalian Sec6 and Sec8 were identified by screening human genome expressed sequence tag (EST) databases with the yeast SEC genes and then cloned from a rat brain cDNA library
(Ting et al., 1995). Similar to the yeast exocyst, mammalian Sec6 and Sec8 co-migrated on a continuous glycerol gradient at 17s, indicating that they are components of a 600- to 700-kDa protein complex. Successively, the rat brain rSec6/8
6 Jianglan Liu THE ROLE OF THE EXOCYST IN EXOCYTOSIS AND CELL MIGRATION Chapter 1 (exocyst) complex was purified from rat brain lysates using antibodies against rSec6 and rSec8 by sequential column chromatography (Hsu et al., 1996). The purified mammalian exocyst complex contained eight proteins, including rSec6 and rSec8, with a stoichiometry of one copy of each protein within the complex. rSec5, rSec15, rExo70 and rExo84 were subsequently identified by peptide sequencing using mass spectrometry and cloned from cDNA database (Hsu et al., 1996; Kee et al., 1997).
The remaining two mammalian exocyst genes, Sec10 and Sec3, were identified through search in EST database and GenBank based on sequence homology to yeast genes, and then cloned from cDNA libraries (Guo et al., 1997; Matern et al., 2001).
Understanding the function of the exocyst at the molecular level will require elucidation of the molecular organization of the complex. To explore the molecular organization of the exocyst complex, the interactions between individual exocyst components have been extensively studied using a variety of methods, including yeast two-hybrid assay, protein binding assays with in vitro translated proteins and purified recombinant proteins (For review, see Munson and Novick, 2006). The interaction map is shown in Figure 1.3, which suggests that most exocyst subunits can interact with multiple exocyst subunits in the complex. A number of interactions, including Sec3-Sec5, Sec5-Sec6, Sec5-Exo84, Sec6-Sec8, Sec6-Secl0, Sec6-Exo70,
Sec8-Exo70, Sec10-Secl5, and Sec10-Exo70, have been found in both yeast and
7 Jianglan Liu THE ROLE OF THE EXOCYST IN EXOCYTOSIS AND CELL MIGRATION Chapter 1 mammalian exocyst suggesting conserved interacting interfaces between different components in the complex.
Whether individual exocyst component is required for exocyst assembly has been examined by in vitro reconstitution assay. Mammalian exocyst components were expressed and purified from E. coli, then mixed in a 1:1 molar ratio to reconstitute the exocyst complex (Wang et al., 2004). Interestingly, the absence of any single exocyst subunit did not affect the assembly of the remaining seven subunits, which suggests that multiple interactions between the exocyst subunits are sufficient for any of the seven subunits to associate with each other to form a complex
(Wang et al., 2004). On the other hand, studies in budding yeast indicate that the integrity of the exocyst is largely perturbed in several exocyst mutants, including sec3-2, sec5-24, sec6-4, sec 10-2 and sec15-1, at the restrictive temperature (TerBush and Novick, 1995). These observations suggest that although no individual exocyst component is essential for complex assembly in vitro, the function of at least several exocyst components is critical for the assembly of the exocyst in vivo.
1.2.2. The structure of the exocyst complex
The recent crystal structure data have provided valuable insights into the structure and molecular organization of the exocyst complex. So far, partial crystal
8 Jianglan Liu THE ROLE OF THE EXOCYST IN EXOCYTOSIS AND CELL MIGRATION Chapter 1 Figure 1.3
Figure 1.3 Protein-protein interactions among the exocyst subunits. Interactions among individual exocyst components has been studied using (1) yeast two-hybrid assay (red), (2) protein binding assay with recombinant proteins purified from E. coli
(green) or (3) protein binding assay with in vitro translated proteins (blue). The diagram was generated based on Munson and Novick, 2006. The interaction map suggests that most exocyst subunit can interact with multiple exocyst subunits in the complex.
9 Jianglan Liu THE ROLE OF THE EXOCYST IN EXOCYTOSIS AND CELL MIGRATION Chapter 1 structures of five exocyst components have been solved. These include the near full-length yeast and mouse Exo70 (Dong et al, 2005; Hamburger et al, 2006; Moore et al, 2007); the C-terminal domains of Drosophila Sec15 (Wu et al, 2005), yeast
Exo84 (Dong et al., 2005) and yeast Sec6 (Sivaram et al., 2006); and the N-terminal domain of yeast Sec3 (Yamashita et al., 2010; Baek et al., 2010). Despite the little sequence homology among the exocyst components, most of their structures shared some common features: they all exhibit rod-like structures consisting of two or more consecutively packed helical bundles, with each bundle composed of three to five
α-helices connected by loops (Figure 1.4). Predictions of the secondary structure suggest that the remaining unsolved domains and exocyst components may also be composed of similar helical folds. The structural similarity among exocyst components suggests that they evolved from an ancient common ancestor. In addition, several subunits of the exocyst have been found to share distant sequence similarity with subunits of two other tethering complexes that function at the Golgi apparatus, the Golgi-associated retrograde protein (GARP) complex and the conserved oligomeric Golgi (COG) complex (Whyte et al., 2001). The crystal structure of the
COG subunit COG2 reveals a six-helix bundle with a general resemblance to that of the exocyst components (Cavanaugh et al., 2007). These findings suggest that the exocyst, the COG and the GARP complexes, which are all constructed from helical
10 Jianglan Liu THE ROLE OF THE EXOCYST IN EXOCYTOSIS AND CELL MIGRATION Chapter 1 Figure 1.4
Figure 1.4 Structural studies of the exocyst subunits. The diagram shows crystal structures of near full length yeast Exo70 (Dong et al, 2005; Hamburger et al, 2006), and the C-terminal domains of Drosophila Sec15 (Wu et al, 2005), yeast Exo84
(Dong et al, 2005), and yeast Sec6 (Sivaram et al, 2006), and N-terminus of yeast
Sec3 (Yamashita et al., 2010; Baek et al., 2010). Except for Sec3N, these exocyst subunits all display rod like structures composed of two or more consecutively packed helical bundles, each consists of three to five a-helices linked by loops. The core domain of Sec3N consists of a semi-open seven-stranded β-barrel. Figure adapted from Munson and Novick, 2006 and Yamashita et al., 2010.
11 Jianglan Liu THE ROLE OF THE EXOCYST IN EXOCYTOSIS AND CELL MIGRATION Chapter 1 bundles, are structurally related and might have diverged from a common ancestor to mediate vesicle tethering at various stages of membrane trafficking.
The structure of individual exocyst components also provides a clue to the assembly of the exocyst complex. For example, Exo70 has an extended rod-shaped structure. Initial mapping of the binding sites of Sec8 and Sec10 on the Exo70 structure indicates that they have an extended interface with Exo70 along the length of the structure, suggesting that Sec8 and Sec10 themselves also have extended rod-like structures (Dong et al., 2005). Therefore, the assembly of the exocyst complex probably involves the packing of at least several rod-shaped subunits in an elongated side-to-side fashion. This is consistent with the previously described
‘Y’-shaped structure of the mammalian exocyst complex after fixation (Hsu et al.,
1998, see later).
It is worth noting that although the distribution of electronic charges is not conserved in yeast and mammalian Exo70 molecules, both of them share a C-terminal electropositive domain. Indeed, the C-terminal domain is the most highly conserved portion of Exo70. In this region, homologous residues form a surface patch of basic residues at the extreme C-terminal tip of the rod, suggesting a conserved function for this last domain. In many cases the association of proteins with the PM can be mediated by interactions with the negatively charged phospholipids in membrane via
12 Jianglan Liu THE ROLE OF THE EXOCYST IN EXOCYTOSIS AND CELL MIGRATION Chapter 1 clusters of basic residues (for reviews, see McLaughlin et al., 2002; Balla, 2005).
Therefore, the C-terminal basic patch of Exo70 suggests a possible interaction with or proximity to the membrane. It would be intriguing to examine whether Exo70 directly interacts with the membrane.
Recently, the crystal structure of the N-terminal domain of yeast Sec3 (Sec3N) was resolved (Yamashita et al., 2010; Baek et al., 2010). Different from the above four rod-like structures, Sec3N consists of a semi-open seven-stranded β-barrel. The barrel is capped at one end by a C-terminal α-helix, whereas at the other end of the barrel, three inter-strand variable loops form the canonical phosphoinositide-binding pocket (Figure 1.4). The structure of Sec3N represents a typical pleckstrin homology
(PH) domain, which belongs to a structural superfamily including the phosphotyrosine-binding, Ena/VASP homology, and Ran-binding domains (Lemmon,
2004). These domains lack sequence similarity, but all share this core structure. The structure of Sec3N suggests that it may directly associate with the plasma membrane through its PH domain.
While the yeast Sec3N (amino acids 1-320) is composed of β-barrel, the region following the PH domain is predicted to be predominantly helical in structure.
Residues 320–470 are predicted with high probability to form a coiled-coil structure; fold recognition programs further suggest that the region starting from residue 610 to
13 Jianglan Liu THE ROLE OF THE EXOCYST IN EXOCYTOSIS AND CELL MIGRATION Chapter 1 the C terminus of Sec3 is composed of a series of long helices separated by short loops (Baek et al., 2010). This is consistent with the signature pattern of the structure of other exocyst subunits -- the packed helical bundles.
The overall organization of the purified mammalian exocyst complex was revealed by Quick-Freeze/Deep-Etch Electron Microscopy (Hsu et al., 1999).
Without fixation with glutaraldehyde, the complex shows variable conformations usually as a set of four or six arms 4-6 nm in width and 10-30 nm in length which radiate outward from a central point. The arms appear to attach to the body through a flexible hinge region, allowing the arms to extend from the body at varying angles.
Following glutaraldehyde fixation, the exocyst complex displays a much more uniform and packed Y-shaped or T-shaped structure, with a 30 nm x 13 nm stem and two 6 nm x l5 nm arms branched away from one end of the central body (Hsu et al,
1998). The difference in the shape of fixed and unfixed complex suggest that the exocyst might undergo conformational changes when it functions in vesicle tethering.
Or it may represent assembled and disassembled complex, respectively. The structure of fixed complex may more closely resemble the native assembled exocyst structure, in which the rod-like subunits pack against one another along their length; whereas the structure represented in the unfixed preparation is in the process of disassembly into individual subunits.
14 Jianglan Liu THE ROLE OF THE EXOCYST IN EXOCYTOSIS AND CELL MIGRATION Chapter 1 To date, the description of the exact subunit organization and structure of the holo-exocyst complex is still incomplete. The elucidation of the structure of other individual subunits and, ultimately, the intact complex at higher resolution will clearly improve our understanding of the function of the exocyst and the molecular mechanism of vesicle tethering.
1.2.3. Subcellular localization of the exocyst
1.2.3.1. In budding yeast
In budding yeast, the exocyst exhibits a cell cycle-dependent localization pattern that represents sites of active exocytosis and cell surface expansion (Finger et al., 1998; Mondesert et al., 1997; TerBush and Novick, 1995) (Figure 1.5). As cells enter the cycle, exocyst components are found in a patch at the pre-bud site. During bud emergence, the exocyst is concentrated at the tip of the bud, corresponding to the apical growth of the daughter cell. When the daughter cell has grown to a certain size and their growth pattern has switched from apical to isotropic, the exocyst proteins are correspondingly redistributed to the entire surface of the growing bud. At the time of cytokinesis, exocyst components are redistributed to the mother-daughter junction, where active exocytosis takes place for the completion of cytokinesis and separation of the two cells (TerBush et al, 1995; Finger et al, 1998). This pattern of localization
15 Jianglan Liu THE ROLE OF THE EXOCYST IN EXOCYTOSIS AND CELL MIGRATION Chapter 1 Figure 1.5
Figure 1.5 Subcellular localization of the exocyst in various cell types. The diagram reveals the localization of the exocyst in budding yeast, developing neurons, polarized epithelial cells, and migrating cells. The plasma membrane domains where the exocyst is localized are marked by red. In budding yeast, the exocyst is concentrated at the bud tip in small-budded cells, and relocates to the mother-daughter junction during cytokinesis. In developing neurons, the exocyst is enriched in axonal branch points, the growth cone of the extending neurites, and discrete puncta along axons where mature synapses will form. In polarized epithelial cells, the exocyst is enriched at the apical portion of the lateral membrane. In migrating cells, the exocyst is recruited to the leading edge of the plasma membrane.
16 Jianglan Liu THE ROLE OF THE EXOCYST IN EXOCYTOSIS AND CELL MIGRATION Chapter 1 mirrors the pattern of deposition of newly synthesized materials at the cell surface.
Consistent with the cortical localization, biochemical analyses reveal that about
20-25% of the exocyst proteins are associated with the plasma membrane in budding yeast (Bowser and Novick, 1991; Bowser et al, 1992).
In yeast, while all the exocyst components are localized to the growing end of the daughter cells, they seem to have different targeting mechanisms. Sec3 is localized to the bud tip independent of actin cables, along which the vesicles are transported (Finger et al., 1998; Boyd et al., 2004). A portion of Exo70 is transported to sites of exocytosis on vesicles, but approximately half also localizes independently of vesicle traffic (Boyd et al., 2004). By contrast, the remaining exocyst components are associated with exocytic vesicles and delivered to sites of exocytosis through actin cables (Boyd et al., 2004; Zajac et al., 2005; Zhang et al., 2005). These results led to the hypothesis that Sec3 and Exo70 associate with the plasma membrane and interact with the rest of the exocyst components on the arriving vesicles, which promotes the assembly of the exocyst complex to tether the secretory vesicles to the plasma membrane. Consistently, recent structural studies have revealed that Exo70 and Sec3 may directly interact with the plasma membrane through basic residues or
PH domain, respectively, which supports the model that Exo70 and Sec3 are potential landmarks in the exocyst complex (Dong et al., 2005; Hamburger et al., 2006; Moore
17 Jianglan Liu THE ROLE OF THE EXOCYST IN EXOCYTOSIS AND CELL MIGRATION Chapter 1 et al., 2007; Yamashita et al., 2010; Baek et al., 2010).
Septins are additional regulators of the localization of the exocyst in yeast.
Septins are a family of GTPases conserved from yeast to mammals that play central roles in cell division and cell polarization (For reviews, see Spiliotis and Nelson,
2006; Barral and Kinoshita, 2008). Septins act as membrane diffusion barriers between different membrane domains and as molecular scaffolds for the localization of many signaling proteins, bud site selection proteins, and chitin synthases. In budding yeast, septins polymerize to form ring-like structure at the bud neck throughout the entire budding process (Longtine and Bi, 2003). The intact septin ring is essential for the spatial restriction of the exocyst in the bud during isotropic growth
(Barral et al, 2000) as well as the recruitment of the exocyst to the bud neck during cytokinesis (Dobbelaere and Barral, 2004). However, how septin undergoes highly-regulated functional transitions from spatial restriction during isotropic growth to protein recruitment during cytokinesis is unknown. Interestingly, the mammalian exocyst was shown to coimmunoprecipitate with septins and microtubules from rat brain extracts (Hsu et al., 1998; Vega and Hsu, 2001; Vega and Hsu, 2003). It remains under investigation whether there is a direct interaction between exocyst and septin, and whether septin mediates the spatial regulation of the exocyst through this interaction.
18 Jianglan Liu THE ROLE OF THE EXOCYST IN EXOCYTOSIS AND CELL MIGRATION Chapter 1 1.2.3.2. In mammalian cells
The subcellular localization of the exocyst in mammalian cells is much more complex than that in yeast cells (Figure 1.5). a) Epithelial cells
The establishment of epithelial polarity involves the initial formation of cell–cell adhesion and the subsequent specialization of structurally and functionally distinct apical and basal-lateral plasma membrane domains. The subcellular distribution of the exocyst complex dramatically changes during development of epithelial polarity. In single polarized Madin-Darby canine kidney (MDCK) cells in contact with the substratum (contact-naive cells), the exocyst components are diffusely distributed in the cytosol. Upon initiation of E-cadherin-mediated cell adhesion, they are rapidly recruited to the plasma membrane at sites of cell–cell contact (Grindstaf et al., 1998; Yeaman et al., 2001; Yeaman et al, 2004). In fully polarized cells, the exocyst is sorted out to the apex of the lateral membrane and co-localized with components of tight junction and nectin complexes, but only partially overlapped with E-cadherin, which also localized to the rest of the forming lateral membrane (Grindstaf et al., 1998; Yeaman et al, 2004). Consistent with this spatiotemporal redistribution, the exocyst co-fractionates with membranes specifically enriched in apical junctional proteins (Yeaman et al, 2004). The plasma
19 Jianglan Liu THE ROLE OF THE EXOCYST IN EXOCYTOSIS AND CELL MIGRATION Chapter 1 membrane localization of the exocyst in polarized epithelial cells is dependent upon the integrity of the cell-cell adhesion (Grindstaff et al, 1998; Yeaman et al, 2004). It was shown that E-cadherin and nectin-2α mediates the recruitment of the exocyst to the site of cell-cell adhesion. The exocyst components were recruited to the cell-cell contacts when E-cadherin and nectin-2α were co-expressed in fibroblasts (Yeaman et al, 2004). Biochemical studies also indicate that exocyst is associated with apical junctional, proteins, including E-cadherin and nectin-2α, in polarized epithelial cells
(Yeaman et al, 2004).
In addition to the plasma membrane localization, the exocyst components were also shown to be localized in membrane-bound structures at the perinuclear region of epithelial cells (Yeaman et al., 2001; Prigent et al., 2003; Folsch et al., 2003; Oztan et al., 2007). However, which intracellular compartment at the prenuclear region does the exocyst associate with is still under investigation. Some studies found that the exocyst complex is present on both TGN and plasma membrane in normal rat kidney
(NRK) cells that formed either fibroblast-(NRK-49F) or epithelial-like (NRK-52E) intercellular junctions (Yeaman et al., 2001). In contrast, other studies showed that the exocyst was associated with the recycling endosomal compartments in MDCK cells. (Prigent et al., 2003; Folsch et al., 2003; Oztan et al., 2007).
The controversy in exocyst localization may reflect the growth conditions of
20 Jianglan Liu THE ROLE OF THE EXOCYST IN EXOCYTOSIS AND CELL MIGRATION Chapter 1 the cells, the degree of cellular polarization, fixation methods, differential recognition of junctional versus intracellular populations of the exocyst subunits by different antibodies (Yeaman et al., 2001, 2004), and the possibility that the exocyst complex may exist in different conformational states (extended or closed) depending on its localization in the cell (Munson and Novick, 2006).
b) Neurons
The localization of the exocyst in neurons is largely dependent on the differentiation state of the neuronal cells. In developing neurons that have not formed synapses or in cultured neurons that are in the process of differentiation, the exocyst is found in the cell body, as well as in axonal branching points, the growth cone of the extending neurites, and discrete puncta along axons where mature synapses will form
(Hsu et al., 1996; Kee et al., 1997; Hazuka et al., 1999; Vega and Hsu, 2001; Brown et al., 2001; Murthy et al., 2003). In contrast, in mature neurons, the exocyst components are largely absent from mature synapses, where synaptic vesicle cycling takes place (Hazuka et al, 1999). The localization pattern suggests that exocyst is involved in synaptogenesis and neurite outgrowth, but not required for the cycling of synaptic vesicles and neurotransmitter release at the presynaptic membranes. On the other hand, exocyst components have been found to be associated with the
21 Jianglan Liu THE ROLE OF THE EXOCYST IN EXOCYTOSIS AND CELL MIGRATION Chapter 1 postsynaptic membrane of mature synapse, and have been implicated in the delivery of glutamate receptors to the postsynaptic membrane for synaptic transmission (Sans et al, 2003; Gerges et al, 2006).
c) Cytokinesis
During cell division, the exocyst is initially localized to centrosome/spindle poles in interphase, then with mitotic spindles in mitosis, and later enriched at the central spindles and midbody during cytokinesis. The localization of the exocyst at the abscission site could also be detected in cells ectopically expressing exocyst components (Chen et al., 2006). The spatial regulation of the exocyst is coupled to the dynamic redistribution of its interacting Ral GTPases (Chen et al., 2006; Cascone et al., 2008). During cytokinesis, the recruitment of the exocyst to the midbody was shown to be mediated by centriolin, a coil-coiled protein that is localized to the midbody and required for abscission. Centriolin directly interacts with the exocyst subunit Sec15 and depleting centriolin disrupts the midbody localization of the exocyst (Gromley et al., 2005).
d) Migrating cells
Cell migration requires the coordination of polarized exocytosis and actin
22 Jianglan Liu THE ROLE OF THE EXOCYST IN EXOCYTOSIS AND CELL MIGRATION Chapter 1 cytoskeleton remodeling to support dynamic reorganization of the plasma membrane at the leading edge. The initial step of cell migration is the formation of cell protrusions in the direction of cell movement. Overexpression of Exo70 induces numerous actin-based protrusive structures resembling filopodia (Wang et al., 2004;
Xu et al., 2005; Zuo et al., 2006). And Exo70 is localized at the tips of filopodia, where it is colocalized with the actin staining. In migrating cells, the exocyst subunits are enriched at the leading edges of plasma membranes free of cell-cell contacts, where they are colocalized with the Arp2/3 complex (Rosse et al., 2006; Zuo et al.,
2006). This localization pattern of the exocyst at the leading edge reflects its role in mediating vesicular traffic to this region as well as its direct interaction with the
Arp2/3 complex (Zuo et al., 2006).
The recruitment of the exocyst to the leading edge may involve different molecular mechanisms. It was shown that RalB expression is required for promoting both exocyst assembly and localization to the leading edge of moving cells (Rosse et al., 2006). Recently, it was reported that the polarized delivery of the exocyst to the leading edge of migrating epithelial cells is dependent on aPKCs (Rosse et al., 2009).
Interestingly, it was shown that aPKC localization at the leading edge is also dependent on the exocyst. Potential molecular links of how these regulators coordinate to mediate the exocyst localization are still under investigation. It is
23 Jianglan Liu THE ROLE OF THE EXOCYST IN EXOCYTOSIS AND CELL MIGRATION Chapter 1 interesting to note that the absence of ongoing membrane traffic did not affect the leading edge localization of the exocyst subunit Exo70, as revealed by the persistence of its leading edge localization in cells treated with Brefeldin A (BFA), which inhibits vesicular transport within the Golgi apparatus (Rosse et al., 2006). This is consistent with the finding in budding yeast that the polarized localization of Exo70 (and Sec3) is independent of actin cytoskeleton and on-going membrane traffic. It remains to be determined whether the leading edge localization of other exocyst subunits is sensitive to BFA treatment.
In migrating prostate tumor cells, the exocyst was shown to be co-localized with cell–substrate paxillin-containing focal complexes that are newly formed within protrusive cellular extensions (pseudopods) at the leading edge (Spiczka et al., 2008).
Biochemical studies also showed that the exocyst is associated with these paxillin-containing complexes within pseudopods. Localization of the exocyst within the pseudopods is dependent on Ral GTPases, which control the association between
Sec5 and paxillin.
e) Primary cilium
The exocyst components have also been detected at the basal body, a centriole derived organelle, of the primary cilium in polarized epithelial cells (Rogers et al.,
24 Jianglan Liu THE ROLE OF THE EXOCYST IN EXOCYTOSIS AND CELL MIGRATION Chapter 1 2004; unpublished data from Guo lab), reminiscent of the colocalization of the exocyst with centrosomes in interphase cells (Chen et al, 2006).
1.2.4. Cellular functions of the exocyst
1.2.4.1. Functions of the exocyst in exocytosis
1. Budding yeast
Most of the exocyst subunits were originally discovered as SEC gene products in budding yeast due to their essential function in secretion (Novick et al., 1980).
Temperature-sensitive mutants of the exocyst components are defective in the secretion of invertase, which results in intracellular accumulation of the enzyme.
Furthermore, electron microscopic studies revealed the accumulation of 80-nm
Golgi-derived secretory vesicles in the mutant cells, suggesting that the exocyst functions at the post-Golgi stage of exocytosis. To further define which step the exocyst functions during post-Golgi trafficking, the vesicle accumulation patterns are compared between the exocyst and myo2 mutants by electron microscopic analysis.
In budding yeast, during the apical and isotropic growth of the daughter cell, post-Golgi secretory vesicles are delivered to the bud through actin cables by Myo2, a yeast type V myosin. In myo2 mutants, secretory vesicles accumulate almost exclusively in the mother cell. In contrast, in the exocyst mutants, the initial
25 Jianglan Liu THE ROLE OF THE EXOCYST IN EXOCYTOSIS AND CELL MIGRATION Chapter 1 accumulation of secretory vesicles occurs within the bud of the cell, suggesting that the transport of vesicles from donor compartments to the plasma membrane is not affected. To further confirm this, the phenotype of the double mutant for Myo2 and exocyst components was examined, which accumulates vesicles in the mother cell, as does the myo2 single mutant (Govindan et al, 1995; Walch-Solimena et al, 1997).
Thus, the exocyst appears to function at a stage subsequent to the delivery of vesicles to the vicinity of the plasma membrane. On the other hand, yeast genetic studies showed that overexpression of Sso1, a yeast homolog of the mammalian t-SNARE protein syntaxin, can suppress mutations in the exocyst mutants (Aalto et al., 1993).
In addition, in exocyst mutants, assembly of the SNARE complex is blocked (Grote et al, 2000). Thus, the exocyst function is required for the assembly of the SNARE complex and the fusion of the vesicles with the plasma membrane. Therefore, the exocyst functions downstream of the vesicle transport machineries, but upstream of the SNARE complex. It has been accepted in the field that the exocyst functions as a molecular tether which mediates the initial contact of the secretory vesicles with the plasma membrane prior to vesicle docking and fusion. This is also consistent with the localization of the exocyst at the site of plasma membrane where active exocytosis and membrane expansion occur.
26 Jianglan Liu THE ROLE OF THE EXOCYST IN EXOCYTOSIS AND CELL MIGRATION Chapter 1 2. Mammalian cells
In animal cells, the role of the exocyst in exocytosis and cell surface expansion has been demonstrated in a variety of cell types. Comparing to yeast cells, the functions of the exocyst in mammalian cells are much more complex. While the yeast exocyst mainly functions at the plasma membrane, the mammalian exocyst seems to functions at multiple stages during exocytosis. Moreover, the mammalian exocyst is not required for all types of exocytic events in animal cells. The following are several examples of the functions of mammalian exocyst: a) Epithelial cells
In fully polarized MDCK epithelial cells, addition of function-blocking Sec8 antibodies to streptolysin-O–permeabilized cells perturbed the membrane targeting of the basolateral membrane protein LDL receptor (LDLR), but not the apical membrane protein p75NTR. This is consistent with the localization of the exocyst to the apex of the lateral membrane. Therefore, it has been proposed that the exocyst specifies the delivery of vesicles to the basolateral domain, but not the apical domain in polarized epithelial cells (Grindstaff et al., 1998). However, this hypothesis has been challenged by the recent findings that the exocyst is involved in the targeting of vesicles to both the apical and basolateral domains of the cell (Oztan et al., 2007;
Cresawn et al, 2007). Introducing Sec8 antibodies to partially permeabilized
27 Jianglan Liu THE ROLE OF THE EXOCYST IN EXOCYTOSIS AND CELL MIGRATION Chapter 1 epithelial cells revealed exocyst requirements for several endocytic pathways including basolateral recycling, apical recycling, and basolateral-to-apical transcytosis by (Oztan et al., 2007). It was also found that expressing a dominant-negative C-terminal fragment of Sec15 affected the exocytosis of newly synthesized apical membrane domain marker endolyn, which is delivered en route through the Rab11-positive apical recycling endosomes to the plasma membrane
(Cresawn et al, 2007). Although it is not clear whether the block of secretion is imposed at the plasma membrane or the donor membrane (see below), these results clearly indicate that the exocyst function is required for the targeting of a subset of apical-targeted cargos.
In epithelial cells, the exocyst components were found to be localized to both the plasma membrane and perinuclear membrane compartments, presumably TGN and/or recycling endosomes (Yeaman et al., 2001; Prigent et al., 2003; Folsch et al.,
2003; Oztan et al., 2007). Blocking distinct pools of the exocyst by specific antibodies resulted in defects in exocytosis at different stages. In non-polarized NRK cells, selective perturbation of the TGN pool of the exocyst disrupted the transit of the vesicular stomatitis virus G (VSVG) proteins to the plasma membrane, which resulted in cargo accumulation in a perinuclear region. In contrast, selectively perturbing the plasma membrane pool of the exocyst did not affect the transport of
28 Jianglan Liu THE ROLE OF THE EXOCYST IN EXOCYTOSIS AND CELL MIGRATION Chapter 1 VSVG from TGN to the plasma membrane, but disrupted the plasma membrane incorporation of the proteins, which resulted in cargo accumulation near the plasma membrane (Yeaman et al., 2001). In polarized MDCK cells, vesicle budding was reconstituted from apical recycling endosomes (ARE) in mechanically perforated cells. Addition of antibodies against Sec8 resulted in a significant inhibition of cargo release from the reconstituted ARE (Oztan et al., 2007), suggesting that the exocyst may also play some role in steps that precede vesicle targeting, possibly including cargo exit or vesicle transport. These results suggest that different pools of the exocyst function in different vesicular trafficking stages: while the plasma membrane pool functions in vesicle tethering, the perinuclear pool might function at an earlier stage at the donor compartment.
b) Adipocytes
Insulin stimulates glucose transport in adipocytes and muscle by inducing the redistribution of glucose transporter 4 (GLUT4) from intracellular locations to the plasma membrane. GLUT4 trafficking is a highly regulated multi-step process that includes translocation, targeting, docking and fusion of GLUT4-containing vesicles with the plasma membrane. The first evidence to reveal the role of the exocyst in
GLUT4 transport is its interaction with the GTPase TC10 (Inoue et al., 2003). TC10
29 Jianglan Liu THE ROLE OF THE EXOCYST IN EXOCYTOSIS AND CELL MIGRATION Chapter 1 interacts with Exo70 and recruits Exo70 as well as Sec6 and Sec8 to the plasma membrane in response to insulin signaling, where the multiprotein complex is assembled (Inoue et al., 2003; Ewart et al., 2005). Knockdowns of exocyst components by RNA interference blocked insulin-stimulated glucose uptake (Inoue et al., 2006). Overexpression of the N-terminus of Exo70 (Exo70-N), which acts as a dominant negative mutant, inhibited the fusion of the Glut4 vesicles with the plasma membrane, without affecting the transport of the vesicles to the cell periphery (Inoue et al., 2003). In addition to TC10, another G protein RalA was also shown to function in glucose transport downstream of insulin signaling through its interaction with the exocyst (Chen et al., 2007). How these regulations are coordinated to mediate the function of the exocyst still needs clarification.
The function of the exocyst in GLUT4 vesicle exocytosis requires its association with lipid rafts. Upon insulin stimulation, the exocyst is recruited to the lipid rafts and then assembled there, which sets up targeting sites for GLUT4 vesicles.
The anchoring of the exocyst at the lipid rafts is likely mediated by the interaction between Sec8 and the PDZ domain-containing protein SAP97, a MAGUKs family protein expressed in lipid rafts. Depleting SAP97, which prevented the lipid raft recruitment of the exocyst, or disrupting the exocyst function, blocked the translocation of Glut4 vesicles to the lipid raft and prevented the membrane
30 Jianglan Liu THE ROLE OF THE EXOCYST IN EXOCYTOSIS AND CELL MIGRATION Chapter 1 incorporation of GLUT4 (Inoue et al, 2006). On the other hand, Exo70 was shown to interact with Snapin, a ubiquitous protein known to associate with SNAP23, a major t-SNARE regulating GLUT4 vesicle trafficking (Rea et al., 1998). Snapin was predominantly targeted to the plasma membrane and depletion of Snapin in adipocytes using RNA interference inhibits insulin-stimulated glucose uptake. Thus, it is proposed that Exo70 associates with SNARE machinery through the interaction with Snapin, which promotes the anchoring of Exo70 at exocytosis sites of GLUT4 vesicles (Bao et al., 2008). Collectively, these studies provide insights into the mechanism of the targeting of the exocyst to GLUT4 vesicle exocytosis sites during glucose uptake.
All the above studies used newly differentiated 3T3-L1 adipose cells to study
GLUT4 transport. Recently, a group investigated the effects of Exo70 on tethering and fusion of GLUT4 vesicles in primary isolated rat adipose cells (Lizunov et al.,
2009). They utilized total internal reflection (TIRF) microscopy to visualize the trafficking of GLUT4 vesicles, in which the tethering and fusion steps can be observed separately. They found that overexpression of Exo70 in the presence of insulin did not further promote the fusion rate and exposure of GLUT4. In sharp contrast to the previous studies, the Exo70-N mutant induced tethering of GLUT4 vesicles independent of insulin, however it did not lead to fusion and exposure of
31 Jianglan Liu THE ROLE OF THE EXOCYST IN EXOCYTOSIS AND CELL MIGRATION Chapter 1 GLUT4 at the plasma membrane. Upon insulin stimulation, the stationary pre-tethered GLUT4 vesicles in Exo70-N mutant cells underwent fusion without relocation. Taken together, their data suggest that fusion of GLUT4 vesicles is the rate-limiting step regulated by insulin downstream of Exo70-mediated tethering.
The secretion of insulin itself is also dependent on the exocyst. In pancreatic β cells, the exocyst is implicated in the secretion of insulin-containing dense core vesicles. Overexpressing truncated, dominant negative exocyst mutants inhibited the docking of the insulin vesicles to the plasma membrane and substantially reduced insulin secretion in response to glucose stimulation. However, the final fusion step was not affected by these mutants (Tsuboi et al., 2005).
c) Neurons
In developing neurons, the exocyst is essential for neurite extension and synaptogenesis, processes that require active plasma membrane addition and remodeling. Overexpressing a dominant negative fragment of Sec10 blocked neurite outgrowth in cultured neuronal cells treated with nerve growth factor (NGF) (Vega and Hsu, 2001). In contrast, the exocyst was revealed to be dispensable for neurotransmitter release from synaptic vesicles at mature synapse (Hazuka et al.,
1999; Brown et al., 2001; Murthy et al., 2003). In sec5 mutants in Drosophila,
32 Jianglan Liu THE ROLE OF THE EXOCYST IN EXOCYTOSIS AND CELL MIGRATION Chapter 1 synaptic transmission continued to be robust despite the decline of maternal Sec5 protein (Murthy et al., 2003). The function of the exocyst in neurite outgrowth and synaptogenesis but not in synaptic vesicle exocytosis well corresponds to the localization of the exocyst in developing neurons.
The molecular mechanisms underlying the function of the exocyst in neurons are still under investigation. RalA was reported to regulate exocyst function in neurite branching (Lalli and Hall, 2005). PAR-3 and atypical protein kinase C (aPKC) were shown to associate with the exocyst in a RalA-dependent manner, which is essential for early stages of neuronal polarization (Lalli, 2009). Moreover, TC10-Exo70 interaction was shown to be essential for membrane expansion and axonal specification in developing neurons (Dupraz et al., 2009).
In neurons, the exocyst is also involved in the dendritic delivery of the NMDA receptor (NMDAR) and AMPA receptors (AMPARs) to postsynaptic membrane/postsynaptic density (PSD), which is essential for synaptic transmission and synaptic plasticity. The role of the exocyst in the delivery of NMDAR is thought to involve interactions between Sec8, which contains a PDZ-binding domain at its
C-terminus, and the PDZ-containing proteins -- postsynaptic density protein-95 and synapse-associated protein 102 (Riefler et al, 2003; Sans et al, 2003). Overexpressing a Sec8 mutant defective in PDZ binding inhibited the surface exposure of the
33 Jianglan Liu THE ROLE OF THE EXOCYST IN EXOCYTOSIS AND CELL MIGRATION Chapter 1 NMDAR (Sans et al, 2003). During AMPARs delivery, the exocyst function is implicated in at least two stages. While overexpression of a dominant negative
C-terminal fragment of Sec8 abolished the transport of AMPARs toward PSD, a dominant negative N-terminal fragment of Exo70 blocked the insertion of the receptors to the plasma membrane (Gerges et al, 2006). This observation is consistent with the notion that the exocyst functions in multiple vesicular traffic stages in exocytosis.
1.2.4.2. Functions of the exocyst in endosomal recycling
In addition to functioning in biosynthetic secretory pathway, the exocyst has also been implicated in the endocytic recycling pathway. Recycling endosomes mediate the transport of the internalized plasma membrane receptors back to the cell surface and are major sources of cargos destined to the plasma membrane in many types of cells. The role of the exocyst in endocytic recycling is consistent with the localization of the mammalian exocyst to the recycling endosomes. Loss of exocyst function blocks the recycling of internalized cargos and results in their accumulation in recycling endosomes. For example, depletion of Sec5 or dominant inhibition of
Sec10 affects the function and the morphology of the recycling pathway (Prigent et al,
2003). In polarized epithelial cells, disrupting the exocyst function by treating
34 Jianglan Liu THE ROLE OF THE EXOCYST IN EXOCYTOSIS AND CELL MIGRATION Chapter 1 permeabilized cells with specific antibodies blocked multiple endocytic recycling pathways, including basolateral recycling, apical recycling, and basolateral-to-apical transcytosis (Oztan et al., 2007). Overexpression of Sec15 also blocked the recycling of transferrin receptors and resulted in its accumulation in the recycling endosomes
(Zhang et al, 2004).
The exocyst has also been shown to be involved in endocytic recycling in other organisms. Disrupting the functions of the Drosophila exocyst components Sec5,
Sec6, and Sec15 in epithelial cells leads to E-Cadherin accumulation in an enlarged
Rab11 recycling endosomal compartment and inhibits E-Cadherin delivery to the membrane (Langevin et al., 2005). In Exo84 mutants at advanced stages of epithelial degeneration in the Drosophila embryo, apical and adherens junction proteins accumulate in an expanded recycling endosome compartment in the Drosophila embryo (Blankenship et al., 2007). Drosophila oocytes homozygous for a sec5 hypomorphic mutant were defective in endocytic recycling of the yolk receptor and blocked yolk uptake (Murthy and Schwarz, 2004; Sommer et al, 2005).
Recycling endosomes was also found to act as intermediates in biosynthetic secretory pathway after the cargos exit the Golgi apparatus (Hoekstra et al, 2004;
Ang et al, 2004). Therefore, the roles of the exocyst in exocytosis and endocytic recycling pathways are likely to be interrelated, and the exocyst could function
35 Jianglan Liu THE ROLE OF THE EXOCYST IN EXOCYTOSIS AND CELL MIGRATION Chapter 1 analogously to mediate the exocytosis of newly synthesized cargos, or cargos internalized from the plasma membrane for recycling.
1.2.4.3. Other functions of the exocyst a) Cell polarity
Membrane traffic is a major contributing factor to cell polarization. Thus, the exocyst, as a key component functioning in exocytosis, is highly involved in cell polarity establishment.
In budding yeast, cell polarity is established through the localization and activation of Cdc42 at the intrinsic bud site (for review, see Park and Bi, 2007).
Cdc42 mediates directional exocytosis and polarized cell growth through its regulations of the components of exocytic pathways, such as the exocyst. In several cdc42 mutants, the exocyst components fail to localize to the bud tip or mother-daughter junction (Zhang et al, 2001). In turn, polarized exocytosis is also required for the robust asymmetric localization of Cdc42, which is thought to be associated with post-Golgi secretory vesicles and delivered to the bud tip through exocytic/recycling endocytic pathways (Ayscough et al., 1997; Wedlich-Soldner et al., 2003; 2004; Irazoqui et al., 2005; Zajac et al., 2005). Mutations in the exocyst component Sec5 not only lead to mislocalization of Cdc42, but also of Bem1, a
36 Jianglan Liu THE ROLE OF THE EXOCYST IN EXOCYTOSIS AND CELL MIGRATION Chapter 1 polarity establishment component which interacts with Cdc42 and its guanine nucleotide exchange factor (GEF) Cdc24 (Zajac et al., 2005). Thus, the exocyst may contribute to yeast cell polarity by reinforcing the localization of polarity determinants, such as Cdc42, to the site of polarization, which in turn regulates polarized exocytosis by controlling the polarity of actin cytoskeleton and the exocyst.
In addition, the exocyst component Sec15 was found to directly interact with Bem1 and recruit Bem1 as well as Cdc42 and Cdc24 to the bud tip (Zajac et al., 2005;
France et al., 2006). The interaction between Sec15 and Bem1 may also contribute to this positive feedback loop. Collectively, these observations suggest that the exocyst and the polarity-establishment complex coordinate to establish and maintain cell polarity.
In multicellular organisms, cell–extracellular matrix (ECM) and cell–cell interactions are important for polarity establishment. E-cadherin is one of the most important junction proteins for the establishment and maintenance of the apical-basolateral asymmetry in epithelial cells (Gumbiner et al. 1988; Uemura et al.
1996). The polarization of the epithelial cells might also apply a cyclical regulatory mechanism analogous to the polarization of the yeast cells: E-cadherin engagement is necessary for localization of the exocyst. Members of the exocyst complex are recruited to the apex of the lateral membrane through the association with junctional
37 Jianglan Liu THE ROLE OF THE EXOCYST IN EXOCYTOSIS AND CELL MIGRATION Chapter 1 complexes containing E-cadherin during the establishment of cell polarity in MDCK cells (Grindstaff et al. 1998; Yeaman et al. 2004). In turn, E-cadherin itself is delivered as a cargo via secretory vesicles to sites of polarity. Proper localization of the exocyst in epithelial cells is necessary for the specific delivery of polarity determinants including E-cadherin to the proper plasma membrane domains
(Grindstaff et al. 1998; Langevin et al. 2005); disruption of exocyst function led to intracellular accumulation of E-cadherin (Langevin et al. 2005; Blankenship et al.
2007). In addition to this cyclical regulatory network, several exocyst components were shown to affect epithelial polarity on their own. Overexpression of GFP-tagged
Exo70 significantly impaired the formation of tight epithelial monolayers, in which the E-cadherin staining is much more diffuse compared with the crisp pattern in parental cells (Matern et al., 2001). On the other hand, MDCK cells overexpressing
Sec10 were significantly taller than control cells when grown as a monolayer. In addition, these cells formed cysts more efficiently and rapidly, and generated more branching tubules from cysts upon HGF stimulation (Lipschutz et al., 2000).
b) Cytokinesis
Due to its role in targeted membrane addition through polarized exocytosis, the exocyst function is essential for cytokinesis. In the budding yeast S. cerevisiae, the
38 Jianglan Liu THE ROLE OF THE EXOCYST IN EXOCYTOSIS AND CELL MIGRATION Chapter 1 exocyst mediates vesicle fusion at the mother-daughter junction during cytokinesis.
Perturbing the exocyst function in anaphase cells resulted in accumulation of secretory vesicles around the bud neck and impairs actomyosin-ring contraction and cell cleavage (Novick et al, 1980; Salminen and Novick, 1989; Dobbelaere and
Barral, 2004; VerPlank and Li, 2005). In the fission yeast S. pombe, exocyst components localize to the actomyosin ring (Wang et al., 2002). Mutants of the exocyst component Sec8 accumulated 100nm secretory vesicles near the division septum and cannot complete extracellular separation of the two daughter cells.
In mammalian cells, disruption of the exocyst function leads to cytokinesis regression and abscission failure (Fielding et al, 2005; Gromley et al, 2005; Chen et al, 2006). Reduction of exocyst levels by RNAi blocked the delivery of the recycling endosome-derived vesicles to the cleavage furrow, a process important for membrane addition during furrow ingression and abscission (Fielding et al, 2005). Disruption of the exocyst also led to accumulation of exocytic vesicles at the midbody during abscission (Gromley et at, 2005). Overall, these results suggest that the exocyst is required for both furrow regression and abscission by mediating membrane incorporation at cleavage furrow or midbody during cytokinesis of the animal cells, which is well consistent with its localization at both sites.
39 Jianglan Liu THE ROLE OF THE EXOCYST IN EXOCYTOSIS AND CELL MIGRATION Chapter 1 c) Cell migration and cell invasion
The exocyst has also been shown to be involved in cell motility. The initial step of cell migration is the formation of cell protrusions in the direction of cell movement.
In mammalian cells, Sec5 was shown to be required for RalA-induced filopodia formation (Sugihara et al., 2002). Overexpression of Exo70 promoted numerous actin-based protrusive structures resembling filopodia (Wang et al., 2004; Zuo et al.,
2006), whereas inhibition of Exo70 by RNA interference (RNAi) or antibody microinjection blocked the formation of actin-based membrane protrusions induced by activated Cdc42 or Racl (Wang et al., 2004; Zuo et al., 2006; Pommereit and
Wouters, 2007). Depletion of Exo70 by RNAi affected various aspects of cell motility:
Exo70 knockdown prevented the recruitment of Arp2/3 complex to the leading edge and impaired the formation of lamellipodia. As a result, the Exo70 RNAi treatment affected cell migration in the wound healing assay and reduced the directional persistence of individual migrating cells (Zuo et al., 2006). Similarly, depletion of other exocyst components, including Sec5, Sec8, Sec 10 or Exo84, also affected cell motility by reducing the velocity of cell migration (Rosse et al., 2006; Rosse et al.,
2009).
How does the exocyst contribute to cell migration is still under investigation.
First, the exocyst may contribute to cell migration via its vesicle tethering function in
40 Jianglan Liu THE ROLE OF THE EXOCYST IN EXOCYTOSIS AND CELL MIGRATION Chapter 1 exocytosis. In prostate tumor cells, interference with exocyst activity impaired the efficient exocytosis of newly synthesized α5-integrin to the plasma membrane and inhibited tumor cell motility and matrix invasiveness (Spiczka and Yeaman, 2008).
On the other hand, it is also likely that the exocyst contributes to cell migration through some novel mechanisms distinct from its principle role in vesicle tethering.
One such mechanism is to coordinate cytoskeleton remodeling with vesicle trafficking. It was shown that Sec5-mediated filopodia can still form in the absence of ongoing membrane trafficking processes (Sugihara et al., 2002). The exocyst has been shown to associate with the Arp2/3 complex through direct interaction between their subunits Exo70 and Arpcl, respectively (Zuo et al., 2006). Based on these observations, it would be intriguing to examine whether the exocyst contributes to cell migration by directly regulating actin dynamics.
d) Ciliogenesis
The exocyst has also been implicated in primary cilia formation. Sec10 overexpression resulted in increased ciliogenesis (Zuo et al., 2009). On the other hand, deletion of Sec10 by short hairpin RNA (shRNA) blocked primary ciliogenesis (Zuo et al., 2009). Similarly, depleting other exocyst components were also shown to disrupt ciliogenesis (unpublished data from Guo lab).
41 Jianglan Liu THE ROLE OF THE EXOCYST IN EXOCYTOSIS AND CELL MIGRATION Chapter 1 1.2.5. Regulation of the exocyst by the Ras family of small GTPases
Exocytosis is highly regulated in eukaryotic cells. Vesicle tethering, as the initial contact of secretory vesicles with the specific domains of the plasma membrane, is thought to confer specificity on the subsequent SNARE-mediated fusion. Therefore, spatial and temporal regulation of the exocyst is crucial to determining where and when exocytosis takes place. Consistent with this notion, the exocyst has been found to be under the control of a number of small GTPases in a variety of cells (for review, see Novick and Guo, 2002; He and Guo, 2009) (Figure
1.6).
1.2.5.1. Rab and Arf GTPases Rab GTPases, constituting one of the most abundant families of Ras-like small
GTPases, are master regulators of membrane trafficking. There are at least 60 Rab proteins and they are associated with distinct membrane compartments along both exocytic and endocytic pathways and regulate vesicular trafficking at distinct stages through their interactions with specific tethering proteins and cytoskeleton elements (for reviews, see Pfeffer, 2001; Behnia and Munro, 2005; Grosshans et al., 2006).
The first GTPase found to interact with the exocyst is Sec4, the founding member of the Rab family (Salminen and Novick, 1987). Sec4 regulates exocytic trafficking from the TGN to the plasma membrane in budding yeast and was found to be associated with secretory vesicles and the plasma membrane (Salminen and Novick, 1987; Goud et al.,
42 Jianglan Liu THE ROLE OF THE EXOCYST IN EXOCYTOSIS AND CELL MIGRATION Chapter 1 1988). Similar to the exocyst, Sec4 also localizes to active growth sites during the yeast cell cycle (Novick and Brennwald, 1993). The exocyst subunit Sec15 was found to directly and specifically interact with the GTP-bound form of Sec4 through its effector domain (Guo et al., 1999). Furthermore, loss of Sec4p function leaves the exocyst in a partially assembled state (Guo et al., 1999). On the other hand, overproduction of Sec4p partially suppressed the secretion defects in sec15 temperature-sensitive mutant
(Salminen and Novick, 1989). These observations support the hypothesis that the exocyst is a downstream effector of Sec4: Sec4 interacts with the exocyst subunit Sec15 and triggers further interactions between Sec15 and other exocyst components to promote exocyst assembly, and eventually leads to targeting of secretory vesicles with specific plasma membrane domains.
In addition to Sec4, Sec15 was also shown to interact with Sec2, the guanine nucleotide exchange factor (GEF) of Sec4 (Medkova et al., 2006). Sec2 directly interacts with the GTP-bound form of Ypt32, a Rab GTPase regulating vesicle budding from the TGN (Jedd et al. 1997), and this interaction recruited Sec2 to secretory vesicles. Sec15 and Ypt32 bind to Sec2 in a competitive manner, which forms a negative feedback loop. A hypothesis has been proposed on the regulation of the Rab signaling cascade: Ypt32 interacts with Sec2, and this interaction mediates the recruitment of Sec2 to the post-Golgi secretory vesicles. Once on the vesicle,
43 Jianglan Liu THE ROLE OF THE EXOCYST IN EXOCYTOSIS AND CELL MIGRATION Chapter 1 Figure 1.6
Figure 1.6 The exocyst is the downstream effector of a variety of Ras family of small GTPases. Summary of the interactions between the exocyst and Ras-like small
GTPases. In budding yeast, the Rab GTPase Sec4 interacts with the Sec15 on the secretory vesicles; Rhol and Cdc42 GTPases interact with Sec3; Rho3 and Cdc42 interact with Exo70. In mammalian cells, the Rab GTPase Rab11 interacts with
Sec15 at the recycling endosomes. The Rho GTPase TC10 interacts with Exo70. The
Ral GTPases, including RalA and RalB, interact with Sec5 and Exo84. Arf6 interacts with Sec10.
44 Jianglan Liu THE ROLE OF THE EXOCYST IN EXOCYTOSIS AND CELL MIGRATION Chapter 1 Sec2 activates Sec4, which in turn recruits Sec4 and its effector Sec15 to the secretory vesicles, thereby releasing Ypt32 from Sec2 (Medkova et al., 2006).
Both in mammalian cells and Drosophila, Sec15 has been found to be a downstream effector of Rab11 (Zhang et al., 2004; Wu et al., 2005; Langevin et al.,
2005; Beronja et al., 2005). Rab11 is a Rab GTPase that regulates vesicular transport from recycling endosomes to the plasma membrane (Ullrich et al., 1996; Ren et al.,
1998). Sec15 directly interacts with the GTP-bound form of Rab11 through a single alpha helix in its most C-terminal helical bundle (Zhang et al., 2004; Wu et al., 2005).
Consistent with the physical interaction, Sec15 and other exocyst components were shown to be localized to Rab11-positive recycling endosomes in both mammalian and
Drosophila cells (Oztan et al., 2007; Langevin et al., 2005). The exocyst plays an important role in endocytic recycling pathways, which likely involves its interaction with Rab11. Disrupting the interaction between endogenous Sec15 and Rab11 by overexpressing a dominant-negative Sec15 C-term fragment abolished transcytosis from the basolateral apical domains in polarized MDCK cells (Oztan et al., 2007).
However, at which step does Rab11 regulate the exocyst function is still under investigation: recruitment of the exocyst to the recycling endosomes or the assembly of the exocyst or cargo release from recycling endosomes?
The homologues of Ypt32, Sec2 and Sec4 in mammalian cells are Rab11,
45 Jianglan Liu THE ROLE OF THE EXOCYST IN EXOCYTOSIS AND CELL MIGRATION Chapter 1 Rabin8 and Rab8, respectively (Walch-Solimena et al., 1997; Hattula et al., 2002).
Recently, it has been shown that the “Rab cascade” is conserved from yeast to mammals: Rab11, in its GTP-bound form, interacts with Rabin8 and kinetically stimulates the guanine nucleotide-exchange activity of Rabin8 toward Rab8 (Knödler et al., 2010). Furthermore, it has been suggested that these Rab GTPases coordinate with each other in the regulation of vesicular trafficking during primary ciliogenesis
(Yoshimura et al., 2007; Nachury et al., 2007; Knödler et al., 2010). Since the exocyst is the downstream effector of Rab11 and has also been implicated in ciliogenesis (Zuo et al., 2009; unpublished data from Guo lab), it will be interesting to elucidate the molecular connection between the Rab proteins and the exocyst complex during primary ciliogenesis.
It is worthy noting that several other tethering factors have also been shown to interact with specific Rab GTPases that regulate the corresponding membrane trafficking stages. The regulation of specific tethering proteins by distinct sets of Rab
GTPases may be a common mechanism that assures membrane traffic fidelity.
The Arf (ADP Ribosylation Factor) proteins comprise a conserved family of the Ras superfamily of small GTPases which act to regulate membrane traffic and organelle structure (For review, see Gillingham and Munro, 2007). The major cellular functions of Arf6 include uptake and recycling of plasma membrane proteins,
46 Jianglan Liu THE ROLE OF THE EXOCYST IN EXOCYTOSIS AND CELL MIGRATION Chapter 1 organization of the actin cytoskeleton and membrane dynamics, which are essential for cell spreading, cell motility and phagocytosis (For review, see D’Souza-Schorey
& Chavrier 2006). It has been shown that the exocyst subunit Sec10 acts as a downstream effector of Arf6 (Prigent et al, 2003). Expression of a constitutively-active Arf6 promotes the redistribution of Sec10 from the recycling endosomes to the plasma membrane during cell spreading, whereas expression of a dominant negative N-terminal Sec10 fragment interferes with Arf6-induced membrane spreading (Prigent et al, 2003). Collectively, these results suggest that
Arf6 recruits the exocyst from the recycling endosomes to the plasma membrane to promote membrane recycling toward specialized plasma membrane regions.
1.2.5.2. Rho GTPases
The Rho GTP-binding proteins are the key regulators of a wide range of cellular processes including cytoskeleton organization, membrane trafficking, cell polarization, cell growth and gene transcription (For review, see Bustelo et al., 2007).
In budding yeast, the Rho GTPase Cdc42 is a central regulator for the establishment of yeast cell polarity during bud formation (For review, see Park and Bi, 2007). In several cdc42 mutants, the exocyst components were depolarized (Zhang et al, 2001).
Biochemical analyses have revealed that Cdc42 in its GTP-bound state directly
47 Jianglan Liu THE ROLE OF THE EXOCYST IN EXOCYTOSIS AND CELL MIGRATION Chapter 1 interacts with the amino terminus of Sec3 and regulates the polarized localization of the exocyst. Disruption of this interaction leads to secretion and polarity defects
(Zhang et al. 2001; Zhang et al. 2008). In addition to Cdc42, the polarity of the exocyst in budding yeast is also regulated by another Rho GTPase, Rho1 (Guo et al,
2001). Disrupting Rhol function led to mislocalization of the exocyst components
(Guo et al, 2001). Sec3 directly interacts with the GTP-bound Rhol through its amino terminus, and functional Rho1 is required both to establish and to maintain the polarized localization of Sec3 (Guo et al, 2001). Rho1 and Cdc42 compete with each other for their binding to Sec3 in vitro (Zhang et al. 2001). In contrast to Cdc42 which functions in the establishment of cell polarity, the function of Rhol has been implicated in the maintenance of polarized cell growth (For review, see Park and Bi,
2007). Therefore, Cdc42 and Rho1 may regulate Sec3 at different cell cycle stages or physiological conditions. However, Sec3 is not the only mediator of the effect of Rho
GTPases on the exocyst, because some members of the complex are correctly targeted independently of the interaction between Rho and Sec3, and no discernable defects in exocytosis or cell growth were found with the deletion of the N-terminal
Rho-binding domain of Sec3 (Guo et al, 2001). These results reveal that additional pathway(s) may also contribute to the polarized localization of the exocyst besides the Rho-Sec3 interaction.
48 Jianglan Liu THE ROLE OF THE EXOCYST IN EXOCYTOSIS AND CELL MIGRATION Chapter 1 While Sec3 is the downstream effector of both Rho1 and Cdc42, Exo70 has been shown to be the downstream effector of another Rho GTPase, Rho3, which was reported to regulate both exocytosis and actin cytoskeleton (Adamo et al, 1999;
Robinson et al, 1999; Dong et al, 2005). However, mutations in Exo70 that disrupt its interaction with Rho3 did not affect polarized localization of exocyst components
(Adamo et al, 1999; Roumanie et al, 2005). The functional implication of
Rho3-Exo70 interaction still remains unclear. It was speculated that in addition to
Rho3, other factor(s) interact with Exo70 and regulate its activity. One such candidate could be Cdc42, because the phenotypes of exo70 mutants and cdc42-6, a particular mutant allele of Cdc42, are quite similar. Both of them are primarily defective in the secretion of a subset of exocytic vesicles, which carry cargos such as the endoglucanase Bgl2 that are needed for plasma membrane expansion and cell wall remodeling. Furthermore, the secretion defect in exo70 and cdc42-6 mutants is most pronounced at the early budding stage (He et al., 2007, Adamo et al., 2001). The phenotypic similarity between cdc42-6 and exo70 mutants suggests that Exo70 functions downstream of Cdc42. However, a physical interaction between activated recombinant Cdc42 and Exo70 was not detected (He et al., 2007a). Interestingly, a recent study found that C-terminal prenylation of Cdc42 as well as Rho3 promotes their interactions with Exo70 (Wu et al., 2010). Gain-of-function mutants in Exo70
49 Jianglan Liu THE ROLE OF THE EXOCYST IN EXOCYTOSIS AND CELL MIGRATION Chapter 1 were also identified that potently suppress mutants in Rho3 and Cdc42 deficient in exocytic function (Wu et al., 2010). Furthermore, several novel loss-of-function mutants in Exo70 were isolated which displayed phenotypes consistent with the notion that Exo70 acts as an effector for both Rho3 and Cdc42 function in polarized exocytosis (Wu et al., 2010).
Rho proteins are also involved in polarized exocytosis in mammalian cells
(Kroschewski et al., 1999; Musch et al., 2001; Rogers et al., 2003). It has been shown that Exo70 is a downstream effector of TC10, a Rho GTPase sharing sequence similarity with Cdc42 (Inoue et al., 2003). Exo70 interacts with GTP-bound TC10 through its N-terminus. Expression of the active form of TC10 in adipocytes recruited
Exo70 from the cytoplasm to the plasma membrane. Overexpression of full-length
Exo70 led to increase in insulin-mediated glucose uptake. On the other hand, overexpression of a dominant-negative N-terminal portion of Exo70 (Exo70-N) blocked glucose uptake. Exo70-N did not affect the translocation of GLUT4. Instead, it probably inhibited the targeting of GLUT4-containing vesicles to the plasma membrane. This study reveals a molecular connection between the exocyst and Rho protein in mammalian cells. The TC10-Exo70 interaction has also been found in neuronal cells in response to NGF stimulation. Both Exo70 and TC10 antagonized the activation of N-WASP by Cdc42, suggesting that the TC10-Exo70 interaction is
50 Jianglan Liu THE ROLE OF THE EXOCYST IN EXOCYTOSIS AND CELL MIGRATION Chapter 1 involved in the regulation of actin cytoskeleton during formation of membrane protrusions (Pommereit and Wouters, 2007). In addition, the TC10–Exo70 complex is also implicated in membrane expansion and axonal specification in developing neurons (Dupraz et al., 2009). In hippocampal pyramidal neurons in culture, membrane expansion at the axonal growth cone is regulated by IGF-1 through a cascade involving TC10 and the exocyst complex. TC10 and Exo70 are essential for the polarized externalization of IGF-1 receptor, which is necessary for axon specification (Dupraz et al., 2009).
1.2.5.3. Ral GTPases
Ral GTPases also belong to the Ras superfamily of small GTPases, which are found only in animal cells and play an important role in signaling pathways. Ral proteins have been implicated in the regulation of a diverse array of cellular processes such as membrane traffic, actin-cytoskeleton dynamics, cell migration, cell proliferation and oncogenesis (For review, see Feig, 2003). Ral has two isoforms:
RalA and RalB, which play distinct roles in the cell. Using yeast two-hybrid screens and pull-down assays, the exocyst components Sec5 and Exo84 have been identified as downstream effectors of RalA (Brymora et al., 2001; Moskalenko et al., 2002;
Polzin et al., 2002; Sugihara et al., 2002). Sec5 and Exo84 also bind to RalB but with
51 Jianglan Liu THE ROLE OF THE EXOCYST IN EXOCYTOSIS AND CELL MIGRATION Chapter 1 a substantially lower affinity compared to RalA (Shipitsin and Feig, 2004).
Biochemical and structural studies indicate that Sec5 and Exo84 share overlapping binding sites on the RalA effector-domain and compete for the binding to RalA
(Fukai et al., 2003; Mott et al., 2003; Jin et al., 2005). Depletion of RalA by siRNA results in destabilization or disassembly of the exocyst complex, suggesting that Ral might regulate exocytosis by promoting the proper assembly of the exocyst complex
(Moskalenko et al., 2002).
The functional implications of the Ral-exocyst interaction in exocytosis have been extensively studied in various cell types. In polarized MDCK cells, inhibition of the interaction between RalA and Sec5 using the Ral-binding domain (RBD) prevented protein targeting to the basolateral domain (Moskalenko et al., 2002).
Expression of constitutively-activated RalA enhanced protein delivery to the basolateral membrane, whereas expression of the RalA mutant which can not bind to the exocyst failed to promote enhanced secretion (Shipitsin and Feig, 2004). Thus, exocyst binding to active RalA is required for the Ral GTPases to regulate cellular secretion. In neuroendocrine PC12 cells, both expression of activated Ral and disruption of RalA-exocyst interaction using Sec5 RBD were found to block the secretion of secretory granules (Moskalenko et al., 2002). In another study, expression of wild type RalA, but not the RalA mutant deficient in Sec5 binding,
52 Jianglan Liu THE ROLE OF THE EXOCYST IN EXOCYTOSIS AND CELL MIGRATION Chapter 1 enhanced GTP-dependent exocytosis. On the other hand, disruption of endogenous
RalA-exocyst interaction by adding Sec5 RBD inhibited GTP-dependent exocytosis
(Wang et al., 2004). In adipocytes, RalA is activated in response to insulin stimulation and regulates the delivery of Glut4 vesicles to the plasma membrane
(Chen et al., 2007). Knockdown of RalA by RNAi or disruption of RalA-Sec5 interaction by overexpressing Sec5 RBD inhibited Glut4 transport (Chen et al., 2007).
Collectively, these findings strongly suggest that RalA binding to the exocyst is required for RalA to regulate exocytosis. Since RalA has been found to be associated with a variety of cellular compartments - the plasma membrane (Ngsee et al., 1991), secretory vesicles (Bielinski et al., 1993; Mark et al., 1996; Chen et al., 2007) or recycling endosomes (Shipitsin and Feig, 2004; Chen et al., 2006), it will be interesting to test where and how RalA regulates the exocyst.
Since many cellular processes require functional exocytosis, the RalA-exocyst interaction has been reported to function in many exocytosis-based cellular processes.
The exocyst has been implicated in epidermal growth factor (EGF)-induced lamellipodia protrusions through RalA activation. Disruption of the Ral-exocyst interaction by overexpressing the dominant negative Sec5 RBD inhibited lamellipodia formation in COS cells in response to EGF stimulation (Yoshizaki et al.,
2006). In differentiating neurons, RalA is needed for cell body spreading and neurite
53 Jianglan Liu THE ROLE OF THE EXOCYST IN EXOCYTOSIS AND CELL MIGRATION Chapter 1 branching, which seems to be mediated by the exocyst. Overexpression of wild type
RalA, but not a RalA mutant specifically defective in exocyst binding, stimulated neurite branching (Lalli and Hall, 2005). In addition, exocyst is also involved in neuronal polarity through interaction with RalA. Expression of a constitutively activated RalA mutant that loses the ability to interact with the exocyst complex resulted in non-polarized neurons (Lalli, 2009). During cytokinesis, the exocyst is colocalized with RalA at the midbody. Knockdown of RalA or Sec8 led to incomplete abscission and cytokinesis failure (Chen et al, 2006). During invasive cell migration, the exocyst is colocalized with focal complex proteins within the pseudopods through the interaction between Sec5 and paxillin which is dependent on
Ral GTPases (Spiczka and Yeaman, 2008). Overexpression of Ral-uncoupled Sec5 mutants or RNAi-mediated depletion of Ral or RalB inhibited the exocyst interaction with paxillin which inhibited tumor cell motility (Spiczka and Yeaman, 2008).
Recently, the Ral-exocyst interaction has been implicated in membrane nanotube formation, which is a new type of cell-cell communication dependent on the formation of thin membranous nanotubes between adjacent cells (Hase et al., 2009).
Further investigations are needed to elucidate how RalA regulates exocyst function during these processes.
In addition, the exocyst has also been implicated in several cellular processes
54 Jianglan Liu THE ROLE OF THE EXOCYST IN EXOCYTOSIS AND CELL MIGRATION Chapter 1 that seem to be exocytosis-independent, such as RalA-induced filopodia formation
(Sugihara et al., 2002), RalA-induced human cell transformation (Lim et al., 2005), and the anti-apoptotic function of RalA during the formation of Drosophila sensory organ (Balakireva et al, 2006). How the exocyst functions in these processes remains puzzled.
Even though RalA and RalB are 80% identical, the interaction of the exocyst and RalB is implicated in distinct cellular processes compared to that of the exocyst and RalA. The RalB-exocyst interaction was reported to function in the activation of the atypical IkappaB kinase TBK1, a key mediator of the host defense upon virus infection (Chien et al, 2006). RalB activation promotes the interaction between Sec5 and TBK1, which leads to the activation of TBK1 to initiate host defense pathway in response to virus exposure. Intriguingly, during oncogenic transformation, the same mechanism has been applied in cancer cells which antagonizes the apoptotic programs and leads to tumor cell survival (Chien et al, 2006). The role of
RalB-exocyst interaction during cytokinesis is still under debate. One study showed that in contrast to RalA, which is required for the recruitment of the exocyst to the cytokinetic furrow in early cytokinesis, RalB is required for the targeting of the exocyst to the midbidy during the late abscission step (Cascone et al., 2008).
However, another study showed that RalA on its own is sufficient for the
55 Jianglan Liu THE ROLE OF THE EXOCYST IN EXOCYTOSIS AND CELL MIGRATION Chapter 1 relocalization of the exocyst proteins through the whole process of cell division
(Chen et al., 2006). The RalB-exocyst interaction has also been implicated in cell migration (Rosse et al, 2006). In contrast to the study from Spiczka and Yeaman as described above, in which RalA and RalB were both found to contribute to cell motility (Spiczka and Yeaman, 2008), this study showed that RalB on its own drives cell migration. They found that RalB activation and the RalB-Sec5 interaction were enhanced under the conditions that promoted cell migration. RalB expression is required for promoting both exocyst assembly and recruitment of the exocyst to the leading edge. In addition, Knockdown of RalB or exocyst components largely inhibited the velocity of cell migration. In contrast, RalA is neither activated by cell motility, nor needed for the leading edge targeting of the exocyst during cell migration (Rosse et al, 2006).
The apparent discrepancies of the roles of RalA and RalB reported by different groups have not been solved yet. Different cell types or growth conditions may partially account for these differences.
1.3. Phosphoinositides in cell regulation and membrane dynamics
1.3.1. Composition and spatial distribution of phosphoinositides
The biological membrane is mostly made of phospholipids, which have a polar
56 Jianglan Liu THE ROLE OF THE EXOCYST IN EXOCYTOSIS AND CELL MIGRATION Chapter 1 head group and two hydrocarbon tails. Among the phospholipids, phosphatidylinositol (PI) is a minor but important component of the inner leaflet
(Hokin 1985; Berridge et al., 1999). The polar head group of PtdIns is inositol, which can be phosphorylated on specific carbons of the inositol ring. Reversible phosphorylation of the inositol ring of PtdIns at positions 3, 4 and 5 results in the generation of seven phosphoinositide species including phosphatidylinositol phosphate -- PI(3)P, PI(4)P, PI(5)P, phosphatidylinositol bisphosphate -- PI(3,4)P2,
PI(3,5)P2, PI(4,5)P2, and phosphatidylinositol trisphosphate PI(3,4,5)P3.
Each of the seven phosphoinositides has a unique subcellular distribution:
PI(4,5)P2 and PI(3,4,5)P3 are mostly found at the plasma membrane, PI(4)P is associated with the Golgi, PI(3)P and PI(3,5)P2 are associated with various types of endosomes, and PI(3,5)P2 and PI(5)P are associated with multi-vesicular bodies (De
Matteis and Godi 2004; Behnia and Munro 2005; Di Paolo and De Camilli 2006).
The differential intracellular distribution of each of these phospholipids not only contributes to defining the identities of these membrane compartments, but also makes these lipids optimal signaling molecules in a diverse array of cellular processes including cytoskeletal dynamics and vesicular trafficking.
The spatial restriction and steady-state levels of specific phosphoinositides are achieved by a combination of localized synthesis by specific kinases and rapid
57 Jianglan Liu THE ROLE OF THE EXOCYST IN EXOCYTOSIS AND CELL MIGRATION Chapter 1 turnover by phosphotases that prevent the lipid spreading between compartments. The localizations of phosphoinositide kinases and phosphatases are tightly controlled. All phosphoinositide kinases are themselves peripheral membrane proteins and, in many cases, their localizations are regulated by organelle-specific GTPases. For example, the endosomal PI-3-OH kinase (PI(3)K) Vps34 that makes PI(3)P is recruited by the endosomal GTPase Rab5 (Murray et al., 2002), whereas the PI(5)K that makes PI
(4,5)P2 is recruited to the plasma membrane by Arf6 (Krauss et al., 2003).
1.3.2. Molecular mechanisms for phosphoinositides in protein targeting
Phosphoinositides are involved in a number of signaling events through the binding of their head groups to cytosolic proteins or cytosolic domains of membrane proteins. Thus, they can regulate the function of integral membrane proteins, or recruit cytoskeletal and signaling components to the plasma membrane. Typically, proteins bind to phosphoinositides through electrostatic interactions with the negative charges of the phosphate(s) on the inositol ring. In some cases, adjacent hydrophobic amino acids enhance the interaction through a partial embedding into the bilayer
(Lemmon, 2003; Balla, 2005). Protein surfaces that interact with phosphoinositides are either composed of folded modules, such as the pleckstrin homology domain
(Lemmon, 2003; Balla, 2005; Hurley and Meyer, 2001), or of clusters of basic
58 Jianglan Liu THE ROLE OF THE EXOCYST IN EXOCYTOSIS AND CELL MIGRATION Chapter 1 residues within unstructured regions, such as those found in many actin regulatory proteins (for example, profilin) (Yin and Janmey, 2003). There is no clear distinction between these two types. Furthermore, unstructured peptides can undergo folding upon binding to phosphoinositide head groups. The number of phosphoinositide-binding modules is rapidly expanding and their diverse properties greatly amplify the signaling potential of phosphoinositides.
1.3.3. Cellular functions of PI(4, 5)P2
PI(4,5)P2, which is enriched in the plasma membrane, is involved in almost all of the cellular events that occur at the cell surface. In addition, it plays a major part in the transduction of extracellular signals, either through its metabolites or the fluctuations of its own levels.
PI(4,5)P2 has an important role in endocytosis owing to its functions as a co-receptor for the recruitment and regulation of endocytic proteins to the plasma membrane (Wenk and Camilli, 2004; Owen et al., 2004). PI(4,5)P2 interacts with all the known endocytic clathrin adaptors such as AP-2 and AP180/CALM as well as many other endocytic factors including dynamin, which controls the fission reaction
(Gaidarov and Keen, 1999; Wenk and De Camilli, 2004; Owen et al., 2004).
PI(4,5)P2 is also implicated in exocytosis because it plays a critical role at a
59 Jianglan Liu THE ROLE OF THE EXOCYST IN EXOCYTOSIS AND CELL MIGRATION Chapter 1 pre-fusion stage, also known as the vesicle priming step. It acts both in cis on plasma membrane proteins and in trans on vesicle proteins (Martin, 1998; Bai et al., 2004).
Importantly, in-trans interactions probably cooperate with SNARE pairing in marking the plasma membrane as the appropriate partner for the fusion of these organelles.
PI(4,5)P2 along with small GTPases, participates in the activation of a variety of actin regulatory proteins at the plasma membrane, thus regulates cell motility, cytokinesis and a wide variety of other processes. A well-characterized mechanism regulating actin assembly that involves PI(4,5)P2 is the Arp2/3-mediated nucleation of branched actin networks. PI(4,5)P2, in concert with the small GTPase Cdc42, binds
N-WASP and releases its autoinhibition which allows its binding to and activation of the Arp2/3 complex (Pohatgi et al., 2000; Pollard and Borisy 2003).
1.4. Cell invasion and invadopodia structures
When cultured on a flat extracellular matrix substratum, invasive tumor or transformed cells extend actin-rich protrusions emanating from their ventral surfaces into the matrix and displaying focalized proteolytic activity towards the substrate.
1.4.1. The identification and general features of invadopodia
Invadopodia were first discovered by Wen-Tien Chen in fibroblast cells
60 Jianglan Liu THE ROLE OF THE EXOCYST IN EXOCYTOSIS AND CELL MIGRATION Chapter 1 transformed by the v-src oncogene, which encodes a constitutively active non-receptor tyrosine kinase (v-Src) (Chen, 1989). Src-transformed fibroblasts grown on fibronectin formed ventral protrusions with degradative properties. Furthermore, vSrc was found to co-localize with the sites of fibronectin degradation (Chen et al.,
1985). In addition to Src-transformed fibroblast cells, invadopodia have also been found in cell lines or primary tumor cells from malignant melanoma (such as
A375MM), breast/mammary carcinoma cells (such as MDA-MB-231) and other malignant tumor cells (Baldassarre et al., 2003; Artym et al., 2006; Chuang et al.,
2004).
There are several criteria to recognize invadopodial structures under the light microscope: 1) roundish actin-rich protrusions at the ventral surface of the cell, 2) sites with the colocalization of cortactin and phosphotyrosine (Bowden et al., 2006),
3) not confined to the cell periphery, and 4) are associated with the degradation of the extracellular matrix (Artym et al., 2006). There are other features that can be used to identify invadopodia at least in some cell lines, such as their localization proximal to the Golgi apparatus (Baldassarre et al., 2003) and their extended half-life of up to 2 h or more (Yamaguchi et al., 2005; Baldassarre et al., 2003) as compared to other protrusive adhesions.
61 Jianglan Liu THE ROLE OF THE EXOCYST IN EXOCYTOSIS AND CELL MIGRATION Chapter 1 1.4.2. Structure of invadopodia
The description of the ultrastructural features of invadopodia still remains incomplete. Through transmission electron microscopy, Chen made an initial observation on the invadopodial structures in transformed fibroblasts and found that they are thin protrusions extending from the plasma membrane into the underlying
ECM (Chen, 1989). Later, a detailed ultrastructural analysis was carried out in the melanoma cell line A375MM using a correlative confocal light electron microscopy technique, through which individual areas of ECM degradation with matching invadopodia were first identified under the light microscope, analyzed using the electron microscope and reconstituted in three dimensions (Baldassarre et al., 2003).
In this study, invadopodia were shown to be originated from profound invaginations of the ventral plasma membrane surface, which averaged 8 µm in width and 2 µm in depth. From these invaginations, many surface protrusions emanated and sometimes penetrated into the matrix, which exhibited lengths of about 500 nm and diameters ranging from hundreds of nanometers to a few micrometers. These protrusions were consistent with the “invading” structures originally described by Chen. However, they seemed to be part of more complex superstructures. More recently, the connections between invadopodial protrusions and the cell body were revealed by electron microscopy tomography experiments (Baldassarre et al., 2006). They found
62 Jianglan Liu THE ROLE OF THE EXOCYST IN EXOCYTOSIS AND CELL MIGRATION Chapter 1 that the Golgi complex is always polarized in a direction towards the invadopodial area, suggesting a tight relationship between proteolytic activity and membrane/ protein transport.
1.4.3. Molecular components of invadopodia
1.4.3.1. Initial events at the cell-ECM interface
It has been accepted that the initiation of invadopodia formation is largely triggered by the engagement of cell surface integrins through ECM components
(Nakahara et al., 1996& 1997; Mueller et al., 1999). α3β1 and α5β1 integrins were the first two pairs shown to be involved in the formation of invadopodia, albeit the specific integrin combination might be cell-type dependent. Integrin activation leads to the activation of the Rho family of GTPases to regulate actin cytoskeleton, thus promoting membrane-protrusive and proteolytic activity, leading to invadopodia formation and cell invasion. In addition to their role in signaling, integrins also spatially and temporally regulate metalloprotease activity, so to focalize the degradation process. For example, integrins can coordinate with the membrane-type matrix metalloprotease MT1-MMP to regulate membrane proteolytic activity through the activation of matrix prometalloproteinase-2 (MMP2) (Deryugina et al., 2001). In some cases, invadopodia formation can also be triggered by activation of the EGF
63 Jianglan Liu THE ROLE OF THE EXOCYST IN EXOCYTOSIS AND CELL MIGRATION Chapter 1 receptor followed by activation of the signaling cascade leading to activation of the actin polymerization machinery (Yamaguchi et al., 2005).
1.4.3.2. Signaling molecules in invadopodia
Many molecular effectors have been characterized in the integrin engagement-initiated signaling cascade in invadopodia, including protein kinases, small GTPases and effector proteins. a) Protein kinases
Invadopodia were initially identified as specialized membrane protrusions enriched in Src non-receptor tyrosine kinase (Src) (Chen 1989). Cells failed to initiate invadopodia formation and matrix degradation when treated with tyrosine phosphorylation inhibitors (Bowden et al., 2006). On the other hand, constitutively-active Src was found to be able to stimulate invadopodia formation in fibroblasts and breast carcinoma cells (Chen et al., 1985; Artym et al., 2006).
Therefore, Src activity is essential for invadopodia formation and matrix degradation.
Recently, several classes of serine/threonine kinases have been implicated in invadopodia formation. For example, protein kinase D (PKD), a family of serine/threonine protein kinases involved in a variety of fundamental functions including vesicular trafficking, cell shape, cell motility and adhesion (Rykx et al.,
64 Jianglan Liu THE ROLE OF THE EXOCYST IN EXOCYTOSIS AND CELL MIGRATION Chapter 1 2003; Wang, 2006), was the first serine/threonine kinase to be implicated in invadopodia activity (Bowden et al., 1999). ERK1/2, part of the mitogen-activated protein kinase (MAPK) pathways, has also been connected to invadopodia biogenesis
(Tague et al., 2004; Ayala et al., 2008). However, how these kinases contribute to invadopodia formation remains to be clarified. b) Small GTPases
Cdc42, Rac and Rho GTPases have all been shown to be involved in invadopodia formation albeit in various cell types. While depletion of Cdc42 by
RNAi or overexpression of a constitutively inactive Cdc42 mutant inhibited invadopodia formation in the metastatic MTLn3 adenocarcinoma cell line
(Yamaguchi et al., 2005), overexpression of a constitutively active mutant of Cdc42, but not Rac or Rho, promoted dot-like degradation in RPMI17951 melanoma cells
(Nakahara et al., 2003). In addition, transfected Cdc42 but not RhoA or Rac, was detected at invadopodia in A375MM melanoma cells through immunofluorescence microscopy. However, In SNB19 and U87MG glioma cells, depletion of Rac1 by siRNA disrupted lamellipodia and invadopodia formation (Chuang et al., 2004). In another study, endogenous active form of RhoA was shown to be localized in invasive protrusions of Src transformed NIH 3T3 fibroblasts (Berdeaux et al., 2004).
Further investigations are required to obtain a clearer understanding of the functions
65 Jianglan Liu THE ROLE OF THE EXOCYST IN EXOCYTOSIS AND CELL MIGRATION Chapter 1 of Rac1, Cdc42 and RhoA during invadopodia formation.
ARF6 has been found to be localized at invadopodia in MDA-MB-231 breast cancer cells (Hashimoto et al., 2004) and in LOX melanoma cells (Tague et al., 2004).
Disruption of Arf6 by RNAi or expression of GTP-binding deficient mutants blocked
ECM degradation and matrigel invasion. Furthermore, ARF6 activity at invadopodia was shown to be dependent on ERK activation (Tague et al., 2004). c) Effector proteins
The multi-domain actin-binding protein cortactin is one of the key effector proteins during invadopodia formation. Cortactin was first found to be associated with invadopodia in a complex with paxillin and PKD (Bowden et al., 1999), and later shown to be essential for invadopodia formation (Artym et al., 2006). Live cell imaging of c-src-expressing carcinoma cells showed that aggregation of cortactin to the invadopodial initiation site is an early step during invadopodia formation, which is followed by MT1-MMP targeting to these sites and the subsequent degradation of underlying matrix substrates (Artym et al., 2006). It was proposed that cortactin may couple actin assembly to the secretory machinery through the regulation of MMP secretion (Clark et al., 2007). A recent study showed that multiple phosphorylation events by Src, ERK1/2 and PAK(s) converge onto cortactin and are crucial for the regulation of invadopodia function (Ayala et al., 2008).
66 Jianglan Liu THE ROLE OF THE EXOCYST IN EXOCYTOSIS AND CELL MIGRATION Chapter 1 Dynamin 2 was recently found at invadopodia-forming sites and colocalized with other invadopodia markers as F-actin, cortactin and phosphotyrosine. It was also shown to be required for invadopodia function (Baldassarre et al., 2003 McNiven et al., 2004). The novel Src substrate Tks5, a scaffold protein containing five SH3 and one PX domains, was recently identified at invadopodia of Src-transformed fibroblasts and several cancer cell lines and shown to be required for invadopodia formation and function (Abram et al., 2003, Seals et al., 2005).
In addition, many components of the actin-based membrane remodeling machinery have been reported to be involved in invadopodia biogenesis, which include the Arp2/3 complex, N-WASP (Wiskott-Aldrich syndrome protein) (Mizutani et al., 2002; Yamaguchi et al., 2005), and the actin depolymerizing factor cofilin
(Yamaguchi et al., 2005).
1.4.3.3. Degradation of the ECM during invadopodia formation
The proteolytic activity of invadopodia is mainly due to members of the matrix metalloprotease (MMP) family. MMPs are synthesized as inactive proenzymes and become activated through proteolytic removal of a prodomain. MMPs are either secreted from the cell (such as MMP-2 and MMP-9) or anchored to the plasma membrane as integral proteins (membrane-type MMPs). MMP-2 was the first MMP
67 Jianglan Liu THE ROLE OF THE EXOCYST IN EXOCYTOSIS AND CELL MIGRATION Chapter 1 found to be enriched at invadopodia which led to the recognition of invadopodia as protease-rich membrane protrusions (Monsky et al., 1993). MT1-MMP has been considered as a master regulator of the degradative activity of invadopodia in a variety of cell types (Nakahara et al., 1997; Clark et al., 2007). MMP-9 was also found at invadopodial structures such as those of the metastatic breast cancer cells
(Bourguignon et al., 1998) and leukemia cells (Redondo-Muñoz et al., 2006).
Metalloproteases are crucial for invadopodia formation and cell invasion.
However, how they are specifically targeted to invadopodia is still unclear. The secretory pathway might play an important role, as persistent ECM degradation requires an active transport of protease-delivering carriers to specific sites of ECM degradation. But it still remains elusive how this transport is organized.
1.4.4. Invadopodia vs podosomes
Another similar structure, termed podosome, is formed by a number of normal cell types including osteoclasts, smooth muscle cells and macrophages (Linder, 2007).
Invadopodia and podosomes share many structural and functional characteristics, including a dependence on actin assembly and Src kinase signaling (Gimona and
Buccione, 2007; Buccione et al., 2004). However, they differ in size, turn-over rate, cell types and molecular components that are involved (Linder, 2007). It is still
68 Jianglan Liu THE ROLE OF THE EXOCYST IN EXOCYTOSIS AND CELL MIGRATION Chapter 1 unclear whether podosomes and invadopodia are distinct structures or whether they are two similar structures of the cellular response to contact with the substratum.
1.5. Thesis overview
The exocyst, as a vesicle tethering machinery, regulates vesicle trafficking at the vicinity of the plasma membrane. Although genetic, cell biological and biochemical studies in various systems have shown important roles of the exocyst in a multitude of developmental processes, many uncertainties still exist in the field. I am asking two fundamental questions in my thesis work: how does the exocyst mediate vesicle tethering to the plasma membrane? What is the role of the exocyst in cell migration and tumor cell invasion?
My thesis has three major parts that describe the studies on exocyst functions in various aspects. Chapter 2 presents a molecular mechanism of how the exocyst associates with the plasma membrane. Chapter 3 describes the role of the exocyst in tumor cell invadopodia formation by mediating the secretion of matrix metalloproteinases at focal degrading sites and regulating Arp2/3-mediated actin dynamics. Chapter 4 further discusses the molecular mechanism of how the exocyst regulates actin assembly and contributes to cell migration and cell invasion.
69 Jianglan Liu THE ROLE OF THE EXOCYST IN EXOCYTOSIS AND CELL MIGRATION Chapter 2 Chapter 2. Phosphatidylinositol 4,5-bisphosphate mediates the targeting of the exocyst to the plasma membrane for exocytosis in mammalian cells
Abstract
The exocyst is an evolutionarily conserved octameric protein complex that mediates the tethering of post-Golgi secretory vesicles at the plasma membrane for exocytosis. To elucidate the mechanism of vesicle tethering, it is important to understand how the exocyst physically associates with the plasma membrane (PM).
In this study, I report that the mammalian exocyst subunit Exo70 associates with the
PM through its direct interaction with phosphatidylinositol 4,5-bisphosphate
(PI(4,5)P2). Furthermore, I have identified key conserved residues at the C-terminus of Exo70 that are crucial for the interaction of Exo70 with PI(4,5)P2. Disrupting
Exo70-PI(4,5)P2 interaction abolished the membrane association of Exo70. I have also found that wild-type Exo70 but not the PI(4,5)P2-binding–deficient Exo70 mutant is capable of recruiting other exocyst components to the PM. Using the ts045 vesicular stomatitis virus glycoprotein trafficking assay, I demonstrate that
Exo70-PI(4,5)P2 interaction is critical for the docking and fusion of post-Golgi secretory vesicles, but not for their transport to the PM.
70 Jianglan Liu THE ROLE OF THE EXOCYST IN EXOCYTOSIS AND CELL MIGRATION Chapter 2 2.1. Introduction
Exocytosis is essential in a variety of cellular functions, ranging from the release of hormones to the incorporation of membrane proteins for cell growth and morphogenesis. The late stage of exocytosis is a multistep process including directional transport, tethering, docking, and fusion of post-Golgi secretory vesicles with the plasma membrane (PM). The tethering step, defined as the initial contact of secretory vesicles with the PM prior to SNARE-mediated docking and fusion (Pfeffer,
1999; Guo et al., 2000; Waters and Hughson, 2000; Whyte and Munro, 2002), is mediated by the exocyst, an evolutionarily conserved octameric complex composed of Sec3, Sec5, Sec6, Sec8, Sec10, Sec15, Exo70, and Exo84 (for review, see Guo et al., 2000; Hsu et al., 2004; Munson and Novick, 2006; Wang and Hsu, 2006). In budding yeast, the exocyst components localize to the growing end of the daughter cell ("bud tip"), where active exocytosis and membrane addition take place (TerBush and Novick, 1995; Finger et al., 1998, Guo et al., 1999). This localization pattern contrasts that of the membrane fusion machine, the t-SNAREs, which are evenly distributed along both the mother and daughter cell membrane (Brennwald et al.,
1994). In mammalian cells, the exocyst components were found in the cytosol, recycling endosomes and trans-Golgi network (Yeaman et al., 2001; Fölsch et al.,
2003; Ang et al., 2004; Langevin et al., 2005). However, they are recruited to the PM
71 Jianglan Liu THE ROLE OF THE EXOCYST IN EXOCYTOSIS AND CELL MIGRATION Chapter 2 during a number of cellular processes. For example, in epithelial cells, the exocyst is recruited to the adherens junction region upon cell–cell contact, where it mediates protein and membrane addition at the basolateral domain (Grindstaff et al., 1998;
Yeaman et al., 2001); in developing neurons, the exocyst is localized to the growing neurites, where it mediates membrane expansion (Hazuka et al., 1999; Vega and Hsu,
2001); during cell migration, the exocyst is recruited to the leading edges of the PM
(Rosse et al., 2006; Zuo et al., 2006).
The mechanism by which the exocyst mediates vesicle tethering to the PM is unclear. One key question yet to be resolved is how the exocyst itself associates with the PM. Using fluorescence recovery after photobleaching (FRAP) analyses and immunoelectron microscopy, Boyd et al. (2004) have shown that Exo70 is stably localized to the yeast bud tip membrane and remains polarized even when the actin cables are disrupted, suggesting that Exo70 is a candidate in this complex involved in membrane targeting of the exocyst. In MDCK cells, extragenically expressed
GFP-tagged Exo70 is localized to the PM near cell–cell contacts, suggesting that
Exo70 may mediate PM association independent of the rest exocyst components in these cells (Matern et al., 2001). Recent structural studies have revealed that Exo70 contains a number of conserved basic residues that cluster on a surface patch at the
C-terminal end of the tertiary structure that may directly bind to the PM (Dong et al.,
72 Jianglan Liu THE ROLE OF THE EXOCYST IN EXOCYTOSIS AND CELL MIGRATION Chapter 2 2005; Hamburger et al., 2006; Moore et al., 2007). In fact, the C-terminal sequence of
Exo70 is the most evolutionarily conserved region of this protein.
Here, I report that mammalian Exo70 directly interacts with PI(4,5)P2 in the
PM through the positively charged residues at its C-terminus. I have also identified key residues in Exo70 that are crucial for this interaction. Finally, using the ts045 vesicular stomatitis virus glycoprotein (VSV-G) trafficking assay, I found that the
Exo70-lipid interaction is critical for PM stages of exocytosis, but not for the trafficking steps through ER and Golgi. My study revealed a molecular mechanism by which the exocyst directly interacts with the PM that is critical for vesicle tethering and exocytosis.
2.2. Results
Association of Exo70 with the Plasma Membrane in HeLa Cells
I have tested the localization of GFP-tagged rat Exo70 in HeLa cells. As shown in Figure 2.1A, GFP-Exo70 was enriched at the PM as revealed by serial optical sectioning from different axes (Figure 2.1A). In addition, cells expressing
GFP-Exo70 exhibited filopodia-like structures as previously reported (Wang et al.,
2004; Xu et al., 2005; Zuo et al., 2006). In order to map the region of Exo70 that is required for its association with the PM, I examined the localization of a series of
73 Jianglan Liu THE ROLE OF THE EXOCYST IN EXOCYTOSIS AND CELL MIGRATION Chapter 2 GFP-tagged Exo70 truncates in HeLa cells. As shown in Figure 2.1B, Exo70 with its
C-terminus deleted (amino acids 1-408; Exo70-∆C) was cytosolic, whereas the
C-terminal domain Exo70 (amino acids 403-653; Exo70-CT) was enriched at the PM
(Figure 2.1B), suggesting a critical role of Exo70 C-terminus in membrane association. The crystal structure of yeast Exo70 revealed that this protein is a long rod composed mainly of α-helices that fold into four domains (named domains A, B,
C, and D; Dong et al., 2005 ; Hamburger et al., 2006). Domain D (the C-terminal
114 amino acids) is the most evolutionarily conserved domain in Exo70 that contains a number of basic residues that cluster into an electro-positive surface patch at the
C-terminal tip of the rod. Because in many cases the association of proteins with the
PM can be mediated by interactions with the negatively charged phospholipids in membrane via clusters of basic residues (for reviews, see McLaughlin et al., 2002;
Balla, 2005), it is likely that mammalian Exo70 directly associates with the PM through those positively charged residues at its C-terminus. I sought to examine whether domain D is required for the PM localization of Exo70 by generating truncations of Exo70, in which the last 95 amino acids (aa 559-653) or the last 45 amino acids (aa 609-653) were deleted. The resulting Exo70 fragments, Exo70 (aa
1-558) and Exo70 (aa 1-608), were both cytosolic (Figure 2.1B), indicating that domain D of mammalian Exo70, especially the last 45 amino acids, is necessary for
74 Jianglan Liu THE ROLE OF THE EXOCYST IN EXOCYTOSIS AND CELL MIGRATION Chapter 2
75 Jianglan Liu THE ROLE OF THE EXOCYST IN EXOCYTOSIS AND CELL MIGRATION Chapter 2 Figure 2.1 Association of Exo70 with the plasma membrane (PM) in HeLa cells.
(A) GFP-Exo70 was detected at the PM. HeLa cells were transfected with
GFP-tagged Exo70, fixed, and observed using a confocal microscope. Serial optical sections in the x-z and y-z planes were taken. (B) Various truncates of Exo70 were tested for their localization at the PM. HeLa cells were transfected with GFP-tagged
Exo70 and Exo70 truncates as diagramed. The cells were fixed, stained, and observed under microscope. Full-length and the C-terminal fragments of Exo70 containing domain D (aa 558-653) are associated with the PM. +, membrane association.
76 Jianglan Liu THE ROLE OF THE EXOCYST IN EXOCYTOSIS AND CELL MIGRATION Chapter 2 the association of Exo70 with the PM. I have also examined the localization of Exo70
(aa 403-558) and Exo70 (aa 403-608), which are C-terminal fragments of Exo70 with the last 95 amino acids (aa 559-653) or the last 45 amino acids (aa 609-653) deleted, and found that both of the fragments were cytosolic (Figure 2.1B). I next tested whether the C-terminal fragments of Exo70 containing aa 403-653 or aa 540-653
(domain D) were able to associate with the PM. As shown in Figure 2.1B, both fragments were localized to the PM, suggesting that domain D of Exo70 is sufficient for its PM localization. A portion of these fragments was also found in the nucleus, probably resulting from nonspecific retention of the GFP fusion. Collectively, these data suggest that the C-terminus of Exo70 is both necessary and sufficient for the PM localization of Exo70.
Exo70 Directly Interacts with Phospholipids through Its C-Terminus
Based on sequence analysis, domain D of Exo70 contains a number of positively charged residues that are well conserved in yeast. Moreover, based on the crystal structure, the basic residues cluster onto a surface patch at the C-terminal tip in the folded Exo70 protein. Therefore, it is likely that Exo70 directly interacts with negative charged phospholipids through this basic patch. To test this hypothesis, I examined the binding between recombinant GST-Exo70 and various phospholipids by
77 Jianglan Liu THE ROLE OF THE EXOCYST IN EXOCYTOSIS AND CELL MIGRATION Chapter 2 cosedimentation assay using the methods as previously described (Papayannopoulos et al., 2005; Hokanson and Ostap, 2006). PI(4,5)P2 is the major phosphatidylinositol species which comprises 1–5% of the total lipids in the PM (for review, see
McLaughlin et al., 2002). Binding assays were carried out with large unilamellar vesicle (LUVs) composed of the neutral PC and PI(4,5)P2 at 5% or the acidic phospholipid PS in molar percentages of 20, 40, and 60%. Because the recombinant
Exo70 protein could only access the outer leaflet of the reconstituted LUV vesicles, the effective PI(4,5)P2 and PS in the binding reaction were only half of the total. As shown in Figure 2.2A, Exo70 bound to LUVs containing 5% PI(4,5)P2, but not to
LUVs containing 100% neutral PC. While Exo70 also bound to PS, the binding was weak unless the molar ratio of PS in the LUVs was raised to 60%. As a control, GST did not bind to LUVs with any lipid composition.
I have also measured the affinity of Exo70 for various lipids (Figure 2.2B). The bound Exo70 was quantified and plotted against the lipid concentration with the equation: B = BmaxX/[Kd + X], where Kd is the dissociation constant and X and B represent the concentrations of the free Exo70 and the bound Exo70, respectively.
The Kd was calculated by nonlinear regression. As shown in Table 1, Exo70 bound to
LUVs composed of 5% PI(4,5)P2 with a Kd of 13.9 ± 3.0 µM and bound to LUVs composed of 60% PS with a Kd of 46.3 ± 9.7 µM. The Kd value for PI(4,5)P2 would
78 Jianglan Liu THE ROLE OF THE EXOCYST IN EXOCYTOSIS AND CELL MIGRATION Chapter 2
79 Jianglan Liu THE ROLE OF THE EXOCYST IN EXOCYTOSIS AND CELL MIGRATION Chapter 2 Figure 2.2 The interaction between Exo70 and phospholipids in vitro. (A)
GST-Exo70 purified from bacteria (0.15 µM) was incubated with liposomes (200 µM) containing 100% PC, 5% PI(4,5)P2, 20% PS, 40% PS, and 60% PS. After centrifugation at 150,000 x g for 30 min, the proteins in supernatant (S) and pellet (P) were subjected to SDS-PAGE and visualized by SYPRORed staining. Exo70 bound to vesicles containing 5% PI(4,5)P2 or 60% PS. It also bound weakly to vesicles containing 40% PS. GST did not bind to liposomes with any lipid composition in the cosedimentation assay. (B and C) The interaction of Exo70 with phospholipids.
Exo70, 0.15 µM, was incubated with increasing concentrations of LUVs composed of
(B) 100% PC, 5% PI(4,5)P2, 20% PS, 40% PS, and 60% PS or (C) PI(3)P, PI(4)P,
PI(3,5)P2, PI(4,5)P2, and PI(3,4,5)P3. The percentage of bound Exo70 was plotted with the increasing liposome concentration with a single rectangular hyperbola equation (B = BmaxX/[Kd + X]) using SigmaPlot. Each point is the average of three measurements. Error bars, SD.
Table 1. Comparison of the affinities of Exo70 and exo70-1 for phospholipids.
LUV Composition Kd (µM) for Exo70 Kd (µM) for exo70-1 100% PC >> 400 >> 400 20% PS >> 400 >> 400 60% PS 46.30±9.7 146.23±14.6
5% PIP2 13.92±3.0 >> 400
* The Kd was obtained by rectangular hyperbolae equation using the SigmaPlot software. For some of the bindings, the Kd was >> 400 because the binding never reached saturation. 80 Jianglan Liu THE ROLE OF THE EXOCYST IN EXOCYTOSIS AND CELL MIGRATION Chapter 2 be much smaller if expressed in terms of pure PI(4,5)P2, because the molar ratio of
PI(4,5)P2 is only 5% of the total lipids in the reconstituted LUVs. Overall, these results suggest that Exo70 may associate with the PM through its direct interaction with PI(4,5)P2.
To determine the specificity of Exo70-PI(4,5)P2 interaction, I have tested the binding of Exo70 to four additional phosphoinositides: PI(3)P, PI(4)P, PI(3,5)P2, and
PI(3,4,5)P3. In mammalian cells, PI(4)P and PI(4,5)P2 are the two most abundant phosphoinositides enriched in the Golgi and PM, respectively (Di Paolo and De
Camilli, 2006). PI(4,5)P2 constitutes more than 95% of the bis-phosphoinositides in mammals (Bonangelino et al., 2002). PI(3)P and PI(3,5)P2 are mainly localized to the endosomal compartments (for reviews, see Behnia and Munro, 2005; Di Paolo and
De Camilli, 2006). PI(3,4,5)P3 is present in negligible amount in mammalian cells at the "resting" state, but is up-regulated at specific PM domains in response to certain stimuli (Insall and Weiner, 2001). Recently, PI(3,4,5)P3 was found to be localized to the basolateral domain of MDCK cells (Gassama-Diagne et al., 2006). As shown in
Figure 2.2C, Exo70 hardly binds to PI(3)P; the affinity of Exo70 for PI(4,5)P2 (Kd =
13.92 ± 3.0 µM) is ~4–5-fold higher than PI(3,5)P2 (Kd = 58.34 ± 9.2 µM) and more than 10-fold higher than PI(4)P (Kd = 173.12 ± 15.5 µM). The affinity of Exo70 for
PI(3,4,5)P3 (Kd = 15.55 ± 4.6 µM) is similar to that of PI(4,5)P2.
81 Jianglan Liu THE ROLE OF THE EXOCYST IN EXOCYTOSIS AND CELL MIGRATION Chapter 2 Mutations in Domain D Disrupt the Plasma Membrane Association of Exo70
The direct binding of Exo70 with PI(4,5)P2 suggests a critical role for the domain D basic residues in the PM targeting of Exo70. To determine the responsible residues that are essential for Exo70 to bind to the PM, the conserved basic residues in domain D of Exo70 (Figure 2.3A) were targeted for mutagenesis and the resulting mutants were tested for their cellular localization (summarized in Table 2). Among the 10 mutants I tested, 6 failed to associate with the PM. The other four mutations had no effect on Exo70 localization, suggesting that not all of the basic residues are required for PI(4,5)P2 binding. I focused on one of the mutants, named exo70-1, in which residues K632 and K635 have been mutated to alanine. As shown in Figure
2.3B, GFP-tagged wild-type Exo70 (GFP-Exo70) was enriched at the PM, whereas
GFP-tagged exo70-1 was distributed diffusely throughout the intracellular regions.
These results indicate that K632 and K635 are necessary for the PM localization of
Exo70.
To directly test whether K632 and K635 are required for the physical association of Exo70 with the PM, I performed subcellular membrane fractionation assay using HeLa cells transfected with GFP-tagged wild-type Exo70 or exo70-1.
Cell lysates were fractionated, and the amount of Exo70 or exo70-1 in the PM, cytosol, and other fractions was examined. As shown in Figure 2.3C, wild-type
82 Jianglan Liu THE ROLE OF THE EXOCYST IN EXOCYTOSIS AND CELL MIGRATION Chapter 2 Exo70 was present in the PM fraction, whereas exo70-1 was mostly found in the cytosol. The amount of Exo70 and exo70-1 in the HDM (mostly ER membrane) and
LDM (mostly Golgi and endosomal compartments) fractions was almost the same.
During my studies, I have noticed that GFP-tagging of Exo70 may promote its translocation from cytosol to the PM. It is likely that GFP-tagging induces conformational changes on Exo70, exposing the lipid-binding site on Exo70; and mutations on the C-terminus of Exo70 may disrupt its membrane-association (see below). That may explain the observed clear PM versus cytosol distribution of
GFP-Exo70 versus GFP-exo70-1 in Figure 2.3. The membrane fractionation data, together with the fluorescence localization results, indicate that K632 and K635 are critical residues in Exo70 that are involved in the physical association of Exo70 with the PM.
Next, I tested the interaction of the mutant exo70-1 protein with phospholipids in the cosedimentation assay. As shown in Figure 2.4 and Table 1, the interaction between exo70-1 and 5% PI(4,5)P2 was almost abolished, suggesting that K632 and
K635 are critical residues for the binding of Exo70 to PI(4,5)P2. Interestingly, the binding of exo70-1 for PS was only partially affected, suggesting that these two residues may confer certain degree of specificity for PI(4,5)P2.
It was previously shown that Exo70 directly interacts with the constitutively
83 Jianglan Liu THE ROLE OF THE EXOCYST IN EXOCYTOSIS AND CELL MIGRATION Chapter 2
84 Jianglan Liu THE ROLE OF THE EXOCYST IN EXOCYTOSIS AND CELL MIGRATION Chapter 2 Figure 2.3 Mutations at Exo70 C-terminus abolish the association between
Exo70 and the PM. (A) Sequence alignment between the C-termini (domain D) of yeast Exo70 and rat Exo70. The mutated residues in the exo70-1 mutant were marked in grey. (B) The localization of the exo70 mutant, exo70-1, in HeLa cells. HeLa cells were transfected with GFP-tagged wild-type Exo70 as a control (left) and exo70-1
(right). The exo70-1 mutant failed to associate with the PM. (C) Membrane fractionation was performed to examine the localization of GFP-Exo70 and
GFP-exo70-1 in HeLa cells. Equal amounts of proteins from each fraction of the cells expressing Exo70 and exo70-1 were loaded on SDS-PAGE. The total proteins in each lane were detected by SYPRORed staining. Exo70 was detected by Western blot.
LDM, low-density microsomal membranes, mostly the Golgi fraction; HDM, high-density microsomal membranes, mostly the ER fraction. Table 2. Exo70 C-terminus mutants. Mutants Mutation sites Localization exo70-1 K632A, K635A Cytoplasm exo70-2 ∆634-636aa Cytoplasm exo70-3 K571A, E572A Cytoplasm D564A, K565A exo70-4 Cytoplasm K571A, E572A exo70-5 ∆571-573aa Cytoplasm K628A, K632A exo70-6 Cytoplasm R573A, K575A exo70-7 K628A, K632A Plasma membrane exo70-8 R620A Plasma membrane exo70-9 K635A Plasma membrane exo70-10 R573A, K575A Plasma membrane 85 Jianglan Liu THE ROLE OF THE EXOCYST IN EXOCYTOSIS AND CELL MIGRATION Chapter 2 activated form of TC10 (Q67L; Inoue et al., 2003). I then examined whether mutations in exo70-1 affect this interaction. Cell lysates transfected with GFP-tagged
Exo70 or exo70-1 were incubated with GST-TC10 (Q67L) conjugated to the glutathione-Sepharose for binding reaction. Exo70 or the exo70 mutant bound to the
Sepharose was detected by Western blot using an anti-GFP antibody. The amount of exo70-1 bound to the TC10 beads was similar to that of the wild-type Exo70 (Figure
2.5A). As a control, neither the wild-type Exo70 nor exo70-1 bound to the GST beads.
The result indicates that the two key basic residues mutated in exo70-1 are not required for its interaction with TC10. Therefore, the loss of PM association of the exo70 mutant is unlikely due to impaired interaction with TC10. The binding results strongly suggest that Exo70 associates with the PM through its direct interaction with
PI(4,5)P2. In addition to TC10, I have also examined the binding of exo70-1 with another exocyst component Sec8. I found that mutations on exo70-1 did not affect its binding with Sec8 in the cell (Figure 2.5B). This result is consistent with the previous reports in mammalian and yeast cells that the interaction of Exo70 with the other exocyst subunits for complex assembly is mediated by the N-terminal domains of
Exo70 (Inoue et al., 2003; Dong et al., 2005).
86 Jianglan Liu THE ROLE OF THE EXOCYST IN EXOCYTOSIS AND CELL MIGRATION Chapter 2
87 Jianglan Liu THE ROLE OF THE EXOCYST IN EXOCYTOSIS AND CELL MIGRATION Chapter 2 Figure 2.4 Mutations at the C-terminus of Exo70 affect its interaction with phospholipids. (A) GST-tagged Exo70 and exo70-1 (0.15 µM) were used in the lipid cosedimentation assay. The binding of exo70-1 to 5% PI(4,5)P2 was abolished, whereas its interaction with 60% PS was reduced. (B) Binding curves of Exo70 and exo70-1 to LUVs containing various phospholipids.
88 Jianglan Liu THE ROLE OF THE EXOCYST IN EXOCYTOSIS AND CELL MIGRATION Chapter 2
89 Jianglan Liu THE ROLE OF THE EXOCYST IN EXOCYTOSIS AND CELL MIGRATION Chapter 2 Figure 2.5 Mutations in the exo70-1 mutant did not affect its interaction with
TC10 (Q75L) or Sec8. (A) Mutations in exo70-1 did not affect its interaction with
TC10 (Q75L). Glutathione Sepharose conjugated with GST or GST-TC10 (Q75L) was incubated with either GFP-tagged wild type Exo70 or the exo70 mutant. The input and the bound Exo70 or exo70 mutant were analyzed by western blot using anti-GFP monoclonal antibody. The lower panel was a Commassie blue stained gel showing the amounts of GST-TC10 and GST (as a control) used in the binding assay.
(B) The exo70-1 mutant binds Sec8. Lysates from HeLa cells expressing GST-tagged
Exo70 or exo70-1 were incubated with Glutathione Sepharose. The binding of Sec8 to Exo70 or exo70-1 were detected by western blotting using an anti-Sec8 monoclonal antibody. GST-tagged Exo70 or exo70-1 bound to the beads was detected using an anti-GST monoclonal antibody. Cell lysates expressing GST was used as a negative control.
90 Jianglan Liu THE ROLE OF THE EXOCYST IN EXOCYTOSIS AND CELL MIGRATION Chapter 2 Exo70 Recruits Sec8 to the Plasma Membrane
I next asked whether Exo70 is involved in recruiting other exocyst components to the membrane. It has been shown that the exocyst subunits were mostly localized in the cytoplasm in cultured HeLa cells (Zuo et al., 2006). I then examined whether an increase of Exo70 at the PM would recruit Sec8 to the membrane. GFP-Exo70 and
GFP-exo70-1 were expressed in HeLa cells, and the localization of Sec8 in these cells was detected by immunofluorescence staining using the anti-Sec8 mAb 2E12. As shown in Figure 2.6, both GFP-Exo70 and Sec8 were detected at the PM. In contrast, in cells expressing GFP-exo70-1 that is defective in binding PI(4,5)P2, Sec8 was found in the cytoplasm and intracellular membrane structures. This result suggests that the wild type, but not the mutant Exo70, is able to recruit other members of the exocyst to the PM. Because exo70-1 maintains its ability to interact with Sec8, the lack of PM association of Sec8 in cells expressing GFP-exo70-1 is unlikely resulted from a defect in exocyst complex assembly.
Exo70 Is Essential for Exocytosis of VSV-G ts045 at the Plasma Membrane
Because the exocyst functions to tether post-Golgi secretory vesicles at the PM, the physical interaction between Exo70 and phospholipids may play an important role in exocytosis. Here I examined the effect of Exo70 depletion on the trafficking of a
91 Jianglan Liu THE ROLE OF THE EXOCYST IN EXOCYTOSIS AND CELL MIGRATION Chapter 2
92 Jianglan Liu THE ROLE OF THE EXOCYST IN EXOCYTOSIS AND CELL MIGRATION Chapter 2 Figure 2.6 Exo70 is required for the PM localization of Sec8. GFP-Exo70 and
GFP-exo70-1 were transfected into HeLa cells. Sec8 in the transfected cells was detected by the anti-Sec8 monoclonal antibody 2E12. Sec8 was detected at the PM in cells expressing GFP-Exo70 (top panel), but not in cells expressing GFP-exo70-1
(bottom panel).
93 Jianglan Liu THE ROLE OF THE EXOCYST IN EXOCYTOSIS AND CELL MIGRATION Chapter 2 temperature-sensitive VSV-G mutant (ts045) to the PM. At the restrictive temperature
(40°C), the VSV-G ts045 mutant is reversibly misfolded and retained in the ER. Upon temperature shift to 32°C, the protein correctly folds and is transported through the
Golgi apparatus to the PM. I treated HeLa cells with EXO70 siRNA specific for human Exo70 (hExo70), and the luciferase siRNA was used as a control. The treatment reduced the level of Exo70 by more than 90% without affecting the level of other exocyst subunits such as Sec8 (Figure 2.7A). Cells treated with EXO70 siRNA or luciferase siRNA were transfected with GFP-tagged VSV-G mutant ts045
(VSV-G-GFP). Cells were then kept at 40°C overnight and shifted to 32°C for various times before being examined by fluorescence microscopy. The translocation of
VSV-G-GFP inside the cells was shown by GFP fluorescence, whereas the incorporation of VSV-G-GFP to the PM (at the 90-min point) was evaluated by immunostaining using the 8G5 mAb against the extracellular domain of VSV-G
(Lefrancois and Lyles, 1982). As shown in Figure 2.7B, in luciferase siRNA-treated cells, VSV-G-GFP was retained in the ER before the cells were shifted from 40 to
32°C (0 min). After the shift, the protein was transported from ER to the Golgi apparatus around 30 min and then to the cell surface at 90 min. The incorporation of
VSV-G-GFP at the PM at 90 min was clearly detected by immunostaining of nonpermeabilized cells with the 8G5 antibody (Figure 2.7B). In EXO70
94 Jianglan Liu THE ROLE OF THE EXOCYST IN EXOCYTOSIS AND CELL MIGRATION Chapter 2
95 Jianglan Liu THE ROLE OF THE EXOCYST IN EXOCYTOSIS AND CELL MIGRATION Chapter 2
96 Jianglan Liu THE ROLE OF THE EXOCYST IN EXOCYTOSIS AND CELL MIGRATION Chapter 2 Figure 2.7 Exocytosis of VSV-G ts045 is blocked in EXO70 siRNA knockdown cells. (A) HeLa cells were treated with EXO70 siRNA and Luciferase siRNA (as control). Exo70 was knocked down as detected by Western blot. The amount of Sec8 in these cells was not affected. The anti-Exo70 monoclonal antibody (13F3) and anti-Sec8 monoclonal antibody (2E12) were used in the Western blot analysis. (B)
Luciferase siRNA-treated HeLa cells were transfected with VSV-G-GFP, kept at 40°C overnight, and shifted to 32°C for 0, 15, 30, 60, and 90 min in the presence of cycloheximide (100 µg/ml). The cells were then fixed and stained as described in
Materials and Methods. VSV-G was transported from ER, to the Golgi, and to the PM.
(C) In EXO70 knockdown cells, VSV-G transport to the PM through the endoplasmic reticulum and Golgi complex was normal. However, the incorporation of VSV-G
(stained by the 8G5 monoclonal antibody recognizing the extracellular domain of
VSV-G) was considerably delayed. Instead of being detected at the surface at 90 min as in control cells, VSV-G was detectable after 180 min from temperature arrest. (D and E) VSV-G association with various intracellular compartments was quantified in cells expressing luciferase siRNA (D) or EXO70 siRNA (E). For the quantification, boundaries of the whole cell, Golgi region and cell periphery region were outlined, and VSV-G fluorescence in these areas was then quantified using ImageJ 1.73v software after subtraction of background outside the cell. (F) Quantification of
97 Jianglan Liu THE ROLE OF THE EXOCYST IN EXOCYTOSIS AND CELL MIGRATION Chapter 2 fluorescence intensity of surface VSV-G signal stained by the 8G5 monoclonal antibody. Boundary of the cell surface was outlined, and average fluorescence intensity of surface VSV-G signal was quantified using ImageJ 1.73v software and then divided by the perimeter of the cell surface. Three independent experiments (20 cells each) were carried out. Error bars, SD. p < 0.01.
98 Jianglan Liu THE ROLE OF THE EXOCYST IN EXOCYTOSIS AND CELL MIGRATION Chapter 2 siRNA-treated cells, the translocation of VSV-G-GFP from the ER to the Golgi apparatus and from the Golgi to the PM followed time courses similar to that in control siRNA-treated cells. However, at 90 min, the extracellular exposure of
VSV-G-GFP was hardly detectable in nonpermeabilized cells; instead, it could only be detected after 180 min (Figure 2.7C), suggesting that the fusion of the exocytic vesicles with the PM was substantially delayed. Quantification of the GFP signal in different intracellular compartments indicates that the cellular distributions of
VSV-G-GFP at various time points after the temperature shift were similar in EXO70 siRNA and luciferase siRNA-treated cells (Figure 2.7, D and E): at 0 min, most of the
VSV-G-GFP was located at the ER (88% for luciferase siRNA treatment and 84% for
EXO70 siRNA treatment); at 30 min, a majority of VSV-G-GFP was located at the
Golgi apparatus (91% for luciferase siRNA and 92% for EXO70 siRNA treatment); and at 90 min, VSV-G-GFP was located at the cell periphery (88% for luciferase siRNA and 74% for EXO70 siRNA treatment). In contrast, quantification of the surface VSV-G signal displayed a different pattern in EXO70 siRNA and luciferase siRNA-treated cells. As shown in Figure 2.7F, at 0-, 15-, and 30-min points, fluorescence intensity of exposed VSV-G was diminutive both in control and EXO70 siRNA-treated cells, which indicates that VSV-G-GFP was translocating in the intracellular compartments; at 60 and 90 min, fluorescence intensity of surface
99 Jianglan Liu THE ROLE OF THE EXOCYST IN EXOCYTOSIS AND CELL MIGRATION Chapter 2 VSV-G was much less in EXO70 siRNA-treated cells than in control siRNA-treated cells; at 180 min, fluorescence intensity of exposed VSV-G in EXO70 siRNA-treated cells began to appear at the cell surface. These results suggest that the incorporation of the exocytic VSV-G vesicles with the PM was delayed in cells treated with EXO70 siRNA. Collectively, these results demonstrated that Exo70 is probably not required for the transport of VSV-G from the intracellular compartments to the cell periphery, but is critical for the efficient tethering or subsequent docking/fusion of
VSV-G–containing exocytic vesicles with the PM.
The Interaction of Exo70 with PI(4,5)P2 Is Important for the Exocytosis of
VSV-G ts045
To examine the role of the Exo70-PI(4,5)P2 binding in exocytosis, it is necessary to specifically disrupt the interaction between Exo70 and phospholipids in vivo. I therefore tested the transport of VSV-G in EXO70 siRNA knockdown cells transfected with the rat exo70 mutant, exo70-1, that is deficient in the PI(4,5)P2 binding. EXO70 siRNA-treated cells transfected wtih wild-type rat Exo70 (rExo70
WT) were used as control. While rat Exo70 is over 90% identical in protein sequence to human Exo70, on the nucleotide level, it cannot be targeted by the EXO70 siRNA oligos used in this study. The level of Exo70 knockdown as well as the expression of
100 Jianglan Liu THE ROLE OF THE EXOCYST IN EXOCYTOSIS AND CELL MIGRATION Chapter 2 GST-rExo70 and GST-rexo70-1 is shown in Figure 2.8A.
In EXO70 siRNA-treated cells, only a negligible amount of VSV-G-GFP was detected on the cell surface after 90 min of growth at the permissive temperature
(Figure 2.8B). Expression of wild-type rat Exo70 restored normal surface incorporation of VSV-G (VSV-G-myc) in EXO70 siRNA knockdown cells (Figure
2.8C); therefore the rat Exo70 can serve as a rescue reagent for the RNAi experiment in HeLa cells. In contrast, in EXO70 siRNA-treated cells expressing the exo70-1 mutant that is deficient in PI(4,5)P2 binding, the surface exposure of VSV-G-myc was disrupted, whereas the translocation of VSV-G-myc from the intracellular compartments to the cell periphery was not affected (Figure 2.8D). VSV-G-myc rather than VSV-G-GFP was used here so that different pairs of proteins in the cells could be stained (GST-rExo70 and myc vs. GST-rExo70 and 8G5). Quantification of cells with surface VSV-G indicates that expressing wild-type rat Exo70 in EXO70 siRNA-treated cells restores VSV-G surface exposure in the majority of the cells
(from 18 to 61%), whereas expressing rat exo70-1 does not have an obvious effect
(Figure 2.8E). These results suggest that the interaction of Exo70 with membrane lipids is essential for the exocytosis of VSV-G at the PM.
101 Jianglan Liu THE ROLE OF THE EXOCYST IN EXOCYTOSIS AND CELL MIGRATION Chapter 2
102 Jianglan Liu THE ROLE OF THE EXOCYST IN EXOCYTOSIS AND CELL MIGRATION Chapter 2
103 Jianglan Liu THE ROLE OF THE EXOCYST IN EXOCYTOSIS AND CELL MIGRATION Chapter 2
104 Jianglan Liu THE ROLE OF THE EXOCYST IN EXOCYTOSIS AND CELL MIGRATION Chapter 2 Figure 2.8 VSV-G ts045 exocytosis defect in EXO70 siRNA knockdown cells expressing the exo70-1 mutant. (A) EXO70 siRNA knockdown cells were transfected with GST-tagged rat Exo70 (GST-rExo70) or rat exo70 mutant
(GST-rexo70-1). The amount of endogenous Exo70 in HeLa cells, and the amount of extragenically expressed GST-tagged rat Exo70 were detected by anti-Exo70 monoclonal antibody (top panel). The total proteins in the cell lysates were detected by SYPRORed staining (bottom panel). (B) VSV-G-GFP trafficking in HeLa cells transfected with EXO70 siRNA. The cells were grown at 40°C overnight after transfection. The cells were then shifted to 32°C for 0, 30, 60, and 90 min in the presence of cycloheximide, fixed, and stained as described in Materials and Methods.
The 8G5 monoclonal antibody was used to detect the surface-incorporated VSV-G. (C and D) VSV-G-myc trafficking was examined in EXO70 siRNA knockdown cells expressing GST-rExo70 (C) or GST-rexo70-1 (D). VSV-G-myc and GST-tagged wild-type rExo70 (C) or exo70-1 (D) were cotransfected into EXO70 knockdown
HeLa cells. VSV-G transport to the PM was rescued in the EXO70 siRNA knockdown cells expressing wild-type rExo70, whereas VSV-G transport was not rescued in the
EXO70 siRNA knockdown cells expressing exo70-1. (E) Quantification of the percentage of cells with surface VSV-G staining.
105 Jianglan Liu THE ROLE OF THE EXOCYST IN EXOCYTOSIS AND CELL MIGRATION Chapter 2 2.3. Discussion
To understand the molecular basis of vesicle tethering at the PM, it is important to clarify how the tethering complex itself, the exocyst, is targeted to the PM. Here I identified a direct interaction between the exocyst component Exo70 and PI(4,5)P2; and demonstrated that this interaction is essential for the recruitment of the exocyst to the PM. Furthermore, disruption of this interaction impaired later stages of exocytosis of post-Golgi secretory vesicles at the PM.
PI(4,5)P2 and PS are the major negatively charged lipids in the PM. The observation that Exo70 binds to both 5% PI(4,5)P2 and 60% PS indicates that the interaction of Exo70 with the phospholipids is electrostatic in nature. This type of interaction has been found in a number of proteins, such as N-WASP and MARCKS
(McLaughlin and Murray, 2005). Comparing the charges of PI(4,5)P2 versus PS at the physiological pH, LUVs composed of 5% PI(4,5)P2 have approximately the same amount of effective charges as LUVs composed of 15–20% PS. However, the interaction of Exo70 with LUVs composed of 20% PS is much weaker. Mutations on exo70-1 that eliminate some of the positive charges in the Exo70 C-terminus nearly abolished the ability of Exo70 to bind PI(4,5)P2; however, the ability of this mutant to bind PS at high concentrations (≥60%) was only partially affected. These data suggest that Exo70 has significant binding specificity for PI(4,5)P2 over PS. I have also
106 Jianglan Liu THE ROLE OF THE EXOCYST IN EXOCYTOSIS AND CELL MIGRATION Chapter 2 examined the interaction of Exo70 with other phosphoinositides and observed its selectivity for PI(4,5)P2 over the stereoisomeric PI(3,5)P2 and other monophosphorylated phosphoinositides. I have also found that Exo70 binds
PI(3,4,5)P3 with an affinity that is comparable to that for PI(4,5)P2. PI(3,4,5)P3 has recently been found to be localized to the basolateral domain in MDCK cells
(Gassama-Diagne et al., 2006), and the exocyst has been implicated in basolateral vesicle targeting (Grindstaff et al., 1998). In other types of mammalian cells, although the concentration of PI(3,4,5)P3 is low at the PM in resting cells, it can be rapidly up-regulated in response to extracellular stimuli. It is therefore possible that Exo70 binds to PI(3,4,5)P3 under certain physiological circumstances or in certain cell types.
In yeast, our lab has found that the amount of the exocyst complex associated with the PM was much lower in the temperature-sensitive mss4 mutant cells, in which the PI(4,5)P2 level in the PM was reduced (data not shown), indicating that
PI(4,5)P2 mediates the membrane targeting of the exocyst. Moreover, the structural analysis of Exo70 provided important insights into the potential mechanism of membrane association of Exo70 (Dong et al., 2005; Hamburger et al., 2006). On the basis of the crystal structure information of yeast Exo70, I have made mutations on the rat Exo70 residues K632 and K635 (exo70-1), which are positively charged amino acids well conserved in the yeast Exo70 sequence. My in vitro vesicle sedimentation
107 Jianglan Liu THE ROLE OF THE EXOCYST IN EXOCYTOSIS AND CELL MIGRATION Chapter 2 experiments demonstrated that these mutations disrupted the PI(4,5)P2-binding.
Furthermore, the VSV-G trafficking analysis further revealed its functional importance in exocytosis at the PM. Later, Moore et al. (2007) resolved the crystal structure of mouse Exo70. Analysis of the structure indicates that the point mutations on exo70-1 are localized on the loop between Helix 18 and 19 on the surface of the mouse Exo70, which is almost identical to that of the yeast Exo70.
Taking advantage of the VSV-G trafficking assay, I was able to assess the role of Exo70 in various stages of membrane traffic. More importantly, using the exo70-1 mutant in the EXO70 RNAi knockdown cells, I was able to specifically examine the functional importance of Exo70-PI(4,5)P2 interaction in VSV-G exocytosis. The exocyst has been found in various cellular compartments, including Golgi and recycling endosomes, in addition to the PM (Yeaman et al., 2001; Fölsch et al., 2003;
Ang et al., 2004; Langevin et al., 2005). Here I found that the Exo70-PI(4,5)P2 interaction is not involved in the early stages of VSV-G trafficking through the endoplasmic reticulum and Golgi. Instead, it is critical for the PM events such as vesicle tethering and fusion. When the Exo70-PI(4,5)P2 interaction was perturbed, the transport of VSV-G to the PM was barely changed. However, the incorporation of
VSV-G into the PM was significantly impaired, as revealed by the 8G5 antibody specifically recognizing the extracellular domain of VSV-G. Similarly the exocyst has
108 Jianglan Liu THE ROLE OF THE EXOCYST IN EXOCYTOSIS AND CELL MIGRATION Chapter 2 been implicated in tethering and fusion of Glut4-containing vesicles in 3T3-L1 adipocytes (Inoue et al., 2003; Ewart et al., 2005; Tsuboi et al., 2005). It is possible that other exocyst components also interact with phospholipids (Moskalenko et al.,
2003). However, specific disruption of Exo70-PM interaction is sufficient to block exocytosis in mammalian cells.
The interaction of the exocyst with the PM is an important step in vesicle tethering. When and where this association takes place may regulate the kinetics and location of exocytosis. In the budding yeast S. cerevisiae, the exocyst complex is specifically concentrated at the growing tip of the daughter cell (the "bud tip"), which is the site of active exocytosis and cell surface expansion. Moreover, Exo70 primarily functions at the early stages of the yeast cell cycle, suggesting a temporal control of
Exo70 function (He et al., 2007a). In mammalian cells, growth factor signaling involving small GTPases may mediate the subunits assembly, translocation of the exocyst from intracellular compartments to, or activation of the exocyst at, the PM
(Sugihara et al., 2002; Moskalenko et al., 2002, 2003; Inoue et al., 2003; Takaya et al., 2004; Zuo et al., 2006). Future work will be focused on the identification and characterization of proteins that temporally and/or spatially regulate Exo70 and other exocyst components using different eukaryotic systems.
109 Jianglan Liu THE ROLE OF THE EXOCYST IN EXOCYTOSIS AND CELL MIGRATION Chapter 2 2.4. Materials and Methods
DNA Plasmid Construction
Wild-type rat Exo70 (rExo70) cDNA and various truncates of Exo70 were cloned in-frame into pEGFP-C1 for expression as green fluorescent protein (GFP) fusions or in pEBG for expression as glutathione S-transferase (GST) fusion in mammalian cells. rExo70 was also cloned into pGEX-KG (a modified form of pGEX-2T, from Amersham Biosciences, Piscataway, NJ) for expression as GST fusion in bacteria. The Exo70 mutants were generated using the QuikChange site-directed mutagenesis kit (Stratagene, La Jolla, CA). All the constructs were confirmed by nucleotide sequencing.
Cell Culture and RNA Interference Experiments
HeLa cells were cultured at 37°C in DMEM supplemented with 10% fetal bovine serum and 100 U/ml penicillin and 100 µg/ml streptomycin in a 5% CO2 incubator. For RNA interference (RNAi) experiments, cells were grown to 50% confluence and transfected with small interfering RNA (siRNA) duplexes using
Oligofectamine (Invitrogen, Carlsbad, CA). The human Exo70 siRNA target sequence is 5'-GGTTAAAGGTGACTGATTA-3'. The control Luciferase GL2 siRNA target sequence is 5'-AACGTACGCGGAATACTTCGA-3'. The efficiency of Exo70
110 Jianglan Liu THE ROLE OF THE EXOCYST IN EXOCYTOSIS AND CELL MIGRATION Chapter 2 knockdown was determined by Western blot.
Confocal Microscopy
Transfected HeLa cells were grown on coverslips, washed with phosphate-buffered saline (PBS), fixed in 4% paraformaldehyde at room temperature for 12 min, washed, permeabilized for 5 min with PBST (PBS-Tween), and blocked for 10 min with 2% bovine serum albumin in PBST. The coverslips were incubated sequentially with primary and secondary antibodies for fluorescence observation using the Leica TCS SL laser-scanning confocal microscope (63x objective; Deerfield,
IL). Images were processed with Adobe Photoshop (Adobe Systems, San Jose, CA; version 7.0).
Large Unilamellar Vesicle Sedimentation Assay
Large unilamellar vesicle (LUV) sedimentation assay was performed as previously described (Hokanson and Ostap, 2006). Phospholipids were purchased from Avanti Polar Lipids (Alabaster, AL). LUVs with a 100-nm diameter were prepared by size extrusion. Various lipids were mixed at different molar ratios, dried with nitrogen stream, and resuspended at a concentration of 2 mM in a buffer containing 12 mM HEPES, pH 7.0, and 176 mM sucrose. The mixed lipids were
111 Jianglan Liu THE ROLE OF THE EXOCYST IN EXOCYTOSIS AND CELL MIGRATION Chapter 2 subjected to five cycles of freeze-thaw and a 1-min bath sonication before being passed through 100-nm filters using a mini-extruder. LUVs were dialyzed overnight in the HNa100 buffer (10 mM HEPES, pH 7.0, 100 mM NaCl, 1 mM EGTA, and 1 mM dithiothreitol [DTT]). The percentages of phosphotidylserine (PS), PI(3)P, PI(4)P,
PI(3,5)P2, PI(4,5)P2, and PI(3,4,5)P3 indicated in the text are the molar percentages of total PS and PIPs with the remainder being phosphatidylcholine (PC). Lipid concentrations are given as total lipid. The binding of Exo70 to LUVs was determined by sedimentation assays in 200 µl total volume using TLA-100 rotor
(Beckman Coulter, Fullerton, CA). Sucrose-loaded LUVs were precipitated at
150,000 x g for 30 min at 25°C. The supernatants and pellets were subjected to 10%
SDS-PAGE and stained with SYPRORed (Invitrogen) for quantification of free and bound materials with the Image Quant software (Molecular Dynamics, Sunnyvale,
CA).
Membrane Fractionation
HeLa cells were plated in 10-cm dishes at 1.5 x 106 cells per dish. The next day cells were transfected with DNA by FuGene6 reagent and incubated at 37°C overnight. Homogenization and subcellular fractionation of the cells to isolate the PM fraction, cytosol, the low-density microsomal fraction (LDM), and the high-density
112 Jianglan Liu THE ROLE OF THE EXOCYST IN EXOCYTOSIS AND CELL MIGRATION Chapter 2 microsomal fraction (HDM) were performed basically as previously described
(Weber et al., 1988). All steps were performed at 4°C in the presence of a protease inhibitor cocktail. The distribution of Exo70 and Sec8 were detected by monoclonal antibodies (kind gifts of Dr. Shu-Chan Hsu, Rutgers University).
GST Pulldown Assay
HeLa cells were transfected with GFP-tagged Exo70 or exo70-1, and the cells were lysed in a buffer containing 20 mM Tris-HCl, pH 7.5, 25 mM KCl, 1 mM
MgCl2, 0.5 mM EGTA, 1 mM DTT, 0.5% Triton X-100, and protease inhibitors. Cell lysates were incubated overnight with glutathione-Sepharose conjugated with GST or
GST-TC10 (Q75L) at 4°C. After incubation, the beads were washed five times with the lysis buffer, and the bound proteins were analyzed by Western blot using an anti-GFP antibody. To detect the interaction of Exo70 and Sec8 in the cell, HeLa cells were transfected with GST-tagged Exo70 or exo70-1, and cell lysates were incubated overnight with glutathione-Sepharose beads at 4°C. After incubation, the beads were washed, and the bound proteins were detected by Western blot using anti-GST or anti-Sec8 monoclonal antibodies.
113 Jianglan Liu THE ROLE OF THE EXOCYST IN EXOCYTOSIS AND CELL MIGRATION Chapter 2 VSV-G Trafficking Assay
HeLa cells were transfected with EXO70 siRNA. luciferase siRNA was used as the negative control. After 24 h of the siRNA treatment, HeLa cells were transfected with VSV-G-45ts-GFP mutant and immediately placed at 40°C. After overnight growth, the cells were shifted to 32°C for 0, 15, 30, 60, and 90 min in the presence of cycloheximide (100 µg/ml). The cells were then fixed for GFP observation or immunofluorescence. The 8G5 mAb against the extracellular domain of VSV-G was kindly provided by Dr. Douglas Lyles (Wake Forest University). No detergent was used in the immunofluorescence procedure. Cells with surface VSV-Gs were quantified, and statistical analyses were performed using Student's t test. In some cases, HeLa cells were transfected with VSV-G-myc and GST-Exo70 or GST-exo70-1 after 24 h of the EXO70 siRNA treatment. The cells were divided into two sets based on their treatments. For Set I, the cells were fixed, permeabilized, and stained with anti-myc mAb (9E10) and anti-GST polyclonal antibody to test the intracellular traffic of VSV-G and to detect the expression of Exo70 or exo70-1 at 0-, 30-, 60-, and
90-min points. For Set II, cells of the 90-min point group were first stained with the
8G5 antibody, then permeabilized, and stained with anti-GST polyclonal antibody.
Anti-mouse Alexa488 and anti-rabbit Alexa594 were used as secondary antibodies for the above experiments. For the quantification of surface VSV-G signals at various
114 Jianglan Liu THE ROLE OF THE EXOCYST IN EXOCYTOSIS AND CELL MIGRATION Chapter 2 points, boundary of the cell surface was outlined, and average fluorescence intensity of surface VSV-G signal was quantified using ImageJ 1.73v software and then divided by the perimeter of the cell surface. For the quantification of VSV-G in different membrane compartments, boundaries of the whole cell, the Golgi, and the cell periphery were outlined, and VSV-G fluorescence in these areas was then quantified using ImageJ 1.73v software after subtraction of background outside the cell using the following equations:
Golgi (%) = FluorescenceGolgi /Fluorescencetotal
Cytoplasm (%) = Fluorescencecytoplasm /Fluorescencetotal.
Cell periphery (%) = (Fluorescencetotal -Fluorescencecytoplasm -FluorescenceGolgi)/Fluorescencetotal.
* This work has been published in Molecular Biology of the Cell in November 2007.
115 Jianglan Liu THE ROLE OF THE EXOCYST IN EXOCYTOSIS AND CELL MIGRATION Chapter 3 Chapter 3. The Role of the Exocyst in Matrix Metalloproteinase Secretion and Actin Dynamics during Tumor Cell Invadopodia Formation
Abstract
Invadopodia are actin-rich membrane protrusions formed by tumor cells that degrade the extracellular matrix for invasion. Invadopodia formation involves membrane protrusions driven by Arp2/3-mediated actin polymerization and secretion of matrix metalloproteinases (MMPs) at the focal degrading sites. The exocyst mediates the tethering of post-Golgi secretory vesicles at the plasma membrane for exocytosis and has recently been implicated in regulating actin dynamics during cell migration. Here, I report that the exocyst plays a pivotal role in invadopodial activity.
With RNAi knockdown of the exocyst component Exo70 or Sec8, MDA-MB-231 cells expressing constitutively active c-Src failed to form invadopodia. On the other hand, overexpression of Exo70 promoted invadopodia formation. Disrupting the exocyst function by siEXO70 or siSEC8 treatment or by expression of a dominant negative fragment of Exo70 inhibited the secretion of MMPs. I have also found that the exocyst interacts with the Arp2/3 complex in cells with high invasion potential; blocking the exocyst-Arp2/3 interaction inhibited Arp2/3-mediated actin polymerization and invadopodia formation. Together, my results suggest that the
116 Jianglan Liu THE ROLE OF THE EXOCYST IN EXOCYTOSIS AND CELL MIGRATION Chapter 3 exocyst plays important roles in cell invasion by mediating the secretion of MMPs at focal degrading sites and regulating Arp2/3-mediated actin dynamics.
3.1. Introduction
One of the most important features of cancer is the ability of the tumor cells to break through tissue barriers and invade into surrounding tissues. The initial step of tumor cell invasion is the formation of cell protrusions in the direction of cell movement. Invadopodia are specialized membrane protrusions formed by invading tumor cells that extend into the extracellular matrix (ECM). At the ultrastructural level, invadopodia are filament-like extensions from the ventral surface of the cells adherent to matrix that range from hundreds of nanometers to a few micrometers in diameter and reach 500 nm in length (Buccione et al., 2004 ; Marx, 2006; Linder, 2007).
Time-lapse image analysis has shown that invadopodia are formed de novo at the cell periphery and their lifetime varies from minutes to several hours (Yamaguchi et al.,
2005). Invadopodial protrusions are actin-based structures enriched in actin-associated proteins, adhesion proteins, matrix proteases such as matrix metalloproteinases
(MMPs) and signaling proteins that regulate the actin cytoskeleton and membrane remodeling. However, how multiple processes including signaling, protease secretion, and actin assembly are coordinated to generate a functional ECM-degrading machine
117 Jianglan Liu THE ROLE OF THE EXOCYST IN EXOCYTOSIS AND CELL MIGRATION Chapter 3 remains poorly understood.
Focal degradation of tissue barriers by MMPs plays a crucial role in tumor invasion. The matrix-degrading capability of invadopodia is largely dependent on
MMPs, including MMP-2, MMP-9, and MT1-MMP (Gimona and Buccione, 2006;
Linder, 2007). MMP-2 was the first MMP found to localize at invadopodia of
Src-transformed fibroblasts (Monsky et al., 1993). MMP-9 was also found at invadopodial structures such as those of the metastatic breast cancer cells
(Bourguignon et al., 1998) and leukemia cells (Redondo-Muñoz et al., 2006).
MT1-MMP is a transmembrane protease essential for invadopodial activity (Itoh and
Seiki, 2006). The secretion of a combination of MMPs is important for effective invasion. However, the exact mechanism by which MMPs are targeted to and exocytosed at invadopodia-forming sites is elusive.
In addition to MMP secretion, the formation of cell protrusions also involves the assembly of branched actin network at the leading edge. The Arp2/3 complex is the core machinery that nucleates actin for the generation of branched filamentous actin networks. During invadopodia formation, the Arp2/3 complex has been shown to play a critical role in the formation of degrading protrusions (Yamaguchi et al.,
2005). However, how actin assembly is controlled during invadapodia formation is still not well understood.
118 Jianglan Liu THE ROLE OF THE EXOCYST IN EXOCYTOSIS AND CELL MIGRATION Chapter 3 The exocyst is an evolutionarily conserved octameric protein complex that mediates the tethering of secretory vesicles to the plasma membrane for exocytosis
(Guo et al., 2000; Hsu et al., 2004; Munson and Novick, 2006; He and Guo, 2009).
The exocyst complex is involved in a number of cellular processes that require polarized exocytosis, including yeast budding, neurite extension, epithelia polarization, and cytokinesis (for review, see Hsu et al., 2004). Here I hypothesize that the exocyst is involved in invadopodial activities via regulating the secretion of
MMPs at the focal degradation sites. Recently, the exocyst has also been shown to be involved in actin-based membrane protrusion and cell migration (Zuo et al., 2006;
Rosse et al., 2006). The exocyst component, Exo70, directly interacts with the Arp2/3 complex, and this interaction is important for the regulation of actin assembly at the leading edge of migrating cells (Zuo et al., 2006). Therefore it is likely that the exocyst also regulates actin dynamics during invadopodia formation through its interaction with the Arp2/3 complex.
Here, I report that the exocyst plays a pivotal role in invadopodial activity. I have found that blocking the exocyst function inhibits invadopodial formation. RNAi knockdown of the exocyst component Exo70 or Sec8 in MDA-MB-231 cells expressing mutant Y527F c-Src abolished the secretion of MMPs, whereas the overexpression of Exo70 promoted MMP secretion. In addition, the exocyst-Arp2/3
119 Jianglan Liu THE ROLE OF THE EXOCYST IN EXOCYTOSIS AND CELL MIGRATION Chapter 3 interaction is important for actin assembly during invadopodia formation. Together, these findings suggest that the exocyst coordinates protease secretion and cytoskeleton dynamics during tumor invasion.
3.2. Results
The exocyst is necessary for invadopodial activity
A matrix degradation assay was applied to study invadopodia formation. This assay involves culturing cells on coverslips coated with thin, fluorochrome-conjugated gelatin matrices. Local proteolytic activity was revealed by the appearance of dark areas lacking fluorescence in the bright fluorescent matrix
(Artym et al., 2006). MDA-MB-231 parental cells were used in the experiments, along with cells stably transfected with the constitutively active c-Src (Y527F c-Src), which display increased invasive potential.
To investigate the role of the exocyst in invadopodia formation, I examined the effect of siRNA-mediated knockdown of endogenous exocyst subunits on invadopodia formation in c-Src–activated cells. Using different siRNA oligos that target EXO70 (siEXO70(1) and siEXO70(2)) and SEC8 (siSEC8), I observed effective knockdown of the target proteins by Western blot (82% for siEXO70(1), 67% for siEXO70(2) and 69% for siSEC8 (Figure 3.1A, left panel). The knockdown cells
120 Jianglan Liu THE ROLE OF THE EXOCYST IN EXOCYTOSIS AND CELL MIGRATION Chapter 3 formed much fewer invadopodia as indicated by fewer matrix degradation sites and a smaller matrix degradation area. Correspondingly, the number of actin puncta localized to sites of matrix degradation was also reduced in the knockdown cells
(Figure 3.1B). I have also tested whether the expression of rat Exo70 is able to rescue the defect of invadopodia formation in siEXO70-treated MDA-MB-231 (Y527F c-Src) cells. While rat Exo70 is more than 90% identical in amino acid sequence to human
Exo70, it is not targeted by the human EXO70 siRNA oligonucleotides used in this study. The level of Exo70 knockdown and the expression of GFP-rExo70 are shown in Figure 3.1A (right panel). I found that the defect of invadopodia formation in siEXO70-treated cells was restored by the expression of rat Exo70 (Figure 3.1C).
Quantification of the results from four independent experiments was carried out in siEXO70, siSEC8, control siRNA-treated cells, and siEXO70-treated cells expressing
GFP-rExo70. The degradation level was calculated by dividing the total area of the degraded zones per cell by the area of the whole cell. Quantification of degradation levels was included in Materials and Methods, and representative photographs were shown in Figure 3.1D. The percentage of cells with different degradation levels was calculated for each treatment. As shown in Figure 3.1E, most of the EXO70 or SEC8 siRNA-treated cells failed to form detectable invadopodia (77% for siEXO70(1), 68% for siEXO70(2) and 72% for siSEC8), whereas most of the control siRNA-treated
121 Jianglan Liu THE ROLE OF THE EXOCYST IN EXOCYTOSIS AND CELL MIGRATION Chapter 3
122 Jianglan Liu THE ROLE OF THE EXOCYST IN EXOCYTOSIS AND CELL MIGRATION Chapter 3
123 Jianglan Liu THE ROLE OF THE EXOCYST IN EXOCYTOSIS AND CELL MIGRATION Chapter 3 Figure 3.1 Knockdown of the exocyst inhibits invadopodial activity. (A)
Expression levels of the exocyst subunits Exo70 and Sec8 in MDA-MB-231 (Y527F c-Src) cells transfected with siRNAs. Lysates were prepared from cells transfected with siRNA oligos targeting Luciferase (Control siRNA), Exo70 (two different oligos: siEXO70(1) and siEXO70(2)), or Sec8 (siSEC8). siRNA treatments led to significant reduction in the amounts of Exo70 and Sec8 in the cells. The levels of Exo70 and
Sec8 in cell lysates were analyzed by Western blot (left panel). In the rescue experiment, siEXO70(1) knockdown cells were transfected with GFP-tagged rat
Exo70 (GFP-rExo70). The amounts of endogenous Exo70 and GFP-rExo70 were detected by Western blot (right panel). The amounts of actin were also examined as loading controls. (B) MDA-MB-231 (Y527F c-Src) cells with RNAi knockdown were cultured on Alexa 568–labeled gelatin film for 4 h. Cells were then fixed and stained with phalloidin-Alexa 488 to label F-actin. Individual and merged images of
F-actin (green) and gelatin (red) were shown. RNAi knockdown of Exo70 or Sec8 decreased the amount of focal degradation (the black dots). (C) Gelatin degradation assay was performed in siEXO70(1) knockdown cells expressing GFP-rExo70.
Individual and merged images of GFP-rExo70 fluorescence (green), F-actin (blue), and gelatin (red) are shown. The expression of GFP-rExo70 in siEXO70(1) knockdown cells rescued invadopodia formation in these cells. (D) Representative
124 Jianglan Liu THE ROLE OF THE EXOCYST IN EXOCYTOSIS AND CELL MIGRATION Chapter 3 photographs showing the quantification of matrix degradation in the cell. a) The original image of the labeling of Alexa 568-gelatin. b) The area of degraded matrix in the field was measured using ImageJ 1.73v software. The image was first converted to 8 bit grayscale files. Then automatic thresholding function of ImageJ was applied to the image, followed by manually adjusting the maximum threshold value by moving the bottom horizontal bar in the “Threshold” window from right to left until the upper threshold line just touches the edge of the histogram while keeping the minimum threshold value zero. This adjustment allows optimal matching to the original fluorescence images taken from the microscope. The AlexaFluor568-gelatin image was thresholded at 0-127 after the above adjustments. c) The boundary of the black dots and the total degradation area were obtained by automatic outlining. There are 41 degrading spots and the total degradation area is 3909.000 pixel. d) The area of the whole cell (37274.7 pixel) was obtained by manually outlining the boundary of the cell surface. Degradation level (10.487%) was then calculated as the total degradation area divided by the total area of the cell. (E) Comparison of focal degradation in cells with exocyst knockdown. Four independent measurements (50 cells each) for each siRNA treatment (left panel) and rescue experiment (right panel) were carried out. Error bars, SD. *p < 0.01. Scale bar, 5 µm.
125 Jianglan Liu THE ROLE OF THE EXOCYST IN EXOCYTOSIS AND CELL MIGRATION Chapter 3 cells formed invadopodia (81%; left panel). The expression of rat Exo70 in siEXO70(1)-treated cells largely rescued the degradation defects in these cells. The percentage of cells that formed invadopodia was restored from 26% to 68%) (Figure
3.1E, right panel).
I have also examined whether the exocyst is able to induce the formation of invadopodia. As shown in Figure 3.2A, in the parental MDA-MB-231 cells, expression of GFP-tagged Exo70 promoted invadopodia formation by increasing both the numbers of invadopodia and areas of degradation in each transfected cell, whereas expression of GFP alone did not cause any change in invadopodia formation
(data not shown). The percentage of cells with different degradation levels in
GFP-Exo70 and control vector-transfected cells was quantified. As shown in Figure
3.2B, the percentage of cells with focal matrix degradation was substantially increased in Exo70-transfected cells (31, 35, and 23% for 0–1, 1–5, and >5% degradation levels, respectively) compared with control cells (18, 10, and 5% for 0–1,
1–5, and >5% degradation levels, respectively). I also examined whether the exocyst is able to induce invadopodia formation in Y527F c-Src–expressing cells. As shown in Figure 3.2, C and D, expression of GFP-tagged Exo70 further promoted invadopodia formation in transfected cells. Expression of GFP alone was used as a negative control. Collectively, these results suggest that the exocyst plays an essential
126 Jianglan Liu THE ROLE OF THE EXOCYST IN EXOCYTOSIS AND CELL MIGRATION Chapter 3
127 Jianglan Liu THE ROLE OF THE EXOCYST IN EXOCYTOSIS AND CELL MIGRATION Chapter 3
128 Jianglan Liu THE ROLE OF THE EXOCYST IN EXOCYTOSIS AND CELL MIGRATION Chapter 3 Figure 3.2 Overexpression of Exo70 stimulates invadopodial activity. (A)
MDA-MB-231 cells were transfected with GFP-tagged Exo70 (GFP-Exo70), plated on fluorescent Alexa 568–labeled gelatin film for 4 h, and then processed for microscopy. Individual and merged images of GFP-Exo70 fluorescence (green),
F-actin (blue), and gelatin (red) are shown. Overexpression of GFP-Exo70 stimulated focal degradation in MDA-MB-231 cells (top panel). Cells transfected with GFP alone were used as negative control (bottom panel). (B) Quantification of degradation areas in cells overexpressing Exo70. Three independent measurements (~70 cells each) for each treatment were carried out. Error bars, SD. *p < 0.01. Scale bar, 5 µm.
(C) MDA-MB-231 (Y527F c-Src) cells were transfected with GFP-tagged Exo70
(GFP-Exo70), plated on fluorescent Alexa 568 labeled-gelatin film for 4 hrs, and then processed for microscopy. Individual and merged images of GFP-Exo70 fluorescence
(green), F-actin (blue) and gelatin (red) were shown. Expression of GFP alone did not change the ability of c-Src-expressing cells to form invadopodia (bottom panel).
Overexpression of GFP-Exo70 stimulated focal degradation in MDA-MB-231
(Y527F c-Src) cells (upper panel) and more invadopodia were formed compared with
GFP-transfected cells. (D) Quantification of degradation areas in cells overexpressing
Exo70. Three independent measurements (~50 cells each) for each treatment were carried out. Error bars, SD. “*” indicates p < 0.01. Scale bar = 5 µm.
129 Jianglan Liu THE ROLE OF THE EXOCYST IN EXOCYTOSIS AND CELL MIGRATION Chapter 3 role in invadopodia formation and invasion.
I also noticed that cells transfected with siRNA against Exo70 have weaker
F-actin staining comparing with control cells or SEC8 siRNA-treated cells. The effect of Exo70 on actin organization will be discussed later.
The localization of the exocyst at invadopodia-forming sites
Next, I tested the localization of exocyst components in the parental
MDA-MB-231 cells using confocal microscopy. In addition to the staining in the cytosol, endogenous Exo70 was also enriched in invadopodia as well as the plasma membrane (Figure 3.3A). Part of the Exo70 staining colocalized with actin puncta at sites of matrix degradation. I have also examined the localization of GFP-tagged
Exo70, expressed at low levels using the pJ3-GFP vector, in MDA-MB-231 cells. As shown in Figure 3.3B, there are partial overlaps of Exo70 and the degrading foci.
Sec8 has a similar pattern of localization to the invasion sites (data not shown;
Sakurai-Yageta et al., 2008). The exocyst components were not always concentrated at the degradation sites. This may be due to the highly dynamic nature of invadopodia and the mobility of the cells. Indeed, as previously demonstrated, F-actin and its regulatory proteins were not always detected at sites of degradation (Artym et al.,
2006). It is possible that the localization of the exocyst at invadopodia-forming sites
130 Jianglan Liu THE ROLE OF THE EXOCYST IN EXOCYTOSIS AND CELL MIGRATION Chapter 3
131 Jianglan Liu THE ROLE OF THE EXOCYST IN EXOCYTOSIS AND CELL MIGRATION Chapter 3 Figure 3.3 Localization of Exo70 at the focal degradation sites. (A)
MDA-MB-231 cells were plated on fluorescent Alexa 568–labeled gelatin (red) for
20 h. After fixation, cells were stained for Exo70 (green). Individual and merged images are shown. Endogenous Exo70 showed colocalization with the "degradation holes" in the fluorescent gelatin matrix, along with some enrichment at the plasma membrane. (B) The localization of GFP-tagged Exo70 expressed at low levels using the pJ3-GFP vector was examined in MDA-MB-231 cells. GFP-tagged Exo70 partially overlapped with the degrading spots. Higher magnification views of the boxed areas are shown underneath each image. Arrows, colocalization of Exo70 and invadopodia-forming sites. Scale bars, 5 µm.
132 Jianglan Liu THE ROLE OF THE EXOCYST IN EXOCYTOSIS AND CELL MIGRATION Chapter 3 is transient. Some of the focal degradation sites may represent regions where the exocyst completed their function.
The exocyst is required for the secretion of MMPs in MDA-MB-231 (Y527F c-Src) cells
The exocyst is involved in the tethering of secretory vesicles at the plasma membrane for exocytosis. It is thus likely that the exocyst mediates the secretion of MMPs at invadopodial sites. To test this hypothesis, I examined the effect of siRNA knockdown of EXO70 and SEC8 on the secretion of MMP-2 and MMP-9 in
MDA-MB-231 (Y527F c-Src) cells. Substrate zymography was performed to detect the secretion levels of MMP-2 and MMP-9. Zymography is the most sensitive and widely used assay for the detection of various forms of gelatinases on the basis of their different molecular weights (Mott and Werb, 2004). It involves the incorporation of substrate gelatin in polyacrylamide gels, the digestion of the “in gel” gelatin by separated gelatinases after electrophoresis, and the detection of gelatinases by the appearance of white areas on a blue background after Coomassie blue staining (Van den Steen et al., 2002). Gelatin zymography analyses of cell culture media detected a dramatic decrease in the levels of MMP-2 and MMP-9 in both EXO70 and SEC8 knockdown cells (Figure 3.4A). I have also tested whether the expression of rat
133 Jianglan Liu THE ROLE OF THE EXOCYST IN EXOCYTOSIS AND CELL MIGRATION Chapter 3 Exo70 is able to rescue the secretion defect in siEXO70-treated MDA-MB-231
(Y527F c-Src) cells. The expression of rat Exo70 did not affect EXO70 siRNA knockdown efficiency on endogenous Exo70 (Figure 3.4A). I found that the MMP secretion defects in siEXO70(1)-treated cells was restored by the expression of rat
Exo70 (Figure 3.4A). In all treatments, quantification of secreted MMP levels was carried out based on three independent experiments for each group. The levels of secreted MMP were represented as normalized secretion index, which was calculated as the percentage of secreted MMP relative to that of the control cells. As shown in
Figure 3.4B, in EXO70 and SEC8 siRNA-treated cells, secretion of MMP-2 decreased to 8% for siEXO70(1), 18% for siEXO70(2), and 17% for siSEC8; secretion of
MMP-9 decreased to 10% for siEXO70(1), 19% for siEXO70(2), and 18% for siSEC8.
The expression of rat Exo70 in siEXO70-treated cells largely rescued the secretion defect of MMPs in these cells (72% for MMP-2 and 81% for MMP-9). These findings strongly suggest that the exocyst complex plays a critical role in the secretion of the key invadopodia-associated MMPs.
Our lab has previously shown that Exo70 directly interacts with phosphatidylinositol 4,5-bisphosphate (PI(4,5)P2) through its C-terminus, and that this interaction is important for the association of Exo70 with the plasma membrane and the targeting of post-Golgi secretory vesicles to the plasma membrane
134 Jianglan Liu THE ROLE OF THE EXOCYST IN EXOCYTOSIS AND CELL MIGRATION Chapter 3
135 Jianglan Liu THE ROLE OF THE EXOCYST IN EXOCYTOSIS AND CELL MIGRATION Chapter 3 Figure 3.4 The exocyst is required for the secretion of MMPs in MDA-MB-231
(Y527F c-Src) cells. (A) Secretion of MMP-2 and MMP-9 was examined in
MDA-MB-231 (Y527F c-Src) cells with Exo70 or Sec8 RNAi knockdown. Cell culture media were collected and concentrated. MMP-2 and MMP-9 activities were analyzed by gelatin zymography. Knockdown of Exo70 or Sec8 led to dramatic decreases of MMP-2 and MMP-9 secreted in the media (top panel). Expression of rat
Exo70 rescued MMP secretion in Exo70 knockdown cells (siEXO70(1) + rExo70).
The amounts of endogenous Exo70 and Sec8, and the amount of extragenically expressed GFP-tagged rat Exo70 were detected by Western blot using anti-Exo70 and
Sec8 monoclonal antibodies (middle panel). Actin was used as the loading control
(bottom panel). A and B are duplicates of each experiment. (B) Quantification of secreted MMP levels was performed based on three independent experiments for each group. Secreted MMPs were represented as normalized secretion index, which was calculated as the amount of secreted MMP relative to that of the control cells. Error bars, SD. *p < 0.01.
136 Jianglan Liu THE ROLE OF THE EXOCYST IN EXOCYTOSIS AND CELL MIGRATION Chapter 3 (He et al., 2007; Liu et al., 2007). Disrupting the interaction between Exo70 and the plasma membrane by overexpressing a C-terminal truncated version of Exo70
(GFP-Exo70∆C, a.a. 1-408) would be expected to impair the secretion of MMPs. As shown in Figure 3.5, A and B, expression of Exo70∆C caused a significant decrease in the amounts of MMP-2 and MMP-9 secreted in the medium (81% decrease for
MMP-2 and 82% decrease for MMP-9) compared with GFP-transfected cells. I also examined whether the overexpression of full-length Exo70 (GFP-Exo70) is able to promote the secretion of MMPs. Gelatin zymography detected elevated levels of secreted MMP-2 (1.6-fold) and MMP-9 (1.7-fold) in cells transfected with
GFP-Exo70 (Figure 3.5, A and B). The observation that overexpression of Exo70∆C led to decreased level of secreted MMPs suggests that the plasma-membrane targeting of the exocyst is crucial for the secretion of MMPs.
The interaction between Exo70 and the Arp2/3 complex is required for invadopodia formation
Invadopodia formation involves membrane protrusions driven by
Arp2/3-mediated actin polymerization (Yamaguchi et al., 2005). Studies from our lab have shown that Exo70 directly interacts with the Arpc1 subunit of the Arp2/3 complex and that this interaction is important for actin-based membrane protrusion
137 Jianglan Liu THE ROLE OF THE EXOCYST IN EXOCYTOSIS AND CELL MIGRATION Chapter 3
138 Jianglan Liu THE ROLE OF THE EXOCYST IN EXOCYTOSIS AND CELL MIGRATION Chapter 3 Figure 3.5 Zymography analyses of MMP-2 and MMP-9 secreted from
MDA-MB-231 (Y527F c-Src) cells overexpressing Exo70∆C (amino acids 1–408) or full-length Exo70. (A) After transfection with GFP-tagged Exo70∆C or Exo70 for
18 h, cell culture media were collected and analyzed by gelatin zymography.
Overexpression of Exo70∆C caused a dramatic decrease in the amounts of MMP-2 and MMP-9 secreted in the media. Overexpression of Exo70 stimulated the secretion of MMPs (bottom panel). Cell lysates were collected for the detection of extragenically expressed GFP-tagged Exo70 proteins (top panel). Actin staining was shown as a loading control (middle panel). (B) Quantification of MMP secretion.
Error bars, SD. *p < 0.01.
139 Jianglan Liu THE ROLE OF THE EXOCYST IN EXOCYTOSIS AND CELL MIGRATION Chapter 3 and cell migration (Zuo et al., 2006). To investigate whether the exocyst-Arp2/3 interaction also plays a role in invadopodial activity, I first examined this interaction in carcinoma cells with different levels of invasiveness. It has been well demonstrated that the CA domain from the WASP family proteins directly interacts the Arp2/3 complex. I then used a recombinant GST-CA fusion protein to pull down the Arp2/3 complex from cells with various levels of invasiveness (HeLa cells, the parental
MDA-MB-231 cells, and the Y527F c-Src cells) and examined the interaction of the exocyst with Arp2/3 in these cells. As shown in Figure 3.6A, a much greater amount of Exo70 was pulled down by GST-CA from Y527F c-Src cells than from parental
MDA-MB-231 cells, and less Exo70 was pulled down from HeLa cells than from parental MDA-MB-231 cells. As a control, similar amounts of Arp3 were pulled down. On the other hand, neither Arp3 nor Exo70 was pulled down by GST. In addition, I did not detect any direct interaction between Exo70 and CA or VCA
(Figure 3.6B). This observation indicated that the Exo70-Arp2/3 interaction was much stronger in Y527F c-Src cells than in parental MDA-MB-231 cells. As the
Y527F c-Src cells are more invasive because of Src activation, this result suggests that the exocyst-Arp2/3 interaction is up-regulated in cells with high invasive potential.
Our lab has previously shown that the exo70 mutant, Exo70∆628–630, is
140 Jianglan Liu THE ROLE OF THE EXOCYST IN EXOCYTOSIS AND CELL MIGRATION Chapter 3
141 Jianglan Liu THE ROLE OF THE EXOCYST IN EXOCYTOSIS AND CELL MIGRATION Chapter 3
142 Jianglan Liu THE ROLE OF THE EXOCYST IN EXOCYTOSIS AND CELL MIGRATION Chapter 3 Figure 3.6 The interaction between Exo70 and the Arp2/3 complex is required for invadopodia formation. (A) The interaction between Exo70 and the Arp2/3 complex was examined in cells with different levels of invasiveness. GST-CA pulldown assay was performed in HeLa cells, MDA-MB-231 parental and c-Src
(Y527F)-transfected cells as described in Materials and Methods. Although
GST–CA–Sepharose (the CA domain of N-WASP) pulled down similar amounts of
Arp2/3 in HeLa, MDA-MB-231 parental and c-Src (Y527F)-transfected cells, more
Exo70 bound to Arp2/3 in c-Src (Y527F)-transfected cells than in MDA-MB-231 parental cells and less Exo70 bound to Arp2/3 in HeLa cells than in MDA-MB-231 parental cells. The inputs and the bound Exo70 and Arp2/3 were analyzed by Western blot using anti-Exo70 monoclonal and anti-Arp3 polyclonal antibodies, respectively.
GST alone was used as a negative control in the binding assay. (B) The interaction between Exo70 and CA/VCA was tested by in vitro GST pulldown assay. Glutathione
Sepharose conjugated with 20 µg of GST or GST-Exo70 (labeled on the top) was incubated with either 15 µg of Hisx6-tagged CA or VCA (labeled at the bottom). The input (1/20 of the total) and bound Hisx6-CA or Hisx6-VCA proteins were analyzed by western blot using an anti-Hisx6 monoclonal antibody. The molecular weights
(“MW”) are marked to the left (in kDa). (C) Matrix degradation assay was performed in MDA-MB-231 (Y527F c-Src) cells transfected with GFP-tagged Exo70∆628–630 or
143 Jianglan Liu THE ROLE OF THE EXOCYST IN EXOCYTOSIS AND CELL MIGRATION Chapter 3 GFP alone. After transfection with GFP-tagged Exo70∆628–630 or GFP for 18 h, cells were cultured on Alexa 568–labeled gelatin for 4 h and then processed for microscopy.
Individual and merged images of GFP-Exo70 fluorescence (green), F-actin (blue), and gelatin (red) were shown. Overexpression of GFP-tagged Exo70∆628–630 in Y527F c-Src cells largely inhibited matrix degradation, whereas transfection with GFP did not affect invadopodia formation. (D) Quantification of percentages of cells with different degradation levels in each treatment in B. Error bars, SD. *p < 0.01.
144 Jianglan Liu THE ROLE OF THE EXOCYST IN EXOCYTOSIS AND CELL MIGRATION Chapter 3 specifically deficient in interacting with the Arp2/3 complex (Zuo et al., 2006). I thus tested whether the overexpression of Exo70∆628–630 would affect invadopodia formation. As shown in Figure 3.6C, overexpression of GFP-tagged Exo70∆628–630
(GFP-Exo70∆628–630) in Y527F c-Src cells largely impaired matrix degradation, whereas transfection with GFP did not affect invadopodia formation. The percentage of cells without any invadopodia was substantially increased in
Exo70∆628–630-transfected cells (Figure 3.6D, 66% for GFP-Exo70∆628–630-transfected cells and 22% for GFP-transfected cells). Overall, the above results suggest that the interaction between the exocyst and the Arp2/3 complex plays an important role in the formation of invadopodia.
Next, I directly tested whether Exo70 affects Arp2/3-mediated actin polymerization in cell lysates using pyrene actin assay (Kouyama and Mihashi, 1981).
In this assay, a fraction of actin monomers were labeled with pyrenyl iodoacetamide on Cys-374. Because the fluorescence of pyrene increases 20 times upon actin polymerization, the fluorescence intensity will be proportional to the concentration of actin polymers. Thus, the kinetics of actin polymerization can be assessed by measuring the fluorescence intensity in real time using a fluorometer.
The lysates of Y527F c-Src cells with various siRNA treatments were collected and mixed with pyrenyl-actin in the presence of the VCA domain of mammalian
145 Jianglan Liu THE ROLE OF THE EXOCYST IN EXOCYTOSIS AND CELL MIGRATION Chapter 3 N-WASP. siRNA knockdown efficiency as well as the amounts of actin and Arp2/3 in cell lysates was examined in all siRNA-treated samples (Figure 3.7A). Similar amounts of actin and Arp3 were observed for all the samples. As shown in Figure
3.7B, actin polymerization was inhibited in lysates from Exo70 knockdown cells compared with control siRNA-treated cells, as revealed by longer lag phase and decreased actin polymerization rate. As a control, cell lysates had little actin polymerization activity in the absence of VCA. SEC8 siRNA-treated cells had a much smaller effect on actin assembly compared with EXO70 siRNA-treated cells. I also examined the final levels of actin polymerization in all siRNA-treated samples after
24 h. As shown in Figure 3.7D, the final level of actin polymerization is similar among all the samples. Moreover, I checked the amount of available actin in each reaction. As shown in Figure 3.7E, the amounts of actin in the pellet (polymerizable actin) and supernatant (unpolymerizable actin) fractions were at the same level in each treatment. This result suggests that the differences in initial actin polymerization rates are not likely due to the difference in the levels of polymerization-competent actin. Quantification was carried out based on normalized polymerization rate.
Polymerization rate was represented as the slope of the linear approximation line of each curve, and the normalized polymerization rate was calculated as the rate of each treatment relative to control siRNA-treated cells. As shown in Figure 3.7C, EXO70
146 Jianglan Liu THE ROLE OF THE EXOCYST IN EXOCYTOSIS AND CELL MIGRATION Chapter 3
147 Jianglan Liu THE ROLE OF THE EXOCYST IN EXOCYTOSIS AND CELL MIGRATION Chapter 3
148 Jianglan Liu THE ROLE OF THE EXOCYST IN EXOCYTOSIS AND CELL MIGRATION Chapter 3
149 Jianglan Liu THE ROLE OF THE EXOCYST IN EXOCYTOSIS AND CELL MIGRATION Chapter 3 Figure 3.7 Exo70 is involved in Arp2/3-mediated actin polymerization in the cell.
(A) Lysates were prepared from MDA-MB-231 (Y527F c-Src) cells transfected with indicated siRNA oligos. Exo70 and Sec8 knockdown levels were examined by
Western blot, along with the amounts of actin and Arp3 in cell lysates. (B) The ability of the lysates to stimulate actin polymerization was analyzed by pyrene actin assay in the presence or absence of the VCA domain of N-WASP as described in Materials and Methods. The initial rate of actin polymerization, an indicator of actin nucleation by Arp2/3, was two fold lower in Exo70 knockdown cells than in the control cells.
Sec8 knockdown slightly decreased the initial rate of actin polymerization. Cell lysates in the absence of VCA had little actin polymerization activity. (C) Normalized initial polymerization rates in each treatment in A were calculated by dividing the actin polymerization rate of each treatment by that of the control siRNA-treated cells.
Three independent measurements for each treatment were carried out. Error bars, SD.
*p < 0.01. (n = 3). (D) The final level of actin polymerization in exocyst knockdown and GFP-Exo70 overexpression samples. Cell lysates from all the treatments were ultracentrifuged and mixed with pyrenyl-actin for the actin polymerization reaction as described in MATERIALS AND METHODS. After 24 hours of incubation at room temperature, the samples were transferred into a cuvette and the fluorescence intensity was read in the fluorometer. The final extent of actin polymerization was
150 Jianglan Liu THE ROLE OF THE EXOCYST IN EXOCYTOSIS AND CELL MIGRATION Chapter 3 shown to be identical for all the samples tested. (E) The amounts of available actin in the reactions of all the samples. Actin polymerization reactions were set up as described above. After 24 hours of incubation for the reactions to reach the steady state, the samples were ultracentrifuged and the pellet / supernatant fractions of each sample were collected. The fractions were then subjected to SDS-PAGE and stained with SYPRO-Red. The amounts of actin in pellet and supernatant fractions were at the same level in each treatment. (F) Actin polymerization kinetics was analyzed in lysates from MDA-MB-231 (Y527F c-Src) cells transfected with GFP-Exo70 and
GFP-Exo70∆628–630 mutant. The levels of Exo70, actin, and Arp3 in cell lysates were detected by Western blot. (G) Lysates from cells expressing GFP-Exo70∆628–630 had a lower initial actin polymerization rate compared with GFP-transfected cells. Cells overexpressing GFP-Exo70 had an increased actin polymerization rate compared with the control cells. (H) Quantification of normalized polymerization rate of each treatment in D. Error bars, SD. *p < 0.01; n = 3.
151 Jianglan Liu THE ROLE OF THE EXOCYST IN EXOCYTOSIS AND CELL MIGRATION Chapter 3 siRNA treatment reduced the normalized actin polymerization rate by more than two fold compared with control siRNA treatment.
I also tested whether overexpression of Exo70 promotes Arp2/3-mediated actin polymerization in cell lysates. As shown in Figure 3.7, G and H, lysates from cells overexpressing GFP-Exo70 showed a decreased lag phase and an increased actin polymerization rate compared with GFP-transfected cells (1.38-fold that of
GFP-transfected cells). In contrast, lysates from cells overexpressing the Arp2/3 binding-deficient mutant of Exo70, GFP-Exo70∆628–630, had an increased lag phase and a lower actin polymerization rate compared with control cells (Figure 3.7, G and
H, with a normalized polymerization rate of 0.52). The final levels of actin polymerization were identical in Exo70 overexpression samples (Figure 3.7, C and D).
The amounts of actin and Arp3 were similar for all the samples (Figure 3.7F).
Collectively, these results suggest that Exo70 plays a positive regulatory role in
Arp2/3-mediated actin polymerization in addition to its function in vesicle tethering and exocytosis during invadopodia formation.
3.3. Discussion
Invadopodia are actin-based membrane protrusive structures formed by tumor cells that degrade the extracellular matrix for invasion. Investigation of the molecular
152 Jianglan Liu THE ROLE OF THE EXOCYST IN EXOCYTOSIS AND CELL MIGRATION Chapter 3 mechanisms of invadopodia formation and regulation is important for the understanding of tumor invasion and metastasis. Here, I demonstrate that the exocyst plays a critical role in invadopodia formation and invasion. The exocyst is enriched at focal degrading sites, and disruption of its function results in a reduction of invadopodial activity.
Focal degradation of the ECM barrier is a key feature of invadopodia, which is achieved by the secretion of proteases that degrade the basement membrane surrounding the tumor. A number of MMPs, including MMP-2 and MMP-9, have been shown to play an important role in degrading ECM during tumor invasion (Boyd,
1996; Chen, 1996; Chen and Wang, 1999; Deryugina and Quigley, 2006; Itoh and
Seiki, 2006; Furmaniak-Kazmierczak et al., 2007). MMPs are delivered to the surface of the tumor cells through exocytosis, and components of membrane traffic machinery have been implicated in tumorigenesis (Palmer et al., 2002; Cheng et al.,
2004; Steffen et al., 2008; Sakurai-Yageta et al., 2008). The exocyst is involved in tethering post-Golgi secretory vesicles to the plasma membrane for exocytosis. Here, using RNAi and dominant-negative mutants to inhibit exocyst function, I demonstrate that the exocyst is required for the secretion of MMP-2 and MMP-9. Our results are also in agreement with recent study showing the involvement of the exocyst in the secretion of MT1-MMP, a transmembrane protease that degrades ECM
153 Jianglan Liu THE ROLE OF THE EXOCYST IN EXOCYTOSIS AND CELL MIGRATION Chapter 3 (Sakurai-Yageta et al., 2008). Together, these results indicate that the exocyst mediates the secretion of different classes of MMPs during tumor invasion.
Although the principal function of the exocyst is to tether secretory vesicles for secretion, recent studies suggest that the exocyst is also involved in actin-based membrane protrusion. It was shown that RalA and RalB, members of the Ras family of small GTP-binding proteins, regulate exocyst function (Brymora et al., 2001;
Moskalenko et al., 2002; Sugihara et al., 2002; Polzin et al., 2002), and the
Ral-exocyst interaction induces filopodia formation through a mechanism that is independent of exocytosis (Sugihara et al., 2002). The exocyst is involved in cell migration (Zuo et al., 2006; Rosse et al., 2006; Spiczka and Yeaman, 2008).
Furthermore, Exo70 was found to directly interact with the Arpc1 subunit of the
Arp2/3 complex; EGF, which promotes cell membrane protrusion, stimulates the interaction between Exo70 and the Arp2/3 complex in HeLa cells (Zuo et al., 2006).
The Arp2/3 complex is the core machinery that nucleates actin for the generation of the branched actin network underneath the leading edges of the plasma membrane for membrane protrusion (Pollard and Borisy, 2003). It has been well established that the
Arp2/3 complex is essential for invadopodia formation (Buccione et al., 2004; Lorenz et al., 2004; Yamaguchi et al., 2005). RNAi-mediated knockdown of members of the
Arp2/3 complex or N-WASP, the activator of Arp2/3, inhibited invadopodia
154 Jianglan Liu THE ROLE OF THE EXOCYST IN EXOCYTOSIS AND CELL MIGRATION Chapter 3 formation (Lorenz et al., 2004; Yamaguchi et al., 2005). Microscopy analyses demonstrated that the Arp2/3 complex is enriched at invadopodia together with
N-WASP (Yamaguchi et al., 2005). Other regulators of the Arp2/3 complex such as
Nck1, Cdc42, cortactin, and WIP have also been shown to be involved in invadopodia formation (Yamaguchi et al., 2005; Clark et al., 2007). In the present study, we found that the exocyst has stronger interaction with the Arp2/3 complex in Src-activated cells compared with parental cells. Overexpression of the exo70 mutant that is defective in its interaction with Arp2/3 inhibited invadopodia formation. How Src kinase regulates the Exo70-Arp2/3 interaction is still under investigation. Because
GTPases such as Rac and Rho function downstream of Src kinase, these GTPases could be potential candidates for this process.
Using the pyrene actin assay, I have found that lysates from Y527F c-Src
MDA-MB-231 cells are capable of stimulating Arp2/3-mediated actin polymerization in the presence of VCA. I further found that cells with Exo70 knockdown by RNAi or cells with overexpression of the exo70 mutant deficient in Arp2/3-binding were less potent in Arp2/3-mediated actin polymerization, whereas lysates prepared from cells with Exo70 overexpression were more potent in stimulating actin polymerization.
These data are consistent with the fluorescence microscope observation that there were fewer and dimmer F-actin foci in the Exo70 knockdown cells (Figure 3.1).
155 Jianglan Liu THE ROLE OF THE EXOCYST IN EXOCYTOSIS AND CELL MIGRATION Chapter 3 These findings suggest that Exo70 plays a positive regulatory role in Arp2/3-mediated actin polymerization in cells. This is consistent with the observation that Exo70 overexpression led to extensive membrane protrusions in many types of cultured cells
(Wang et al., 2004; Xu et al., 2005; Zuo et al., 2006).
Overall, my study suggests that the exocyst is involved in invadopodia through mediating MMP secretion and regulating Arp2/3-mediated actin polymerization. The results suggest a coordination of protease secretion and cytoskeleton dynamics during tumor invasion. Future studies will focus on the molecular mechanisms by which
Exo70 regulates actin dynamics and how the exocyst is regulated by upstream signaling molecules in the cell.
3.4. Materials and Methods
Plasmids and Antibodies
Full-length rat Exo70 (rExo70 FL) cDNA was cloned in-frame in pEGFP-C1 and pJ3-GFP vectors for expression as enhanced green fluorescent protein (EGFP) or
GFP fusions. Exo70∆C (amino acids 1-408) was cloned in-frame in pEGFP-C1. The exo70 mutant, Exo70∆628–630, was generated using the QuikChange site-directed mutagenesis kit (Stratagene, La Jolla, CA) with GFP-Exo70 in pEGFP-C1 as a template (Zuo et al., 2006). Constructs were confirmed by restriction enzyme analysis
156 Jianglan Liu THE ROLE OF THE EXOCYST IN EXOCYTOSIS AND CELL MIGRATION Chapter 3 and sequencing. Monoclonal antibodies against Exo70 and Sec8 were generously provided by Dr. Shu-Chan Hsu (Rutgers University, Piscataway, NJ). Mouse anti-actin mAb (MAB 1501, clone C4) was purchased from Chemicon, Millipore
(Bedford, MA).
Cell Culture and RNA Interference Treatment
Human breast carcinoma MDA-MB-231 cells and stable lines of parental cells transfected with Y527F constitutively active c-Src were maintained at 37°C in
DMEM (Invitrogen, Carlsbad, CA) supplemented with 10% FBS, 2 mmol/L
L-glutamine, 100 U ml–1 penicillin, and 100 µg ml–1 streptomycin in a 5% CO2 incubator. Cell transfections were carried out using Lipofectamine 2000 (Invitrogen,
Carlsbad, CA). For RNA interference (RNAi), cells were grown to 50% confluence and transfected with small interfering RNA (siRNA) duplexes using Lipofectamine
2000. The human EXO70(1) siRNA target sequence is
5'-GGTTAAAGGTGACTGATTA-3'. The human EXO70(2) siRNA target sequence is
5'-GACCTTCGACTCCCTGATA-3'. The human SEC8 siRNA target sequence is 5'-
AGAACCTGCTTTCATGCAA-3'. The control Luciferase GL2 siRNA target sequence is 5'-AACGTACGCGGAATACTTCGA-3'. The efficiency of the knockdown was determined by Western blot.
157 Jianglan Liu THE ROLE OF THE EXOCYST IN EXOCYTOSIS AND CELL MIGRATION Chapter 3 Fluorescent Gelatin Degradation Assay
AlexaFluor 568–conjugated gelatin was prepared by labeling porcine gelatin
(Sigma, St. Louis, MO) with AlexaFluor 568 (Molecular Probes, Eugene, OR) according to the manufacturer's instructions. Coverslips (18 mm) were precleaned with 20% nitric acid for 30 min followed by extensive washing and ethanol sterilization. The coverslips were coated with 50 µg/ml poly-L-Lysine (Sigma) for 20 min at room temperature, washed with PBS, and fixed with 0.5% glutaraldehyde (Ted
Pella, Irvine, CA) for 15 min followed by extensive washing. The coverslips were then inverted on a drop of gelatin matrix (0.2% gelatin and AlexaFluor 568–labeled gelatin at an 8:1 ratio) and incubated for 10 min at room temperature. After washing with PBS, coverslips were incubated in 5 mg/ml sodium borohydride for 15 min, washed three times in PBS, and finally incubated in 2 ml of DMEM for 2 h before adding the cells.
To examine the ability of cells to form invadopodia and degrade matrix, 4 x
105 cells were plated on coverslips coated with AlexaFluor 568 and incubated at
37°C for 4 h. Cells were then fixed and prepermeabilized with 10% Formalin/0.1%
Triton X-100 in PBS for 15 min at room temperature. After three washes, cells were postpermeabilized with 0.5% Triton X-100 for 5 min. Cells were then washed with
PBS, labeled with primary antibodies for 2 h, and followed by labeling with
158 Jianglan Liu THE ROLE OF THE EXOCYST IN EXOCYTOSIS AND CELL MIGRATION Chapter 3 secondary fluorochrome-conjugated antibodies for 1 h. Actin filaments were visualized with Alexa-phalloidin (Molecular Probes). Cells were imaged with the
Leica DM IRB microscope (Deerfield, IL; 100xobjective), a high-resolution CCD camera (model ORCA-ER, Hamamatsu Photonics, Bridgewater, NJ) and the Leica
TCS SL laser-scanning confocal microscope (63x objective, Deerfield, IL). Images were processed with Adobe Photoshop (Adobe Systems, San Jose, CA; ver. 7.0). For quantification of degradation, percentage of cells with different degradation levels was calculated. Degradation levels of individual cells are reported as the total area of the degraded zones per cell relative to the area of the whole cell. The area of degraded matrix in the fields was measured using ImageJ 1.73v software
(http://rsb.info.nih.gov/ij/). Dark spots on the bright, fluorescent gelatin matrix were inverted and thresholded into black dots on white background, followed by automatic outlining of the dots (Figure 3.1D). The area of the whole cell was measured by manually outlining the cell boundary (Figure 3.1D).
Zymography
The "in-gel" zymography was used for the detection of MMPs on the basis of their different molecular weights (Van den Steen et al., 2002; Mott and Werb, 2004).
After treatments, cells were cultured in serum-free DMEM for 48 h. Cell culture
159 Jianglan Liu THE ROLE OF THE EXOCYST IN EXOCYTOSIS AND CELL MIGRATION Chapter 3 media were concentrated 50 times by filtration on Microcon concentrators (Millipore).
Samples were mixed with SDS loading buffer (10% SDS, 50% glycerol, 0.4 M Tris, pH 6.8, and 0.1% bromophenol blue) and separated on 8% polyacrylamide/0.3% gelatin gels. Gels were then washed in 2.5% Triton X-100, 30 min each time, and incubated in reaction buffer (50 mM Tris, pH 8.0, and 5 mM CaCl2) at 37°C for
24–48 h. After the reaction, gels were stained with staining buffer (0.12% Coomassie blue R-250, 50% methanol, and 20% acetic acid) for 1 h and destained overnight with destaining buffer (22% methanol and 10% acetic acid). Gels were scanned using
CanoScan 4400F. Gel loadings were normalized to total protein measured with a
Bio-Rad Protein Assay (Richmond, CA).
Glutathione S-Transferase-CA (Cofilin and Acidic Domains of N-WASP)
Pulldown Assay
MDA-MB-231 parental and c-Src (Y527F)-transfected cells were lysed in the lysis buffer (20 mM Tris-HCl, pH 7.5, 25 mM KCl, 1 mM MgCl2, 0.5 mM EGTA, 1 mM DTT, 0.5% Triton X-100, and protease inhibitors). A high-speed centrifugation
(12,000 rpm, 15 min) was carried out, and 1 ml of precleared cell lysates (1.5 mg total protein) was mixed with 20 µl (50% vol/vol) of glutathione Sepharose conjugated with 10 µg glutathione S-transferase (GST)-CA (cofilin and acidic
160 Jianglan Liu THE ROLE OF THE EXOCYST IN EXOCYTOSIS AND CELL MIGRATION Chapter 3 domains of N-WASP) proteins. After incubation at 4°C overnight, the beads were washed five times with the lysis buffer, and the bound proteins were analyzed by
Western blot using antibodies against Exo70 and Arp3. GST alone was used as a negative control in the experiment.
Pyrene Actin Assay
Cell lysates were collected in buffer B (20 mM Tris-HCl, pH 7.5, 25 mM KCl,
1 mM MgCl2, 0.5 mM EGTA, 0.1 mM ATP, and protease inhibitor cocktail [Sigma
P8340, 4-(2-aminoethyl) benzenesulfonyl fluoride, pepstatin A, E-64, bestatin, leupeptin, and aprotinin], and 1 mM DTT) and spun successively at 16,000 x g for 15 min and 80,000 rpm in a Beckman TLA-100.3 rotor (Fullerton, CA) for 20 min at
4°C. The resulting high-speed supernatant (HSS) was used for later experiments.
Pyrenyl-actin was dissolved in column buffer (TEA, 0.3 mM CaCl2, 0.1 mM EDTA,
0.7 mM ATP, and 6.25 mM NaN3) for 1 h, spun at 80,000 rpm in a Beckman
TLA-100.3 rotor for 20 min at 4°C to remove F-actin, and mixed with Mg2+ converting buffer for 5 min to convert Ca2+-actin to Mg2+-actin. Mg2+-pyrenl-actin was then diluted in the polymerization buffer (60 mM KCl, 2.5 mM NaCl, 0.6 mM
MgCl2, 5 mM Tris-HCl, pH 7.5, 2.5 mM HEPES, pH 7.1, 0.5 mM EGTA, 30 µM
CaCl2, 0.2 mM ATP, and 0.3 mM NaN3) to a final concentration of 1.5 µM and
161 Jianglan Liu THE ROLE OF THE EXOCYST IN EXOCYTOSIS AND CELL MIGRATION Chapter 3 immediately mixed with HSS, which contained 2.4–2.6 µM unlabeled G-actin as estimated by Western blot (data not shown) in the presence of 0.2 mM ATP and 50 nM GST-tagged verprolin, cofilin, and acidic (VCA) domain of mammalian N-WASP.
The mixture was quickly transferred into a cuvette, and the fluorescence intensity was read every 5 s in a fluorometer. Three independent measurements were carried out for each treatment. Polymerization curves and rates were obtained using Excel
(Microsoft, Redmond, WA). The polymerization rate was represented as the maximal slope of the elongation phase of each curve, and the normalized polymerization rate was calculated as the rate of each treatment relative to the control treatments. Each polymerization curve was first smoothed using Sigmaplot software to elicit trends from noisy data. Then the slope of each point on the curve was calculated as the slope of the line between this point and the point 60 s ahead. The slopes of all the points on each curve were used to create a new plot. The maximal value of this slope curve is regarded as the actin polymerization rate.
* This work has been published in Molecular Biology of the Cell in June 2009.
162 Jianglan Liu THE ROLE OF THE EXOCYST IN EXOCYTOSIS AND CELL MIGRATION Chapter 4 Chapter 4. The role of Exo70 in Arp2/3-mediated actin assembly during cell migration
Abstract
Efficient cell migration requires the coordination of actin reorganization and membrane trafficking. The Arp2/3 complex is a key nucleating factor for the generation of branched actin network at the leading edge of migrating cells. The exocyst is an octameric protein complex essential for tethering secretory vesicles to specific domains of the plasma membrane for exocytosis. It has been shown that the exocyst component Exo70 interacts with the Arp2/3 complex via Arpc1 (a subunit of the Arp2/3 complex) and plays an important role in cell migration. However, the molecular implication of Exo70-Arp2/3 interaction is unclear. In this study, I found that Exo70 enhances Arp2/3-mediated actin polymerization in concert with WAVE2.
TIRFM examination of actin filament dynamics further suggests that Exo70 acts by promoting Arp2/3-mediated F-actin branching in the presence of WAVE2. In addition, I have examined how the Exo70-Arp2/3 interaction is regulated in the cell downstream of EGF signaling. Overall, these results suggest that Exo70 synergizes with WAVE2 to promote dendritic actin nucleation through a direct interaction with the Arp2/3 complex, which may reveal a novel mechanism for regulating actin polymerization and branching during cell migration. 163 Jianglan Liu THE ROLE OF THE EXOCYST IN EXOCYTOSIS AND CELL MIGRATION Chapter 4 4.1. Introduction
Cell migration plays critical roles in the development and maintenance of living organisms. Cell migration is a highly integrated process that includes a series of steps: establishment of cell polarity, extension of membrane protrusions at the leading edge, formation of the adhesion with extracellular matrix (ECM), contraction of the cell body and release from the ECM at the cell rear (Ridley et al., 2003; Sheetz et al.,
1999). The completion of efficient cell migration requires the coordination of actin dynamics and membrane trafficking. However, how they are functionally coupled is still under investigation.
To migrate, a cell first forms membrane protrusions at the leading edge, which is driven by the assembly of a branched network of actin filaments. The Arp2/3 complex plays a crucial role in the initial nucleation step for the generation of branched actin networks, a rate-limiting step in actin polymerization. The Arp2/3 complex is thought to mimic an actin dimer to function as the “seed” for the initiation of a new actin filament that emerges from an existing filament in a 70° y-branch configuration.
Purified Arp2/3 complex has low actin nucleation activity, but can be activated by a number of nucleation-promoting factors (NPFs). The major NPFs in mammals are the WASP and SCAR/WAVE family proteins, which include the two WASP
164 Jianglan Liu THE ROLE OF THE EXOCYST IN EXOCYTOSIS AND CELL MIGRATION Chapter 4 family proteins WASP and N-WASP, and the three WAVE family proteins WAVE1,
WAVE2 and WAVE3. All of them share a conserved carboxyl-terminal VCA
(verprolin homology, central, and acidic) domain, which is the region required for
Arp2/3 activation. The V and C regions bind G-actins, and the CA region mediates a direct interaction with the Arp2/3 complex which promotes a conformational change that activates the complex. The activated Arp2/3 complex along with the actin monomer delivered by NPF forms an Arp2-Arp3-actin trimer that functions as the nucleation core for the elongation of the new daughter filament. As a result, the daughter filament is anchored to the side of the mother filament as a branch.
The organization of the N-terminal region, which consists of domains that are thought to provide a link with regulatory proteins, is quite different among
WASP/WAVE family proteins. WASP and N-WASP contain a WASP homology 1
(WH1) domain, also known as an Ena/VASP homology 1 (EVH1) domain, and a
CRIB domain, which binds to the small GTPase Cdc42. The WH1 domain interacts with the WASP-interacting protein (WIP) family of proteins, which is thought to inhibit the activity of WASP or N-WASP. By contrast, the SCAR/WAVE proteins contain a SCAR homology domain (SHD), and do not contain any type of
GTPase-binding motif. Both WASPs and SCARs/WAVEs contain a basic region,
which mediates the binding to PtdIns(4,5)P2 and PtdIns(3,4,5)P3, respectively, and
165 Jianglan Liu THE ROLE OF THE EXOCYST IN EXOCYTOSIS AND CELL MIGRATION Chapter 4 localize the proteins to the plasma membrane (Rohatgi et al., 2000; Oikawa et al.,
2004). Thus, the N-terminal domains determine the regulation and subcellular localization of WASP and SCAR/WAVE proteins.
In resting cells, WASP and N-WASP are predominantly found in an auto-inhibited conformation in which intramolecular interactions between GBD and the C region obscure the regions that are required for Arp2/3 activation. This auto-inhibition is released by the competitive binding of the small GTPase Cdc42 and
PI (4,5)P2 (Kim et al., 2000; Rohatgi et al., 2000; Prehoda et al., 2000). Unlike
WASPs, WAVE proteins are not auto-inhibited and they form a complex with four other proteins – PIR121, Nap1, Abi and HSPC300 in the cell (Eden et al., 2002).
Upon stimulation, the small GTPase Rac associates with the complex via direct interaction with the PIR121 subunit and leads to WAVE activation (Ibarra et al.,
2006). Activation of the WAVE2 complex requires simultaneous interactions with prenylated Rac-GTP and acidic phospholipids, as well as a specific state of phosphorylation (Lebensohn and Kirschner, 2009).
WASP and WAVE family proteins are implicated in distinct cellular functions.
WASP is specifically expressed in hematopoietic cells and plays a role in phagocytosis. N-WASP is universally expressed, and functions in a variety of cellular processes, including endocytosis, organelle motility, invadopodia formation, and
166 Jianglan Liu THE ROLE OF THE EXOCYST IN EXOCYTOSIS AND CELL MIGRATION Chapter 4 vesicular trafficking (Merrifield et al., 2004; Lommel, et al., 2001; Yanagawa et al.,
2001; Yamaguchi et al., 2005; Gasman et al., 2004). WAVE2 and WAVE3 are essential for lamellipodia formation and directional cell migration, whereas WAVE1 is required for the formation of dorsal ruffles and functions to stabilize lamellipodia protrusions (Suetsugu et al., 2003; Yamazaki et al., 2003 & 2005).
In addition to regulation by WASP and WAVE proteins, recent studies have indicated that the Arp2/3 complex is phosphorylated by serine/threonine and tyrosine kinases. For example, the p21-activated kinase (PAK) phosphorylates Arpc1 on
Thr21 (Vadlamudi et al., 2004); the MARK-activated protein kinase 2 phosphorylates
Ser77 in Arpc5 (Singh et al. 2003); mass spectrometry revealed phosphorylated
Thr237 and Thr238 in Arp2 (LeClaire et al., 2008). Although phosphorylation of the
Arp2/3 complex has been shown to regulate its actin nucleating activity and increase cell motility, the molecular mechanisms downstream of this phosphorylation are still unknown.
The p21-activated kinases (PAK) were the first Rho family GTPase-regulated kinases to be identified in a screen for specific Rho GTPase binding partners in rat brain cytosol (Manser et al., 1994). Pak kinases are serine/threonine protein kinases that function as important effectors of Rac and Cdc42 GTPases in response to extracellular stimuli such as EGF. Paks play fundamental roles in a wide range of
167 Jianglan Liu THE ROLE OF THE EXOCYST IN EXOCYTOSIS AND CELL MIGRATION Chapter 4 cellular processes, including cytoskeletal dynamics, cell motility, gene transcription, cell-cycle progression, cell death and survival signaling (Sells et al., 1997;
Slack-Davis et al., 2003; Beeser et al., 2005; Eblen et al., 2002). The most well-characterized function of the Pak kinases is its regulation of cytoskeletal organization and cell motility. Immunofluorescence analysis using Pak1-specific antibody have shown that PAK1 redistributed from the cytosol into cortical actin structures after cell stimulation (Dharmawardhane et al., 1997). Overexpression of
Pak induced the formation of lamellipodia, filopodia and membrane ruffles (Sells et al., 1997). The molecular basis for these activities is not completely understood, but a number of known Pak substrates may account for the effects of Pak on the cytoskeletal structure. Arpc1 is a newly discovered substrate of PAK1 kinase and the phosphorylation of Arpc1 by PAK1 was indicated to be required for both constitutive and growth-factor-induced cell motility (Vadlamudi et al., 2004). However, the exact molecular mechanism of how the Pak1-phosphorylation on Arpc1 regulates actin dynamics is still unclear.
In addition to actin reorganization, cell migration also requires polarized exocytosis for the localized delivery of membrane components to the front of the cell.
The exocyst is an evolutionarily conserved octameric protein complex that mediates the targeting of post-Golgi and endocytic recycling vesicles at specific domains of the
168 Jianglan Liu THE ROLE OF THE EXOCYST IN EXOCYTOSIS AND CELL MIGRATION Chapter 4 plasma membrane for exocytosis (Guo et al., 2000; Hsu et al., 2004). The exocyst complex is required for secretion, thus is involved in many cellular processes that require polarized exocytosis including yeast budding, cell-cell adhesion, cytokinesis and neurite extension (TerBush and Novick, 1995; Matern et al., 2001; Hazuka et al.,
1999; Chen et al., 2006). Furthermore, the exocyst has also been implicated in several cellular events through mechanisms that are beyond its role in exocytosis. Recently, the exocyst has been shown to be involved in cell migration through its direct interaction with the Arp2/3 complex (Zuo et al., 2006). The exocyst component
Exo70 was shown to directly bind to Arpc1, a component of the Arp2/3 complex.
Arpc1 is an essential subunit for the nucleating activity of Arp2/3, which interacts with both the CA region of NPFs and the F-actin filaments during the formation of actin branches (Kelly et al, 2006; Rouiller et al, 2008). The Exo70-Arpc1 interaction is regulated by epidermal growth factor (EGF) signaling and is important for the regulation of actin assembly at the leading edge of migrating cells. However, the molecular function and the cellular regulation of the Exo70-Arpc1 interaction are still under investigation.
Here, I have identified Exo70 as a positive regulator for the activity of Arp2/3 and WAVE2 in dendritic actin nucleation. It suggests a potential molecular connection between machineries that control exocytosis and actin organization. I have
169 Jianglan Liu THE ROLE OF THE EXOCYST IN EXOCYTOSIS AND CELL MIGRATION Chapter 4 also examined the possible mechanisms of the molecular basis of the stimulatory effect of Exo70 in actin assembly. In addition, I have examined how the
Exo70-Arp2/3 interaction is regulated in the cell downstream of EGF signaling.
4.2. Results rExo70 enhances Arp2/3-mediated actin polymerization
Exo70 has a direct interaction with the Arp2/3 complex. Moreover, Exo70 has been shown to colocalize with Arp2/3 at the leading edge of the migrating cells and is required for the formation of lamellipodia (Zuo et al., 2006). Based on these observations, I hypothesize that Exo70 promotes the nucleation activity of Arp2/3 in actin assembly. The activity of Arp2/3 and its activators in actin polymerization was examined using the standard pyrene actin assay (Kouyama and Mihashi, 1981). In this assay, a fraction of actin monomers were labeled with pyrenyl iodoacetamide on
Cys-374. Because the fluorescence of pyrene increases 20 times upon actin polymerization, the fluorescence intensity will be proportional to the concentration of actin polymers. Thus, the kinetics of actin polymerization can be assessed by measuring the fluorescence intensity in real time using a fluorometer.
Mammalian Arp2/3 complex was purified from cell lysates by affinity chromatography using recombinant GST-CA immobilized on glutathione
170 Jianglan Liu THE ROLE OF THE EXOCYST IN EXOCYTOSIS AND CELL MIGRATION Chapter 4 Sepharose-4B (Figure 4.1A). rExo70 was purified from E. coli as a GST fusion protein and then treated with Prescission Protease to cleave the GST tag.
Recombinant GST-WAVE2 was purified from E. coli (Figure 4.1A). To examine actin assembly, Arp2/3 and proteins to be tested were mixed and pre-incubated at 4°C for 30 min prior to addition to the actin assembly mix. As shown in Figure 4.1B, the purified Arp2/3 has a low activity in promoting actin polymerization. Addition of
WAVE2 enhanced the actin nucleation activity of Arp2/3 as previously reported. In contrast, addition of rExo70 alone did not affect the activity of the Arp2/3 complex. rExo70 does not promote or inhibit the spontaneous actin nucleation (Figure 4.1B).
Thus, Exo70 neither nucleates actin polymerization like an actin nucleator, nor functions like NPFs to activate the Arp2/3 complex.
Next, I examined whether Exo70 affects the activity of Arp2/3 in the presence of WAVE2. As shown in Figure 4.1B, addition of rExo70 in the presence of Arp2/3 and WAVE2 further promoted actin assembly, as indicated by a shortened lag phase and increased actin polymerization rate compared to samples without Exo70. As a control, GST had no effect on the kinetics of actin polymerization mediated by Arp2/3 and WAVE2 (Figure 4.1B). The effect of Exo70 on actin assembly was quantified by measuring the time needed to reach half maximal polymerization and the rate of the actin polymerization at the elongation phase (Figure 4.1C). And the effect of rExo70
171 Jianglan Liu THE ROLE OF THE EXOCYST IN EXOCYTOSIS AND CELL MIGRATION Chapter 4
172 Jianglan Liu THE ROLE OF THE EXOCYST IN EXOCYTOSIS AND CELL MIGRATION Chapter 4 Figure 4.1 rExo70 enhances actin polymerization mediated by the Arp2/3 complex and WAVE2. (A) Proteins used in the pyrene actin assay. The molecular weights (“MW”) are marked to the left (in kDa). (B) Actin polymerization kinetics for 2 µM monomeric rabbit muscle actin (20% pyrenyl-labeled) in the presence of proteins to be tested including Arp2/3, WAVE2, GST and rExo70. The amounts of various proteins added in the reactions were indicated below the curve. (C) Diagram showing the time to reach half maximal polymerization (left panel) or the rate of actin polymerization during the elongation phase (right panel) for reactions described in
(B). Error bars represents standard deviation. n=3. (D) Dose-dependent enhancement of the Arp2/3 activity by rExo70. The graph shows the assembly of 2 µM G-actin in the presence of 15 nM Arp2/3, 25 nM WAVE2, and 0-2000 nM rExo70. The amounts of rExo70 were indicated below the curve.
173 Jianglan Liu THE ROLE OF THE EXOCYST IN EXOCYTOSIS AND CELL MIGRATION Chapter 4 in Arp2/3-WAVE2 mediated actin polymerization was dose-dependent, which saturated at an Exo70 concentration below 2 µM (Figure 4.1D).
Exo70 promotes actin nucleation in synergy with Arp2/3 and WAVE2
Since Exo70 synergizes with WAVE2 in activating Arp2/3, examining the morphology of the filaments, such as the length of filaments, or the number of branches, may provide insights to the molecular mechanism of the action of Exo70.
In order to directly visualize the dynamics of individual actin filaments in real time, total internal reflection fluorescence microscopy (TIRFM) was applied. With TIRFM, only filaments captured at the surface of the flow chamber will be illuminated, while the background fluorescence from the solution was largely eliminated (Amann and
Pollard, 2001; Kuhn and Pollard, 2005). Experiments were carried out in flow chambers treated with NEM-myosin to capture the actin filaments at the surface of the coverslip. Arp2/3 and proteins to be tested were mixed in the actin polymerization buffer and incubated at 4°C for 30 min. G-actin (8% Rhodamine labeled) was then mixed with the pre-incubated samples which contain (1) Arp2/3 + WAVE2, (2)
Arp2/3 + WAVE2 + rExo70 and immediately transferred into the flow chamber for
TIRF microscopic observation. Images were recorded in real time at 1.14 sec per frame after the polymerization started. As shown in Figure 4.2, A and B, more actin branches were formed in the presence of rExo70 (marked by red dots). Quantification
174 Jianglan Liu THE ROLE OF THE EXOCYST IN EXOCYTOSIS AND CELL MIGRATION Chapter 4
175 Jianglan Liu THE ROLE OF THE EXOCYST IN EXOCYTOSIS AND CELL MIGRATION Chapter 4
176 Jianglan Liu THE ROLE OF THE EXOCYST IN EXOCYTOSIS AND CELL MIGRATION Chapter 4 Figure 4.2. Elongation and branching of actin filaments visualized by TIRFM.
(A, B) G-Actin (0.8 µM, 8% rhodamine-labeled) was polymerized in fluorescence buffer (60 mM KCl/2.5 mM NaCl/0.6 mM MgCl2/5 mM Tris, pH 7.5/2.5 mM
HEPES, pH 7.1/0.5 mM EGTA/30 µM CaCl2/0.2 mM ATP/0.3 mM NaN3/1% glucose/100 µg/ml glucose oxidase/20 µg/ml catalase/0.5% Methylcellulose) in a flow chamber with 6 nM Arp2/3, 10 nM WAVE2, in the presence (B) or absence (A) of 1 µM rExo70. The coverslip of the flow chamber was coated with 6 nM
NEM-myosin for 1.5 min, and blocked with 1% BSA for 4 min before use. Exposures of 1 sec were collected every 1.14 sec for 10 min. Shown are frames representing the time course of elongation and branching. Red dots indicate the branch points. (C)
Images captured for samples with (right panel) or without (left panel) rExo70 at
663sec after the polymerization reaction started. Actin branches are labeled with red dots. (D) Quantification of branch numbers in each treatment. Three movies were analyzed for each category. Error bars stand for standard errors. (E) Total length of
F-actin filaments was measured for each treatment. Three movies were analyzed for each category. Error bars stand for standard errors.
177 Jianglan Liu THE ROLE OF THE EXOCYST IN EXOCYTOSIS AND CELL MIGRATION Chapter 4 of the branch numbers was carried out based on three independent experiments for each group. Each branch nucleation event of every 100 seconds was counted during the first ~600 seconds from the beginning of the polymerization reaction. As shown in Figure 4.2, C and D, the branch numbers of samples with rExo70 was 2-3-fold higher than samples without rExo70. The total length of F-actin filaments in the field was also measured for each treatment (Materials and Methods). As shown in Figure
4.2E, the sample with rExo70 has similar amount of total F-actin filaments as compared to the sample without rExo70, which suggests that the difference in branch numbers between the treatments is not likely due to dfferent amount of total F-actin filaments in each treatment. As a control, GST had no effect on actin branching in the experiment (data not shown). Overall, these observations suggest that Exo70 facilitates Arp2/3-WAVE2 mediated actin polymerization by promoting the generation of branched actin filaments.
Examine whether Exo70 directly interacts with WAVE2
Since Exo70 promotes actin assembly synergistically with Arp2/3 and WAVE2, it is possible that Exo70 directly binds to WAVE2 and regulates its activity through direct interaction. Recombinant GST-WAVE2 was conjugated to Glutathione Sepharose and incubated with HeLa cell lysates or the cell lysates expressing GFP-rExo70 or
178 Jianglan Liu THE ROLE OF THE EXOCYST IN EXOCYTOSIS AND CELL MIGRATION Chapter 4
179 Jianglan Liu THE ROLE OF THE EXOCYST IN EXOCYTOSIS AND CELL MIGRATION Chapter 4 Figure 4.3 GST-WAVE2 pulled down endogenous and GFP/myc-tagged rExo70 from HeLa cells. HeLa cell lysates alone or the cell lysates transfected with
GFP-rExo70 or myc-rExo70 were incubated with glutathione Sepharose conjugated with GST-WAVE2. The inputs and the bound endogenous, GFP and myc-tagged
Exo70 were analyzed by Western blot using anti-Exo70 (A), anti-GFP or anti-myc (B) monoclonal antibodies, respectively. GST alone was used as a negative control in the binding assay.
180 Jianglan Liu THE ROLE OF THE EXOCYST IN EXOCYTOSIS AND CELL MIGRATION Chapter 4 myc-rExo70. The bound proteins were analyzed by Western blot using antibodies against Exo70, GFP or myc tag. As shown in Figure 4.3, GST-WAVE2, but not GST alone, was able to pull-down endogenous (Figure 4.3A), GFP-Exo70 and myc-Exo70
(Figure 4.3B) from HeLa cell lysates, respectively. Further investigations on
Exo70-WAVE2 interaction will be discussed in the Discussion part.
Examine whether Pak1 phosphorylation stimulates the interaction between
Exo70 and Arpc1 using the in vitro binding assay.
While the interaction between Exo70 and the Arp2/3 complex has been shown to be regulated by epidermal growth factor (EGF) signaling, the signaling pathway downstream of EGF that mediates the interaction still remains unclear. Since PAK1 kinase phosphorylates Arpc1 and is downstream of EGF signaling, it is likely that
Pak1 phosphorylation promotes Exo70-Arpc1 interaction in response to EGF stimuli.
To test whether Pak1 phosphorylation promotes Exo70-Arpc1 interaction, an
Arpc1 mutant, in which the site of Pak1 phosphorylation (threonine 21) is mutated to glutamic acid (T21E) was used in the experiment. Threonine 21 is the only site in
Arpc1 that is phosphorylated by Pak1, and the Arpc1T21E mutant has been shown to functionally mimic the phosphorylated form of Arpc1 in promoting cell motility
(Vadlamudi et al., 2004). In vitro binding assay was carried out using GST-tagged
181 Jianglan Liu THE ROLE OF THE EXOCYST IN EXOCYTOSIS AND CELL MIGRATION Chapter 4
182 Jianglan Liu THE ROLE OF THE EXOCYST IN EXOCYTOSIS AND CELL MIGRATION Chapter 4 Figure 4.4. Examine the in vitro interaction of GST-tagged rExo70 and
His(6)-tagged Arpc1 wt or Arpc1 (21E) mutant. Glutathione Sepharose conjugated with GST or GST-rExo70 was incubated with either His(6)-tagged Arpc1 wt or
Arpc1(T21E) mutant. The input and the bound Arpc1 proteins were analyzed by western blot using anti-His(6) monoclonal antibody.
183 Jianglan Liu THE ROLE OF THE EXOCYST IN EXOCYTOSIS AND CELL MIGRATION Chapter 4 rExo70 and His-tagged Arpc1 T21E or wild type Arpc1, which were all purified from
E.coli. GST-rExo70 was conjugated to the glutathione Sepharose and then incubated with increasing concentrations of His-tagged Arpc1 T21E or wild type Arpc1. The bound proteins were analyzed by Western blot using antibodies against His tag. As shown in Figure 4.4, Arpc1 (T21E) mutant bound slightly stronger to Exo70 than the wild type Arpc1. Further investigations on the regulation of Exo70-Arpc1 interaction by Pak1 in vivo are included in the Discussion part.
4.3. Discussion and future perspectives
Exo70 is an essential component of the exocyst complex, which is the key regulator of the tethering step in exocytosis mediating the initial contact of the secretory vesicles with the plasma membrane. Exo70 has also been implicated in cell migration through direct interaction with the Arp2/3 complex. However, the molecular basis of its function in cell migration is unknown. In this study, I found that in addition to functioning in exocytosis, Exo70 also functions in regulating
Arp2/3-mediated actin polymerization. Even though Exo70 is not able to promote actin nucleation on its own or activate Arp2/3 like an NPF, it enhances Arp2/3 activation in concert with WAVE2. TIRFM examination of actin filament dynamics suggests that Exo70 acts by promoting the generation of branched actin filaments in
Arp2/3-WAVE2-mediated actin polymerization. Overall, these results suggest that
184 Jianglan Liu THE ROLE OF THE EXOCYST IN EXOCYTOSIS AND CELL MIGRATION Chapter 4 Exo70 synergizes with WAVE2 to promote dendritic actin nucleation through a direct interaction with the Arp2/3 complex, which may reveal a novel mechanism for regulating actin dynamics.
4.3.1. Examine the molecular mechanisms for the positive effect of Exo70 in
Arp2/3-WAVE2-mediated actin polymerization
The stimulatory effect of Exo70 in Arp2/3-mediated actin polymerization suggests that Exo70 is a novel positive regulator of the Arp2/3 complex. Despite its direct interaction with Arp2/3, Exo70 does not activate Arp2/3 on its own, which is distinct from typical NPFs. Instead, Exo70 functions in synergy with WAVE2 to activate Arp2/3. Exo70 acts, at least partially, by promoting the generation of actin branches. However, how Exo70 facilitates WAVE2 in Arp2/3 activation still remains elusive.
Since Exo70 cannot promote actin assembly on its own, it is unlikely that
Exo70 activates actin polymerization by directly increasing the rate of actin nucleation or accelerating the barbed end elongation, or severing the newly formed actin filaments, which generates more barbed ends for elongation. Rather, since the positive effect of Exo70 on actin polymerization is mediated by Arp2/3 and WAVE2, it may function through the following mechanisms:
185 Jianglan Liu THE ROLE OF THE EXOCYST IN EXOCYTOSIS AND CELL MIGRATION Chapter 4 1. Exo70 may promote the interaction between Arp2/3 and WAVE2, which in turn
facilitates the activation of Arp2/3 by WAVE2.
To test whether Exo70 regulates the function of WAVE2 through direct interaction, I examined the interaction between Exo70 and WAVE2 as the first step.
By GST pulldown assay, I found that recombinant GST-WAVE2 could pull down
Exo70 from the cell lysates, which suggests that Exo70 interacts with WAVE2.
However, it is possible that this association is through other proteins. To further test whether there is a direct interaction between Exo70 and WAVE2, an in vitro binding assay using purified proteins will be carried out. If Exo70 directly interacts with
WAVE2, the domain of Exo70 that binds to WAVE2 will be further mapped.
Furthermore, the in vivo functional implication of Exo70-WAVE2 interaction will be examined. Since WAVE2 and Exo70 are both required for lamellopodia formation, the interaction between Exo70 and WAVE2 will be disrupted by a dominant-negative exo70 mutant that does not bind WAVE2 and the formation of lamellopodia will be examined.
To directly test whether Exo70 promotes the interaction between Arp2/3 and
WAVE2, I will examine the interaction in the presence or absence of Exo70.
GST-tagged WAVE2 purified from E.coli will be immobilized on Sepharose 4B and tested for binding with purified Arp2/3 complex in the presence or absence of Exo70.
186 Jianglan Liu THE ROLE OF THE EXOCYST IN EXOCYTOSIS AND CELL MIGRATION Chapter 4 2. Exo70 may enhance the association between Arp2/3 and F-actin
Exo70 may directly bind to F-actin and function as an additional connection between Arp2/3 and F-actin filaments to reinforce their interaction. I have tested this by measuring the affinity between Exo70 and F-actin. rExo70 was mixed with preformed F-actin and centrifuged at 80,000 rpm in a TLA 100.3 rotor for 20 min.
Only negligible amounts of Exo70 were found to co-precipitate with F-actin (data not shown).
Alternatively, Exo70 may facilitate the binding between Arp2/3 and F-actin without direct interacting with F-actin. Thus, I will directly test the effect of Exo70 on the binding of activated Arp2/3 to the F-actin filament. Purified Arp2/3 and
WAVE2 will be mixed with F-actin in the presence or absence of Exo70, followed by ultra-speed centrifugation to precipitate F-actin. The amount of Arp2/3 associated with F-actin in the pellet will be determined by Western blot.
3. Exo70 may directly bind to G-actin and facilitate the association of G-actin with the Arp2/3 complex
Exo70 may directly bind to G-actin and facilitate the association of G-actin with the Arp2/3 complex thus promote the formation of nucleation core. GST pull-down assay was carried out to test the interaction between Exo70 and G-actin. GST-rExo70 and GST alone were conjugated to Glutathione Sepharose and then incubated with 187 Jianglan Liu THE ROLE OF THE EXOCYST IN EXOCYTOSIS AND CELL MIGRATION Chapter 4 0.05µM G-actin, which is below the critical concentration for barbed filament ends.
Beads were washed and the bound proteins were analyzed by Western blot using anti-actin antibody. GST-rExo70 did not pull down any G-actin (data not shown).
However, it is possible that the conditions used in the experiment did not favor the binding of Exo70 to G-actin. Therefore, the binding assay will be repeated in the future, in which a positive control, such as the VCA domain of N-WASP, will be included.
4. Exo70 may function in stabilizing the existing actin branch structures
Whether Exo70 stabilizes actin branches can be tested by the following debranching assay, which was used before to characterize the role of Cortactin in stabilizing actin branches (Weaver et al, 2001). Actin will be polymerized in the presence of Arp2/3 and WAVE2 to generate branched actin filaments. Right after polymerization reaches plateau, Exo70 or the control buffer will be added to the actin mix. Aliquots will be taken after various periods of time and immediately incubated with fluorescently-labeled phalloidin to stabilize actin filaments and stop debranching.
Phalloidin decorated F-actin will be diluted and visualized under fluorescence microscope. The effect of Exo70 on actin branch stability can be determined by quantifying the rate of debranching in the presence or absence of Exo70.
188 Jianglan Liu THE ROLE OF THE EXOCYST IN EXOCYTOSIS AND CELL MIGRATION Chapter 4 Overall, these studies will be informative for clarifying the molecular basis of the stimulatory effect of Exo70 on Arp2/3-WAVE2-mediated actin polymerization, and may reveal novel mechanisms for Arp2/3 activation.
4.3.2. Determine the specificity of the effect of Exo70 on different NPFs In addition to functioning synergistically with WAVE2, Exo70 may also be involved in the activation of Arp2/3 in the presence of other NPFs. Therefore, it is necessary to determine whether the stimulatory effect of Exo70 is specific to WAVE2.
I have tested the effect of Exo70 in Arp2/3-mediated actin polymerization in the presence of the VCA domain of rat N-WASP or ∆EVH1 N-WASP, an N-WASP truncation mutant lacking the WH1/EVH1 domain that exhibits increased autoinhibition. Exo70 did not show a clear stimulatory effect in Arp2/3 activation with either of them (data not shown). In addition, rExo70 has no effect on N-WASP activation of Arp2/3 as well (unpublished data from Dr. Jeff Peterson). These results suggest that there is some selectivity in the effect of Exo70 on NPFs. Because
WAVE2 plays a critical role in lamellipodia formation and cell migration, whereas
N-WASP is largely involved in endocytosis, it strongly suggests that Exo70 is specifically involved in Arp2/3-mediated actin dynamics during cell migration.
The interaction between Exo70 and Arpc1 (Arc40 in yeast) is conserved in yeast (unpublished data from Guo lab). Furthermore, it was found that the yeast
189 Jianglan Liu THE ROLE OF THE EXOCYST IN EXOCYTOSIS AND CELL MIGRATION Chapter 4 Exo70 also promotes the nucleation of branched actin filaments mediated by Arp2/3 and Las 17, the sole WASP homologue in budding yeast (Li, 1997; Madania et al,
1999; Winter et al, 1999). Interestingly, the stimulatory effect of yeast Exo70 in
Arp2/3-mediated actin polymerization is more potent than that of the mammalian counterpart (data not shown). Additional regulatory factors may be lacking in actin reactions with mammalian Exo70.
4.3.3. Regulation and functional implications of the Exo70-Arpc1 interaction
1. Further examine whether the Exo70-Arp2/3 interaction is regulated by PAK1 phosphorylation on Arpc1.
The in vitro binding result suggests that PAK1 phosphorylation on Arpc1 may enhance its interaction with Exo70. I also tested the interaction between Exo70 and
Arpc1/Arpc1 (T21E) using in vivo binding assays. HeLa cells expressing myc-tagged
Arpc1/Arpc1(T21E) were lysed and incubated with GST-CA conjugated to
Glutathione Sepharose beads. The bound proteins were analyzed by Western blot using antibodies against the myc tag and Exo70. Endogenous Exo70 bound to
Arpc1(T21E) is at similar level compared with that bound to wt Arpc1 (data not shown). In addition, I tested the interaction between myc-Arpc1/Arpc1(T21E) and overexpressed GFP-Exo70. The binding affinity of GFP-Exo70 and ArpC1(T21E) is comparable to that of GFP-Exo70 and ArpC1 (data not shown). Moreover, the
190 Jianglan Liu THE ROLE OF THE EXOCYST IN EXOCYTOSIS AND CELL MIGRATION Chapter 4 recombinant GST-rExo70 purified from E.coli was used to pull-down myc-Arpc1/
Arpc1(T21E) from HeLa cells. And the bound myc-Arpc1/ Arpc1(T21E) was detected by Western blot. The interaction between GST-Exo70 and ArpC1(T21E) is at similar level compared with that between GST-Exo70 and Arpc1 (data not shown).
From these results, I did not see a clear enhancement of Exo70-Arpc1 interaction upon the phosphorylation of Arpc1.
To further examine the regulation of Exo70-Arpc1 interaction by Pak1 phosphorylation, Arpc1 (T21A) mutant will also be used. The Pak1 phosphorylation site was mutated to alanine in this mutant, which completely abolishes Pak1 phosphorylation (Vadlamudi et al., 2004). In vitro and in vivo binding assays will be carried out to test the interaction between Exo70 and Arpc1/Arpc1(T21A).
It is also possible that Pak1 indirectly regulates Exo70-Arpc1 interaction.
Therefore, the NIH-3T3 variant S2-6 cell lines inducibly expressing various forms of
Pak1 will be used here. The exocyst-Arp2/3 interaction will be examined in the cells expressing active Pak1(E423) by GST-CA pull-down assay before and after the induction. As a control, the kinase-dead form of Pak1 (Pak1R299) will also be used in the experiments. On the other hand, the Pak auto-inhibitory peptide (“PID”, amino acid 83-149), which has been shown to effectively inhibit the activity of Paks in cells
(Beeser et al., 2005) can be used in the experiments as well. As a control, an
191 Jianglan Liu THE ROLE OF THE EXOCYST IN EXOCYTOSIS AND CELL MIGRATION Chapter 4 inactivated form of PID (the Phe107 mutant) (Zhao et al., 1998; Beeser et al., 2005) will be included.
2. Determine whether the positive effect of Exo70 in actin assembly is curvature-dependent
Dynamic rearrangements of membrane shape occur frequently during the formation of cell protrusions and cell migration. It has been shown that rExo70 can induce tubulated membranes invaginations when it binds to PI(4,5)P2-rich liposomes in vitro (unpublished data from our lab). However, the functional implication of the role of Exo70 in generating membrane curvature is still unknown. Recent work from
Lappalainen Pekka’s lab discovered that “I-BAR” proteins, which are known to be involved in the Arp2/3-mediated actin filament assembly, can bend membranes and further couple actin polymerization and membrane deformation during cell migration
(Mattila et al., 2007; Saarikangas et al., 2009). Thus, it would be intriguing to examine whether the effect of Exo70 in Arp2/3-mediated actin assembly is curvature dependent. In pyrene actin assay and TIRFM, rExo70 and PI(4,5)P2-containing liposomes with different sizes will be simultaneously added to the actin polymerization reaction mixture containing Arp2/3, WAVE2 and actin. The obtained stimulatory effect will be compared with the samples with either rExo70 or the liposomes alone. The effects between different sizes of liposomes will also be
192 Jianglan Liu THE ROLE OF THE EXOCYST IN EXOCYTOSIS AND CELL MIGRATION Chapter 4 compared. Liposomes with different composition, such as PC alone, or PC/PS will also be included as controls. If membrane curvature can further enhance the stimulatory effect of Exo70 in Arp2/3-WAVE2-mediated actin polymerization, I will further examine its molecular basis. One of the most plausible mechanisms for curvature-dependent actin polymerization by rExo70 is the increased interaction between Exo70 and the Arp2/3 complex. Thus, Exo70-Arp2/3 interaction will also be tested in the presence or absence of PI(4,5)P2-containing liposomes.
3. Examine whether the interaction with GTPases regulates the function of Exo70 in
Arp2/3-mediated actin assembly
A number of small GTP-binding proteins interact with components of the exocyst and regulate the assembly, localization, and function of this complex. It has been shown that TC10 directly interacts with Exo70 (Inoue et al., 2003). However, the cellular function of this interaction is still unclear. It is possible that TC10 regulates the function of Exo70 in actin assembly. In pyrene actin assay and TIRFM,
GTP-bound TC10 will be added in the reaction mixture containing Arp2/3, WAVE2,
Exo70 and actin. GDP-bound or wild type TC10 will be included as controls. I will also examine whether TC10 regulates the interaction between Exo70 and the Arp2/3 complex.
193 Jianglan Liu THE ROLE OF THE EXOCYST IN EXOCYTOSIS AND CELL MIGRATION Chapter 4 Overall, these studies will largely help to elucidate the molecular mechanisms of the function of Exo70 in actin assembly.
4.4. Materials and Methods
Purification of mammalian Arp2/3 complex
Mammalian Arp2/3 complex was purified from cell lysates extracted from mammalian cells by affinity chromatography using recombinant GST-CA immobilized on glutathione Sepharose 4B beads. Cell lysates with 20-25mg total proteins were collected and spun successively at 16,000 g for 10 min and 80, 000 rpm in a Beckman TLA-100.3 rotor (Beckman Coulter Inc., Fullerton, CA) for 15 min.
The resulting high speed supernatant (HSS) was mixed with GST-CA beads (-10 mg proteins) for 1 hr at 4°C. The beads were transferred to a column and washed successively with 20 ml Buffer B (25 mM KC1, 1 mM MgCl2, 0.5 mM EGTA, 0.1 mM ATP, and 1 mM DTT) and 20 ml Buffer B plus 200 mM KC1. Additional washes were performed, if necessary, until no proteins were detected in the flow through. The bound Arp2/3 proteins were eluted from the beads with 5 ml Buffer B plus 200 mM MgCl2. The 1.5 ml fractions with peak protein concentration were pooled and dialyzed against Buffer B. Finally, the Arp2/3 proteins were concentrated to 0.5 ml, supplied with 0.2 M sucrose, snap frozen in liquid nitrogen and stored at
194 Jianglan Liu THE ROLE OF THE EXOCYST IN EXOCYTOSIS AND CELL MIGRATION Chapter 4 -80°C.
Actin polymerization assay
Kinetics of actin filament polymerization was monitored by pyrene fluorescence with excitation of 370 nm and emission at 410 nm. Proteins to be tested for each reaction were mixed and diluted in 90µl polymerization buffer (60 mM KC1,
2.5 mM NaCl, 0.6 mM MgCl2, 5 mM Tris-HCl, pH 7.5, 2.5 mM HEPES, pH 7.1, 0.5 mM EGTA, 30 uM CaCl2, 0.2 mM ATP, and 0.3 mM NaN3), then pre-incubated for
30min at 4°C. 11.5µl G-actin (20.9 µM, 20% pyrenyl-labeled) in G-actin buffer were converted to Mg -G-actin by mixing with 18.5µl Mg2+ converting buffer (1.6 mM
Tris-HCl, pH8, 0.18 mM EGTA, and 0.51 mM MgCl2) for 5 min. The converted actin mix was then diluted into 90µl protein mix, and immediately transferred into a cuvette and monitored for the increase in fluorescence at 410 nm in a fluorescence spectrophotometer. The pyrenyllabeled and unlabeled rabbit skeletal actin used in the assay was from Cytoskeleton (Cytoskeleton Inc., Denver, CO). Three independent measurements were carried out for each treatment. Polymerization curves and rates were obtained using Excel (Microsoft, Redmond, WA). The polymerization rate was represented as the maximal slope of the elongation phase of each curve.
195 Jianglan Liu THE ROLE OF THE EXOCYST IN EXOCYTOSIS AND CELL MIGRATION Chapter 4 Visualization of actin polymerization in real time by TIRFM
Experiments were carried out in 10-20µl flow chambers assembled by mounting a precleaned coverslip to a standard glass slide using two strips of double-sided adhesive tape as spacers. Before use, the flow chambers were coated with NEM-myosin by flowing in 50 nM NEM-myosin in high salt buffer (500 mM
KC1) and incubating for 1.5 min. The NEM-myosin functions to capture the actin filaments at the surface of the chamber for TIRF illumination. The flow chambers were then treated with 1% BSA for 4 min to prevent the nonspecific binding to the surface. Arp2/3 and proteins to be tested were mixed in the actin polymerization buffer and incubated at 4°C for 30 mins. 0.8µM G-actin (8% Rhodamine labeled) was converted to Mg-G-actin, then mixed with the pre-incubated samples which contain
(1)6 nM Arp2/3 + 10 nM WAVE2, or (2) 6 nM Arp2/3 + 10 nM WAVE2 + 1 uM
Exo70, and immediately transferred into the flow chamber for TIRF microscopic observation. Total internal reflection excitation was generated on a Nikon TE2000 inverted microscope by projecting a laser beam at 532 nm (Crystal Laser) on the back focal plane of a Nikon 100x, 1.49 NA objective lens. Fluorescence emission was collected by the objective and imaged on an EMCCD camera (Cascade 11:512,
Photometries). Images were recorded for 10 min at 1.14 sec per frame after the polymerization started. The total length of F-actin filaments was measured using
196 Jianglan Liu THE ROLE OF THE EXOCYST IN EXOCYTOSIS AND CELL MIGRATION Chapter 4 ImageJ 1.73v software (http://rsb.info.nih.gov/ij/). White filaments in the dark field were inverted and thresholded into black filaments in the white field. All the particles were then skeletonized and the total length of filaments was obtained.
GST pulldown assay HeLa cells alone or the cells expressing GFP-rExo70 or myc-rExo70 were scraped from 10cm culture plates and lysed in the lysis buffer (20 mM Tris-HCl, pH
7.5, 25 mM KCl, 1 mM MgCl2, 0.5 mM EGTA, 1 mM DTT, 0.5% Triton X-100, and protease inhibitors). A high-speed centrifugation (12,000 rpm, 15 min) was carried out, and 1 ml of precleared cell lysates (1.5 mg total protein) was mixed with 20 µl
(50% vol/vol) of glutathione Sepharose conjugated with 10 µg GST-WAVE2. After incubation at 4°C overnight, the beads were washed five times with the lysis buffer, and the bound proteins were analyzed by Western blot using antibodies against Exo70,
GFP or myc tag. GST alone was used as a negative control in the experiment.
In vitro protein binding assays His(6)-tagged Arpc1 wt and Arpc1(T21E) mutant were expressed as
His(6)-tagged fusion proteins. rExo70 was expressed as GST-tagged fusion protein
(GST-rExo70). The recombinant fusion proteins were purified from E. coli. and GST or GST-rExo70 (3 µg) was immobilized on glutathione Sepharose 4B beads. The
GST or GST-rExo70 beads were incubated with His(6)-tagged Arpc1 wt or
197 Jianglan Liu THE ROLE OF THE EXOCYST IN EXOCYTOSIS AND CELL MIGRATION Chapter 4 Arpc1(T21E) mutant with various amounts (0 - 7.2 µg) for 2 hrs at room temperature in the binding buffer containing 20 mM Tris-HCl, pH 7.4, 100 mM NaCl, 1 mM
EGTA, 1 mM DTT, 10 mM MgCl2 and 1% Triton X-100. The beads were then washed five times with the binding buffer and the HIS-Arpc1 fusion proteins bound to the beads were detected by Western blot with anti-HIS monoclonal antibody.
198 Jianglan Liu THE ROLE OF THE EXOCYST IN EXOCYTOSIS AND CELL MIGRATION Chapter 5 Chapter 5. Discussion and Future Perspectives
5.1. Impact of my research in the field
My studies have revealed crucial roles of the exocyst in exocytosis and tumor cell migration. Firstly, I have elucidated a molecular mechanism by which the exocyst directly interacts with the PM that is critical for vesicle tethering and exocytosis. I identified a direct interaction between the exocyst component Exo70 and PI(4,5)P2; and demonstrated that this interaction is essential for the recruitment of the exocyst to the PM. Furthermore, disruption of this interaction impaired later stages of exocytosis of post-Golgi secretory vesicles at the PM. Secondly, I have found that the exocyst plays a pivotal role in the formation of invadopodia, a degradative membrane protrusive structures formed by tumor cells. Blocking the exocyst function inhibits invadopodial formation. RNAi knockdown of the exocyst components abolished the secretion of MMPs, whereas the overexpression of Exo70 promoted MMP secretion. In addition, the exocyst-Arp2/3 interaction is important for actin assembly during invadopodia formation. Finally, I have found that Exo70 promotes the generation of branched actin filaments in Arp2/3-WAVE2-mediated actin polymerization, which suggests that Exo70 is a novel positive regulator of the exocytosis and actin reorganization for effective cell migration and cell invasion.
199 Jianglan Liu THE ROLE OF THE EXOCYST IN EXOCYTOSIS AND CELL MIGRATION Chapter 5 5.2. The molecular mechanism of exocyst targeting to the plasma membrane
The exocyst components are recruited to designated regions of the plasma membrane where active exocytosis and membrane expansion occur. However, in most cell types, PI(4,5)P2 distributes throughout the whole plasma membrane.
Therefore, there must be additional factors that contribute to the polarized localization of the exocyst. The exocyst is a downstream effector of a variety of small GTPases
(see Chapter 1). In mammalian cells, growth factor signaling involving small
GTPases may mediate translocation of the exocyst from intracellular compartments to, or activation of the exocyst at, the PM (Sugihara et al., 2002; Moskalenko et al., 2002,
2003; Inoue et al., 2003; Takaya et al., 2004). Phopshoinositides and small GTPases may coordinate to regulate the recruitment of the exocyst to specific sites on the plasma membrane. The observed polarization of the exocyst may in fact be a result of both physical recruitment and kinetic reinforcement. Future work will be focused on the identification and characterization of proteins that spatially regulate exocyst components using different eukaryotic systems.
Similar to its mammalian counterpart, yeast Exo70 also interacts with PI(4,5)P2 through conserved regions (He et al., 2007b). Other tethering factors, such as the
HOPS complex that functions in yeast homotypic vacuolar fusion, have also been found to interact with phosphoinositides (Stroupe et al., 2006). It may be a
200 Jianglan Liu THE ROLE OF THE EXOCYST IN EXOCYTOSIS AND CELL MIGRATION Chapter 5 generalized mechanism that phosphoinositides function to recruit the tethering proteins to specific membrane compartments for vesicle tethering.
5.3. A crosstalk between actin cytoskeleton and exocytosis machineries during cell migration The relationship between actin dynamics and exocytosis has been widely studied in different systems. In several neuron cells such as hippocampal neurons, the actin cytoskeleton has inhibitory roles in neurotransmitter release (Morales et al.,
2000; Ohnishi et al., 2001). In their study, they showed that latrunculin A but not cytochalasin D increased neurotransmission (Morales et al., 2000). However, cell migration requires the coordination of actin reorganization and membrane remodeling.
The leading edges of the migrating cells are generated by a branched network of filamentous actin that pushes the membrane. To accommodate actin “pushing”, exocytosis is involved in the recycling of membranes and proteins to the leading edges. How are actin remodeling and secretion linked during cell migration? In my study, I found that the exocyst directly interacts with the Arp2/3 complex and promotes actin polymerization in addition to its fundamental role in exocytosis. It thus functionally couples actin reorganization and membrane addition at the leading edges for effective directional cell migration.
201 Jianglan Liu THE ROLE OF THE EXOCYST IN EXOCYTOSIS AND CELL MIGRATION Chapter 5 5.4. The functions of the exocyst in many cellular processes
The exocyst plays important roles in a variety of cellular processes including cell migration, epithelial polarity establishment, cytokinesis and ciliogenesis. Why is the exocyst involved in so many cellular processes? Exocytosis is one of the most fundamental cellular events in the cell. It is involved in a number of processes that requires secretion. This may explain the universal role of the exocyst in the cell.
5.5. Future perspectives
Recent decade has witnessed exciting progresses toward our understanding of the exocyst. However, a number of important questions remain unsolved: What are the kinetics of exocyst assembly and disassembly? How are the exocyst components associated with secretory vesicles? While it is thought that the exocyst serves as a vesicle tether, can the tethering step be observed and characterized by microscopy? Is the exocyst also involved in the early steps of vesicular trafficking such as vesicle budding and cargo sorting at the donor compartments? Recent studies demonstrate the roles of the exocyst in a number of developmental and physiological processes.
Future studies are needed to elucidate how the cellular functions of the exocyst are manifested at the multicellular organismal level.
202 Jianglan Liu THE ROLE OF THE EXOCYST IN EXOCYTOSIS AND CELL MIGRATION References References
Aalto, M.K., Ronne, H., and Keranen, S. (1993). Yeast syntaxins Sso1p and Sso2p
belong to a family of related membrane proteins that function in vesicular
transport. Embo J 12, 4095-4104.
Abram, C.L., Seals, D.F., Pass, I., Salinsky, D., Maurer, L., Roth, T.M., and
Courtneidge, S.A. (2003). The adaptor protein fish associates with members of
the ADAMs family and localizes to podosomes of Src-transformed cells. J Biol
Chem 278, 16844-16851.
Adamo, J.E., Moskow, J.J., Gladfelter, A.S., Viterbo, D., Lew, D.J., and Brennwald,
P.J. (2001). Yeast Cdc42 functions at a late step in exocytosis, specifically during
polarized growth of the emerging bud. J Cell Biol 155, 581-592.
Adamo, J.E., Rossi, G., and Brennwald, P. (1999). The Rho GTPase Rho3 has a
direct role in exocytosis that is distinct from its role in actin polarity. Mol Biol
Cell 10, 4121-4133.
Amann, K.J., and Pollard, T.D. (2001). Direct real-time observation of actin filament
branching mediated by Arp2/3 complex using total internal reflection
fluorescence microscopy. Proc Natl Acad Sci U S A 98, 15009-15013.
Andrews, H.K., Zhang, Y.Q., Trotta, N., and Broadie, K. (2002). Drosophila sec10 is
203 Jianglan Liu THE ROLE OF THE EXOCYST IN EXOCYTOSIS AND CELL MIGRATION References required for hormone secretion but not general exocytosis or neurotransmission.
Traffic 3, 906-921.
Ang, A.L., Taguchi, T., Francis, S., Folsch, H., Murrells, L.J., Pypaert, M., Warren,
G., and Mellman, I. (2004). Recycling endosomes can serve as intermediates
during transport from the Golgi to the plasma membrane of MDCK cells. J Cell
Biol 167, 531-543.
Artym, V.V., Zhang, Y., Seillier-Moiseiwitsch, F., Yamada, K.M., and Mueller, S.C.
(2006). Dynamic interactions of cortactin and membrane type 1 matrix
metalloproteinase at invadopodia: defining the stages of invadopodia formation
and function. Cancer Res 66, 3034-3043.
Ayala, I., Baldassarre, M., Caldieri, G., and Buccione, R. (2006). Invadopodia: a
guided tour. Eur J Cell Biol 85, 159-164.
Ayala, I., Baldassarre, M., Giacchetti, G., Caldieri, G., Tete, S., Luini, A., and
Buccione, R. (2008). Multiple regulatory inputs converge on cortactin to control
invadopodia biogenesis and extracellular matrix degradation. J Cell Sci 121,
369-378.
Ayscough, K.R., Stryker, J., Pokala, N., Sanders, M., Crews, P., and Drubin, D.G.
(1997). High rates of actin filament turnover in budding yeast and roles for actin
in establishment and maintenance of cell polarity revealed using the actin
204 Jianglan Liu THE ROLE OF THE EXOCYST IN EXOCYTOSIS AND CELL MIGRATION References inhibitor latrunculin-A. J Cell Biol 137, 399-416.
Baek, K., Knodler, A., Lee, S.H., Zhang, X., Orlando, K., Zhang, J., Foskett, T.J.,
Guo, W., and Dominguez, R. (2010). Structure-function study of the N-terminal
domain of exocyst subunit Sec3. J Biol Chem 285, 10424-10433.
Bai, J., Tucker, W.C., and Chapman, E.R. (2004). PIP2 increases the speed of
response of synaptotagmin and steers its membrane-penetration activity toward
the plasma membrane. Nat Struct Mol Biol 11, 36-44.
Baldassarre, M., Ayala, I., Beznoussenko, G., Giacchetti, G., Machesky, L.M., Luini,
A., and Buccione, R. (2006). Actin dynamics at sites of extracellular matrix
degradation. Eur J Cell Biol 85, 1217-1231.
Baldassarre, M., Pompeo, A., Beznoussenko, G., Castaldi, C., Cortellino, S.,
McNiven, M.A., Luini, A., and Buccione, R. (2003). Dynamin participates in
focal extracellular matrix degradation by invasive cells. Mol Biol Cell 14,
1074-1084.
Balla, T. (2005). Inositol-lipid binding motifs: signal integrators through protein-lipid
and protein-protein interactions. J Cell Sci 118, 2093-2104.
Bao, Y., Lopez, J.A., James, D.E., and Hunziker, W. (2008). Snapin interacts with the
Exo70 subunit of the exocyst and modulates GLUT4 trafficking. J Biol Chem 283,
324-331.
205 Jianglan Liu THE ROLE OF THE EXOCYST IN EXOCYTOSIS AND CELL MIGRATION References Barral, Y., Mermall, V., Mooseker, M.S., and Snyder, M. (2000).
Compartmentalization of the cell cortex by septins is required for maintenance of
cell polarity in yeast. Mol Cell 5, 841-851.
Beeser, A., Jaffer, Z.M., Hofmann, C., and Chernoff, J. (2005). Role of group A
p21-activated kinases in activation of extracellular-regulated kinase by growth
factors. J Biol Chem 280, 36609-36615.
Behnia, R., and Munro, S. (2005). Organelle identity and the signposts for membrane
traffic. Nature 438, 597-604.
Berdeaux, R.L., Diaz, B., Kim, L., and Martin, G.S. (2004). Active Rho is localized
to podosomes induced by oncogenic Src and is required for their assembly and
function. J Cell Biol 166, 317-323.
Beronja, S., Laprise, P., Papoulas, O., Pellikka, M., Sisson, J., and Tepass, U. (2005).
Essential function of Drosophila Sec6 in apical exocytosis of epithelial
photoreceptor cells. J Cell Biol 169, 635-646.
Berridge, M.J., and Irvine, R.F. (1989). Inositol phosphates and cell signalling.
Nature 341, 197-205.
Bielinski, D.F., Pyun, H.Y., Linko-Stentz, K., Macara, I.G., and Fine, R.E. (1993).
Ral and Rab3a are major GTP-binding proteins of axonal rapid transport and
synaptic vesicles and do not redistribute following depolarization stimulated
206 Jianglan Liu THE ROLE OF THE EXOCYST IN EXOCYTOSIS AND CELL MIGRATION References synaptosomal exocytosis. Biochim Biophys Acta 1151, 246-256.
Blankenship, J.T., Fuller, M.T., and Zallen, J.A. (2007). The Drosophila homolog of
the Exo84 exocyst subunit promotes apical epithelial identity. J Cell Sci 120,
3099-3110.
Bonangelino, C.J., Nau, J.J., Duex, J.E., Brinkman, M., Wurmser, A.E., Gary, J.D.,
Emr, S.D., and Weisman, L.S. (2002). Osmotic stress-induced increase of
phosphatidylinositol 3,5-bisphosphate requires Vac14p, an activator of the lipid
kinase Fab1p. J Cell Biol 156, 1015-1028.
Bourguignon, L.Y., Gunja-Smith, Z., Iida, N., Zhu, H.B., Young, L.J., Muller, W.J.,
and Cardiff, R.D. (1998). CD44v(3,8-10) is involved in cytoskeleton-mediated
tumor cell migration and matrix metalloproteinase (MMP-9) association in
metastatic breast cancer cells. J Cell Physiol 176, 206-215.
Bowden, E.T., Barth, M., Thomas, D., Glazer, R.I., and Mueller, S.C. (1999). An
invasion-related complex of cortactin, paxillin and PKCmu associates with
invadopodia at sites of extracellular matrix degradation. Oncogene 18,
4440-4449.
Bowden, E.T., Onikoyi, E., Slack, R., Myoui, A., Yoneda, T., Yamada, K.M., and
Mueller, S.C. (2006). Co-localization of cortactin and phosphotyrosine identifies
active invadopodia in human breast cancer cells. Exp Cell Res 312, 1240-1253.
207 Jianglan Liu THE ROLE OF THE EXOCYST IN EXOCYTOSIS AND CELL MIGRATION References Bowser, R., Muller, H., Govindan, B., and Novick, P. (1992). Sec8p and Sec15p are
components of a plasma membrane-associated 19.5S particle that may function
downstream of Sec4p to control exocytosis. J Cell Biol 118, 1041-1056.
Bowser, R., and Novick, P. (1991). Sec15 protein, an essential component of the
exocytotic apparatus, is associated with the plasma membrane and with a soluble
19.5S particle. J Cell Biol 112, 1117-1131.
Boyd, C., Hughes, T., Pypaert, M., and Novick, P. (2004). Vesicles carry most
exocyst subunits to exocytic sites marked by the remaining two subunits, Sec3p
and Exo70p. J Cell Biol 167, 889-901.
Boyd, D. (1996). Invasion and metastasis. Cancer Metastasis Rev 15, 77-89.
Brennwald, P., Kearns, B., Champion, K., Keranen, S., Bankaitis, V., and Novick, P.
(1994). Sec9 is a SNAP-25-like component of a yeast SNARE complex that may
be the effector of Sec4 function in exocytosis. Cell 79, 245-258.
Brown, D.L., Heimann, K., Lock, J., Kjer-Nielsen, L., van Vliet, C., Stow, J.L., and
Gleeson, P.A. (2001). The GRIP domain is a specific targeting sequence for a
population of trans-Golgi network derived tubulo-vesicular carriers. Traffic 2,
336-344.
Brymora, A., Valova, V.A., Larsen, M.R., Roufogalis, B.D., and Robinson, P.J.
(2001). The brain exocyst complex interacts with RalA in a GTP-dependent
208 Jianglan Liu THE ROLE OF THE EXOCYST IN EXOCYTOSIS AND CELL MIGRATION References manner: identification of a novel mammalian Sec3 gene and a second Sec15 gene.
J Biol Chem 276, 29792-29797.
Buccione, R., Orth, J.D., and McNiven, M.A. (2004). Foot and mouth: podosomes,
invadopodia and circular dorsal ruffles. Nat Rev Mol Cell Biol 5, 647-657.
Bustelo, X.R., Sauzeau, V., and Berenjeno, I.M. (2007). GTP-binding proteins of the
Rho/Rac family: regulation, effectors and functions in vivo. Bioessays 29,
356-370.
Carr, C.M., Grote, E., Munson, M., Hughson, F.M., and Novick, P.J. (1999). Sec1p
binds to SNARE complexes and concentrates at sites of secretion. J Cell Biol 146,
333-344.
Cascone, I., Selimoglu, R., Ozdemir, C., Del Nery, E., Yeaman, C., White, M., and
Camonis, J. (2008). Distinct roles of RalA and RalB in the progression of
cytokinesis are supported by distinct RalGEFs. Embo J 27, 2375-2387.
Cavanaugh, L.F., Chen, X., Richardson, B.C., Ungar, D., Pelczer, I., Rizo, J., and
Hughson, F.M. (2007). Structural analysis of conserved oligomeric Golgi
complex subunit 2. J Biol Chem 282, 23418-23426.
Chen, W.T. (1996). Proteases associated with invadopodia, and their role in
degradation of extracellular matrix. Enzyme Protein 49, 59-71.
Chen, W.T., Chen, J.M., Parsons, S.J., and Parsons, J.T. (1985). Local degradation of
209 Jianglan Liu THE ROLE OF THE EXOCYST IN EXOCYTOSIS AND CELL MIGRATION References fibronectin at sites of expression of the transforming gene product pp60src.
Nature 316, 156-158.
Chen, W.T., and Wang, J.Y. (1999). Specialized surface protrusions of invasive cells,
invadopodia and lamellipodia, have differential MT1-MMP, MMP-2, and TIMP-2
localization. Ann N Y Acad Sci 878, 361-371.
Chen, X.W., Inoue, M., Hsu, S.C., and Saltiel, A.R. (2006). RalA-exocyst-dependent
recycling endosome trafficking is required for the completion of cytokinesis. J
Biol Chem 281, 38609-38616.
Chen, X.W., Leto, D., Chiang, S.H., Wang, Q., and Saltiel, A.R. (2007). Activation of
RalA is required for insulin-stimulated Glut4 trafficking to the plasma membrane
via the exocyst and the motor protein Myo1c. Dev Cell 13, 391-404.
Cheng, K.W., Lahad, J.P., Kuo, W.L., Lapuk, A., Yamada, K., Auersperg, N., Liu, J.,
Smith-McCune, K., Lu, K.H., Fishman, D., Gray, J.W., and Mills, G.B. (2004).
The RAB25 small GTPase determines aggressiveness of ovarian and breast
cancers. Nat Med 10, 1251-1256.
Chi, K.H., Chang, Y.C., Guo, W.Y., Leung, M.J., Shiau, C.Y., Chen, S.Y., Wang,
L.W., Lai, Y.L., Hsu, M.M., Lian, S.L., Chang, C.H., Liu, T.W., Chin, Y.H., Yen,
S.H., Perng, C.H., and Chen, K.Y. (2002). A phase III study of adjuvant
chemotherapy in advanced nasopharyngeal carcinoma patients. Int J Radiat Oncol
210 Jianglan Liu THE ROLE OF THE EXOCYST IN EXOCYTOSIS AND CELL MIGRATION References Biol Phys 52, 1238-1244.
Chuang, Y.Y., Tran, N.L., Rusk, N., Nakada, M., Berens, M.E., and Symons, M.
(2004). Role of synaptojanin 2 in glioma cell migration and invasion. Cancer Res
64, 8271-8275.
Clark, E.S., Whigham, A.S., Yarbrough, W.G., and Weaver, A.M. (2007). Cortactin
is an essential regulator of matrix metalloproteinase secretion and extracellular
matrix degradation in invadopodia. Cancer Res 67, 4227-4235.
De Matteis, M.A., and Godi, A. (2004). PI-loting membrane traffic. Nat Cell Biol 6,
487-492.
Deryugina, E.I., and Quigley, J.P. (2006). Matrix metalloproteinases and tumor
metastasis. Cancer Metastasis Rev 25, 9-34.
Deryugina, E.I., Ratnikov, B., Monosov, E., Postnova, T.I., DiScipio, R., Smith, J.W.,
and Strongin, A.Y. (2001). MT1-MMP initiates activation of pro-MMP-2 and
integrin alphavbeta3 promotes maturation of MMP-2 in breast carcinoma cells.
Exp Cell Res 263, 209-223.
Dharmawardhane, S., Sanders, L.C., Martin, S.S., Daniels, R.H., and Bokoch, G.M.
(1997). Localization of p21-activated kinase 1 (PAK1) to pinocytic vesicles and
cortical actin structures in stimulated cells. J Cell Biol 138, 1265-1278.
Di Paolo, G., and De Camilli, P. (2006). Phosphoinositides in cell regulation and
211 Jianglan Liu THE ROLE OF THE EXOCYST IN EXOCYTOSIS AND CELL MIGRATION References membrane dynamics. Nature 443, 651-657.
Dobbelaere, J., and Barral, Y. (2004). Spatial coordination of cytokinetic events by
compartmentalization of the cell cortex. Science 305, 393-396.
Dong, G., Hutagalung, A.H., Fu, C., Novick, P., and Reinisch, K.M. (2005). The
structures of exocyst subunit Exo70p and the Exo84p C-terminal domains reveal a
common motif. Nat Struct Mol Biol 12, 1094-1100.
Drees, B.L., Sundin, B., Brazeau, E., Caviston, J.P., Chen, G.C., Guo, W., Kozminski,
K.G., Lau, M.W., Moskow, J.J., Tong, A., Schenkman, L.R., McKenzie, A., 3rd,
Brennwald, P., Longtine, M., Bi, E., Chan, C., Novick, P., Boone, C., Pringle,
J.R., Davis, T.N., Fields, S., and Drubin, D.G. (2001). A protein interaction map
for cell polarity development. J Cell Biol 154, 549-571.
D'Souza-Schorey, C., and Chavrier, P. (2006). ARF proteins: roles in membrane
traffic and beyond. Nat Rev Mol Cell Biol 7, 347-358.
Dupraz, S., Grassi, D., Bernis, M.E., Sosa, L., Bisbal, M., Gastaldi, L., Jausoro, I.,
Caceres, A., Pfenninger, K.H., and Quiroga, S. (2009). The TC10-Exo70 complex
is essential for membrane expansion and axonal specification in developing
neurons. J Neurosci 29, 13292-13301.
Eblen, S.T., Slack, J.K., Weber, M.J., and Catling, A.D. (2002). Rac-PAK signaling
stimulates extracellular signal-regulated kinase (ERK) activation by regulating
212 Jianglan Liu THE ROLE OF THE EXOCYST IN EXOCYTOSIS AND CELL MIGRATION References formation of MEK1-ERK complexes. Mol Cell Biol 22, 6023-6033.
Ewart, M.A., Clarke, M., Kane, S., Chamberlain, L.H., and Gould, G.W. (2005).
Evidence for a role of the exocyst in insulin-stimulated Glut4 trafficking in
3T3-L1 adipocytes. J Biol Chem 280, 3812-3816.
Feig, L.A. (2003). Ral-GTPases: approaching their 15 minutes of fame. Trends Cell
Biol 13, 419-425.
Fielding, A.B., Schonteich, E., Matheson, J., Wilson, G., Yu, X., Hickson, G.R.,
Srivastava, S., Baldwin, S.A., Prekeris, R., and Gould, G.W. (2005). Rab11-FIP3
and FIP4 interact with Arf6 and the exocyst to control membrane traffic in
cytokinesis. Embo J 24, 3389-3399.
Finger, F.P., Hughes, T.E., and Novick, P. (1998). Sec3p is a spatial landmark for
polarized secretion in budding yeast. Cell 92, 559-571.
Folsch, H., Pypaert, M., Maday, S., Pelletier, L., and Mellman, I. (2003). The AP-1A
and AP-1B clathrin adaptor complexes define biochemically and functionally
distinct membrane domains. J Cell Biol 163, 351-362.
France, Y.E., Boyd, C., Coleman, J., and Novick, P.J. (2006). The
polarity-establishment component Bem1p interacts with the exocyst complex
through the Sec15p subunit. J Cell Sci 119, 876-888.
Friedrich, G.A., Hildebrand, J.D., and Soriano, P. (1997). The secretory protein Sec8
213 Jianglan Liu THE ROLE OF THE EXOCYST IN EXOCYTOSIS AND CELL MIGRATION References is required for paraxial mesoderm formation in the mouse. Dev Biol 192,
364-374.
Fukai, S., Matern, H.T., Jagath, J.R., Scheller, R.H., and Brunger, A.T. (2003).
Structural basis of the interaction between RalA and Sec5, a subunit of the sec6/8
complex. Embo J 22, 3267-3278.
Furmaniak-Kazmierczak, E., Crawley, S.W., Carter, R.L., Maurice, D.H., and Cote,
G.P. (2007). Formation of extracellular matrix-digesting invadopodia by primary
aortic smooth muscle cells. Circ Res 100, 1328-1336.
Gaidarov, I., and Keen, J.H. (1999). Phosphoinositide-AP-2 interactions required for
targeting to plasma membrane clathrin-coated pits. J Cell Biol 146, 755-764.
Gasman, S., Chasserot-Golaz, S., Malacombe, M., Way, M., and Bader, M.F. (2004).
Regulated exocytosis in neuroendocrine cells: a role for subplasmalemmal
Cdc42/N-WASP-induced actin filaments. Mol Biol Cell 15, 520-531.
Gassama-Diagne, A., Yu, W., ter Beest, M., Martin-Belmonte, F., Kierbel, A., Engel,
J., and Mostov, K. (2006). Phosphatidylinositol-3,4,5-trisphosphate regulates the
formation of the basolateral plasma membrane in epithelial cells. Nat Cell Biol 8,
963-970.
Gerges, N.Z., Backos, D.S., Rupasinghe, C.N., Spaller, M.R., and Esteban, J.A.
(2006). Dual role of the exocyst in AMPA receptor targeting and insertion into
214 Jianglan Liu THE ROLE OF THE EXOCYST IN EXOCYTOSIS AND CELL MIGRATION References the postsynaptic membrane. Embo J 25, 1623-1634.
Giansanti, M.G., Farkas, R.M., Bonaccorsi, S., Lindsley, D.L., Wakimoto, B.T.,
Fuller, M.T., and Gatti, M. (2004). Genetic dissection of meiotic cytokinesis in
Drosophila males. Mol Biol Cell 15, 2509-2522.
Gillingham, A.K., and Munro, S. (2007). The small G proteins of the Arf family and
their regulators. Annu Rev Cell Dev Biol 23, 579-611.
Gimona, M., and Buccione, R. (2006). Adhesions that mediate invasion. Int J
Biochem Cell Biol 38, 1875-1892.
Goley, E.D., and Welch, M.D. (2006). The ARP2/3 complex: an actin nucleator
comes of age. Nat Rev Mol Cell Biol 7, 713-726.
Goud, B., Salminen, A., Walworth, N.C., and Novick, P.J. (1988). A GTP-binding
protein required for secretion rapidly associates with secretory vesicles and the
plasma membrane in yeast. Cell 53, 753-768.
Gournier, H., Goley, E.D., Niederstrasser, H., Trinh, T., and Welch, M.D. (2001).
Reconstitution of human Arp2/3 complex reveals critical roles of individual
subunits in complex structure and activity. Mol Cell 8, 1041-1052.
Govindan, B., Bowser, R., and Novick, P. (1995). The role of Myo2, a yeast class V
myosin, in vesicular transport. J Cell Biol 128, 1055-1068.
Grindstaff, K.K., Yeaman, C., Anandasabapathy, N., Hsu, S.C., Rodriguez-Boulan, E.,
215 Jianglan Liu THE ROLE OF THE EXOCYST IN EXOCYTOSIS AND CELL MIGRATION References Scheller, R.H., and Nelson, W.J. (1998). Sec6/8 complex is recruited to cell-cell
contacts and specifies transport vesicle delivery to the basal-lateral membrane in
epithelial cells. Cell 93, 731-740.
Gromley, A., Yeaman, C., Rosa, J., Redick, S., Chen, C.T., Mirabelle, S., Guha, M.,
Sillibourne, J., and Doxsey, S.J. (2005). Centriolin anchoring of exocyst and
SNARE complexes at the midbody is required for secretory-vesicle-mediated
abscission. Cell 123, 75-87.
Grosshans, B.L., Ortiz, D., and Novick, P. (2006). Rabs and their effectors: achieving
specificity in membrane traffic. Proc Natl Acad Sci U S A 103, 11821-11827.
Grote, E., Carr, C.M., and Novick, P.J. (2000). Ordering the final events in yeast
exocytosis. J Cell Biol 151, 439-452.
Gumbiner, B., Stevenson, B., and Grimaldi, A. (1988). The role of the cell adhesion
molecule uvomorulin in the formation and maintenance of the epithelial
junctional complex. J Cell Biol 107, 1575-1587.
Guo, A., Han, M., Martinez, T., Ketchem, R.R., Novick, S., Jochheim, C., and
Balland, A. (2008). Electrophoretic evidence for the presence of structural
isoforms specific for the IgG2 isotype. Electrophoresis 29, 2550-2556.
Guo, L.R., Chu, M.W., Tong, M.Z., Fox, S., Myers, M.L., Kiaii, B., Quantz, M.,
McKenzie, F.N., and Novick, R.J. (2008). Does the trainee's level of experience
216 Jianglan Liu THE ROLE OF THE EXOCYST IN EXOCYTOSIS AND CELL MIGRATION References impact on patient safety and clinical outcomes in coronary artery bypass surgery?
J Card Surg 23, 1-5.
Guo, W., Grant, A., and Novick, P. (1999). Exo84p is an exocyst protein essential for
secretion. J Biol Chem 274, 23558-23564.
Guo, W., and Novick, P. (2004). The exocyst meets the translocon: a regulatory
circuit for secretion and protein synthesis? Trends Cell Biol 14, 61-63.
Guo, W., Roth, D., Gatti, E., De Camilli, P., and Novick, P. (1997). Identification and
characterization of homologues of the Exocyst component Sec10p. FEBS Lett
404, 135-139.
Guo, W., Roth, D., Walch-Solimena, C., and Novick, P. (1999). The exocyst is an
effector for Sec4p, targeting secretory vesicles to sites of exocytosis. Embo J 18,
1071-1080.
Guo, W., Sacher, M., Barrowman, J., Ferro-Novick, S., and Novick, P. (2000).
Protein complexes in transport vesicle targeting. Trends Cell Biol 10, 251-255.
Guo, W., Tamanoi, F., and Novick, P. (2001). Spatial regulation of the exocyst
complex by Rho1 GTPase. Nat Cell Biol 3, 353-360.
Hamburger, Z.A., Hamburger, A.E., West, A.P., Jr., and Weis, W.I. (2006). Crystal
structure of the S.cerevisiae exocyst component Exo70p. J Mol Biol 356, 9-21.
Hase, K., Kimura, S., Takatsu, H., Ohmae, M., Kawano, S., Kitamura, H., Ito, M.,
217 Jianglan Liu THE ROLE OF THE EXOCYST IN EXOCYTOSIS AND CELL MIGRATION References Watarai, H., Hazelett, C.C., Yeaman, C., and Ohno, H. (2009). M-Sec promotes
membrane nanotube formation by interacting with Ral and the exocyst complex.
Nat Cell Biol 11, 1427-1432.
Hashimoto, S., Onodera, Y., Hashimoto, A., Tanaka, M., Hamaguchi, M., Yamada,
A., and Sabe, H. (2004). Requirement for Arf6 in breast cancer invasive activities.
Proc Natl Acad Sci U S A 101, 6647-6652.
Hattula, K., Furuhjelm, J., Arffman, A., and Peranen, J. (2002). A Rab8-specific
GDP/GTP exchange factor is involved in actin remodeling and polarized
membrane transport. Mol Biol Cell 13, 3268-3280.
Hauck, C.R., Hsia, D.A., Ilic, D., and Schlaepfer, D.D. (2002). v-Src SH3-enhanced
interaction with focal adhesion kinase at beta 1 integrin-containing invadopodia
promotes cell invasion. J Biol Chem 277, 12487-12490.
Hazuka, C.D., Foletti, D.L., Hsu, S.C., Kee, Y., Hopf, F.W., and Scheller, R.H.
(1999). The sec6/8 complex is located at neurite outgrowth and axonal
synapse-assembly domains. J Neurosci 19, 1324-1334.
Hazuka, C.D., Hsu, S.C., and Scheller, R.H. (1997). Characterization of a cDNA
encoding a subunit of the rat brain rsec6/8 complex. Gene 187, 67-73.
He, B., and Guo, W. (2009). The exocyst complex in polarized exocytosis. Curr Opin
Cell Biol 21, 537-542.
218 Jianglan Liu THE ROLE OF THE EXOCYST IN EXOCYTOSIS AND CELL MIGRATION References He, B., Xi, F., Zhang, J., TerBush, D., Zhang, X., and Guo, W. (2007a). Exo70p
mediates the secretion of specific exocytic vesicles at early stages of the cell cycle
for polarized cell growth. J Cell Biol 176, 771-777.
He, B., Xi, F., Zhang, X., Zhang, J., and Guo, W. (2007b). Exo70 interacts with
phospholipids and mediates the targeting of the exocyst to the plasma membrane.
EMBO J 26, 4053-4065.
He, G.W., Liu, X.C., Kong, X.R., Liu, L.X., Yan, Y.Q., Chen, B.J., Li, Z.X., Jing,
W.B., Wang, Z.Q., Wang, K., Zhang, W., Chen, T.N., Wang, P.S., Lu, W.L.,
Zhang, J.L., Guo, Z.P., Xue, L.G., Zhu, Y.X., Wang, X.L., and Xi, L. (2008). The
current strategy of repair of tetralogy of Fallot in children and adults. Cardiol
Young 18, 608-614.
He, Q., Zhang, J., Shi, J., Zhu, Z., Zhang, L., Bu, W., Guo, L., and Chen, Y. (2010).
The effect of PEGylation of mesoporous silica nanoparticles on nonspecific
binding of serum proteins and cellular responses. Biomaterials 31, 1085-1092.
Hoekstra, D., Tyteca, D., and van, I.S.C. (2004). The subapical compartment: a traffic
center in membrane polarity development. J Cell Sci 117, 2183-2192.
Hokanson, D.E., and Ostap, E.M. (2006). Myo1c binds tightly and specifically to
phosphatidylinositol 4,5-bisphosphate and inositol 1,4,5-trisphosphate. Proc Natl
Acad Sci U S A 103, 3118-3123.
219 Jianglan Liu THE ROLE OF THE EXOCYST IN EXOCYTOSIS AND CELL MIGRATION References Hokin, L.E. (1985). Receptors and phosphoinositide-generated second messengers.
Annu Rev Biochem 54, 205-235.
Hsu, J.F., Guo, Y.L., Liu, C.H., Hu, S.C., Wang, J.N., and Liao, P.C. (2007). A
comparison of PCDD/PCDFs exposure in infants via formula milk or breast milk
feeding. Chemosphere 66, 311-319.
Hsu, S.C., Hazuka, C.D., Foletti, D.L., and Scheller, R.H. (1999). Targeting vesicles
to specific sites on the plasma membrane: the role of the sec6/8 complex. Trends
Cell Biol 9, 150-153.
Hsu, S.C., Hazuka, C.D., Roth, R., Foletti, D.L., Heuser, J., and Scheller, R.H. (1998).
Subunit composition, protein interactions, and structures of the mammalian brain
sec6/8 complex and septin filaments. Neuron 20, 1111-1122.
Hsu, S.C., TerBush, D., Abraham, M., and Guo, W. (2004). The exocyst complex in
polarized exocytosis. Int Rev Cytol 233, 243-265.
Hsu, S.C., Ting, A.E., Hazuka, C.D., Davanger, S., Kenny, J.W., Kee, Y., and
Scheller, R.H. (1996). The mammalian brain rsec6/8 complex. Neuron 17,
1209-1219.
Hurley, J.H., and Meyer, T. (2001). Subcellular targeting by membrane lipids. Curr
Opin Cell Biol 13, 146-152.
Ibarra, N., Blagg, S.L., Vazquez, F., and Insall, R.H. (2006). Nap1 regulates
220 Jianglan Liu THE ROLE OF THE EXOCYST IN EXOCYTOSIS AND CELL MIGRATION References Dictyostelium cell motility and adhesion through SCAR-dependent and
-independent pathways. Curr Biol 16, 717-722.
Inoue, M., Chang, L., Hwang, J., Chiang, S.H., and Saltiel, A.R. (2003). The exocyst
complex is required for targeting of Glut4 to the plasma membrane by insulin.
Nature 422, 629-633.
Inoue, M., Chiang, S.H., Chang, L., Chen, X.W., and Saltiel, A.R. (2006).
Compartmentalization of the exocyst complex in lipid rafts controls Glut4 vesicle
tethering. Mol Biol Cell 17, 2303-2311.
Insall, R.H., and Weiner, O.D. (2001). PIP3, PIP2, and cell movement--similar
messages, different meanings? Dev Cell 1, 743-747.
Irazoqui, J.E., Howell, A.S., Theesfeld, C.L., and Lew, D.J. (2005). Opposing roles
for actin in Cdc42p polarization. Mol Biol Cell 16, 1296-1304.
Itoh, Y., and Seiki, M. (2006). MT1-MMP: a potent modifier of pericellular
microenvironment. J Cell Physiol 206, 1-8.
Jafar-Nejad, H., Andrews, H.K., Acar, M., Bayat, V., Wirtz-Peitz, F., Mehta, S.Q.,
Knoblich, J.A., and Bellen, H.J. (2005). Sec15, a component of the exocyst,
promotes notch signaling during the asymmetric division of Drosophila sensory
organ precursors. Dev Cell 9, 351-363.
Jedd, G., Mulholland, J., and Segev, N. (1997). Two new Ypt GTPases are required
221 Jianglan Liu THE ROLE OF THE EXOCYST IN EXOCYTOSIS AND CELL MIGRATION References for exit from the yeast trans-Golgi compartment. J Cell Biol 137, 563-580.
Jin, R., Junutula, J.R., Matern, H.T., Ervin, K.E., Scheller, R.H., and Brunger, A.T.
(2005). Exo84 and Sec5 are competitive regulatory Sec6/8 effectors to the RalA
GTPase. Embo J 24, 2064-2074.
Kee, Y., Yoo, J.S., Hazuka, C.D., Peterson, K.E., Hsu, S.C., and Scheller, R.H.
(1997). Subunit structure of the mammalian exocyst complex. Proc Natl Acad Sci
U S A 94, 14438-14443.
Kelly, A.E., Kranitz, H., Dotsch, V., and Mullins, R.D. (2006). Actin binding to the
central domain of WASP/Scar proteins plays a critical role in the activation of the
Arp2/3 complex. J Biol Chem 281, 10589-10597.
Kim, A.S., Kakalis, L.T., Abdul-Manan, N., Liu, G.A., and Rosen, M.K. (2000).
Autoinhibition and activation mechanisms of the Wiskott-Aldrich syndrome
protein. Nature 404, 151-158.
Knodler, A., Feng, S., Zhang, J., Zhang, X., Das, A., Peranen, J., and Guo, W. (2010).
Coordination of Rab8 and Rab11 in primary ciliogenesis. Proc Natl Acad Sci U S
A 107, 6346-6351.
Kouyama, T., and Mihashi, K. (1981). Fluorimetry study of
N-(1-pyrenyl)iodoacetamide- labelled F-actin. Local structural change of actin
protomer both on polymerization and on binding of heavy meromyosin. Eur J
222 Jianglan Liu THE ROLE OF THE EXOCYST IN EXOCYTOSIS AND CELL MIGRATION References Biochem 114, 33-38.
Krauss, M., Kinuta, M., Wenk, M.R., De Camilli, P., Takei, K., and Haucke, V.
(2003). ARF6 stimulates clathrin/AP-2 recruitment to synaptic membranes by
activating phosphatidylinositol phosphate kinase type Igamma. J Cell Biol 162,
113-124.
Kreitzer, G., Schmoranzer, J., Low, S.H., Li, X., Gan, Y., Weimbs, T., Simon, S.M.,
and Rodriguez-Boulan, E. (2003). Three-dimensional analysis of post-Golgi
carrier exocytosis in epithelial cells. Nat Cell Biol 5, 126-136.
Kuhn, J.R., and Pollard, T.D. (2005). Real-time measurements of actin filament
polymerization by total internal reflection fluorescence microscopy. Biophys J 88,
1387-1402.
Lalli, G. (2009). RalA and the exocyst complex influence neuronal polarity through
PAR-3 and aPKC. J Cell Sci 122, 1499-1506.
Lalli, G., and Hall, A. (2005). Ral GTPases regulate neurite branching through
GAP-43 and the exocyst complex. J Cell Biol 171, 857-869.
Langevin, J., Morgan, M.J., Sibarita, J.B., Aresta, S., Murthy, M., Schwarz, T.,
Camonis, J., and Bellaiche, Y. (2005). Drosophila exocyst components Sec5,
Sec6, and Sec15 regulate DE-Cadherin trafficking from recycling endosomes to
the plasma membrane. Dev Cell 9, 365-376.
223 Jianglan Liu THE ROLE OF THE EXOCYST IN EXOCYTOSIS AND CELL MIGRATION References Lebensohn AM, Kirschner MW. (2009). Activation of the WAVE complex by
coincident signals controls actin assembly. Mol Cell. 36(3), 512-24.
LeClaire, L.L., 3rd, Baumgartner, M., Iwasa, J.H., Mullins, R.D., and Barber, D.L.
(2008). Phosphorylation of the Arp2/3 complex is necessary to nucleate actin
filaments. J Cell Biol 182, 647-654.
Lefrancois, L., and Lyles, D.S. (1982). The interaction of antibody with the major
surface glycoprotein of vesicular stomatitis virus. II. Monoclonal antibodies of
nonneutralizing and cross-reactive epitopes of Indiana and New Jersey serotypes.
Virology 121, 168-174.
Leisner, T.M., Liu, M., Jaffer, Z.M., Chernoff, J., and Parise, L.V. (2005). Essential
role of CIB1 in regulating PAK1 activation and cell migration. J Cell Biol 170,
465-476.
Lemmon, M.A. (2003). Phosphoinositide recognition domains. Traffic 4, 201-213.
Lemmon, M.A. (2004). Pleckstrin homology domains: not just for phosphoinositides.
Biochem Soc Trans 32, 707-711.
Lim, K.H., Baines, A.T., Fiordalisi, J.J., Shipitsin, M., Feig, L.A., Cox, A.D., Der,
C.J., and Counter, C.M. (2005). Activation of RalA is critical for Ras-induced
tumorigenesis of human cells. Cancer Cell 7, 533-545.
Linder, S. (2007). The matrix corroded: podosomes and invadopodia in extracellular
224 Jianglan Liu THE ROLE OF THE EXOCYST IN EXOCYTOSIS AND CELL MIGRATION References matrix degradation. Trends Cell Biol 17, 107-117.
Lipschutz, J.H., Guo, W., O'Brien, L.E., Nguyen, Y.H., Novick, P., and Mostov, K.E.
(2000). Exocyst is involved in cystogenesis and tubulogenesis and acts by
modulating synthesis and delivery of basolateral plasma membrane and secretory
proteins. Mol Biol Cell 11, 4259-4275.
Lipschutz, J.H., Lingappa, V.R., and Mostov, K.E. (2003). The exocyst affects
protein synthesis by acting on the translocation machinery of the endoplasmic
reticulum. J Biol Chem 278, 20954-20960.
Liu, C.Y., Guo, C.W., Chang, Y.F., Wang, J.T., Shih, H.W., Hsu, Y.F., Chen, C.W.,
Chen, S.K., Wang, Y.C., Cheng, T.J., Ma, C., Wong, C.H., Fang, J.M., and Cheng,
W.C. (2010). Synthesis and evaluation of a new fluorescent transglycosylase
substrate: lipid II-based molecule possessing a dansyl-C20 polyprenyl moiety.
Org Lett 12, 1608-1611.
Liu, J., Zuo, X., Yue, P., and Guo, W. (2007). Phosphatidylinositol 4,5-bisphosphate
mediates the targeting of the exocyst to the plasma membrane for exocytosis in
mammalian cells. Mol Biol Cell 18, 4483-4492.
Lizunov, V.A., Lisinski, I., Stenkula, K., Zimmerberg, J., and Cushman, S.W. (2009).
Insulin regulates fusion of GLUT4 vesicles independent of Exo70-mediated
tethering. J Biol Chem 284, 7914-7919.
225 Jianglan Liu THE ROLE OF THE EXOCYST IN EXOCYTOSIS AND CELL MIGRATION References Lommel, S., Benesch, S., Rottner, K., Franz, T., Wehland, J., and Kuhn, R. (2001).
Actin pedestal formation by enteropathogenic Escherichia coli and intracellular
motility of Shigella flexneri are abolished in N-WASP-defective cells. EMBO
Rep 2, 850-857.
Longtine, M.S., and Bi, E. (2003). Regulation of septin organization and function in
yeast. Trends Cell Biol 13, 403-409.
Lorenz, M., Yamaguchi, H., Wang, Y., Singer, R.H., and Condeelis, J. (2004).
Imaging sites of N-wasp activity in lamellipodia and invadopodia of carcinoma
cells. Curr Biol 14, 697-703.
Manser, E., Leung, T., Salihuddin, H., Zhao, Z.S., and Lim, L. (1994). A brain
serine/threonine protein kinase activated by Cdc42 and Rac1. Nature 367, 40-46.
Mark, B.L., Jilkina, O., and Bhullar, R.P. (1996). Association of Ral GTP-binding
protein with human platelet dense granules. Biochem Biophys Res Commun 225,
40-46.
Martin, T.F. (1998). Phosphoinositide lipids as signaling molecules: common themes
for signal transduction, cytoskeletal regulation, and membrane trafficking. Annu
Rev Cell Dev Biol 14, 231-264.
Marx, J. (2006). Cell biology. Podosomes and invadopodia help mobile cells step
lively. Science 312, 1868-1869.
226 Jianglan Liu THE ROLE OF THE EXOCYST IN EXOCYTOSIS AND CELL MIGRATION References Matern, H.T., Yeaman, C., Nelson, W.J., and Scheller, R.H. (2001). The Sec6/8
complex in mammalian cells: characterization of mammalian Sec3, subunit
interactions, and expression of subunits in polarized cells. Proc Natl Acad Sci U S
A 98, 9648-9653.
Mattila, P.K., Pykalainen, A., Saarikangas, J., Paavilainen, V.O., Vihinen, H.,
Jokitalo, E., and Lappalainen, P. (2007). Missing-in-metastasis and IRSp53
deform PI(4,5)P2-rich membranes by an inverse BAR domain-like mechanism. J
Cell Biol 176, 953-964.
McLaughlin, S., and Murray, D. (2005). Plasma membrane phosphoinositide
organization by protein electrostatics. Nature 438, 605-611.
McLaughlin, S., Wang, J., Gambhir, A., and Murray, D. (2002). PIP(2) and proteins:
interactions, organization, and information flow. Annu Rev Biophys Biomol
Struct 31, 151-175.
McNiven, M.A., Baldassarre, M., and Buccione, R. (2004). The role of dynamin in
the assembly and function of podosomes and invadopodia. Front Biosci 9,
1944-1953.
Medkova, M., France, Y.E., Coleman, J., and Novick, P. (2006). The rab exchange
factor Sec2p reversibly associates with the exocyst. Mol Biol Cell 17, 2757-2769.
Mehta, S.Q., Hiesinger, P.R., Beronja, S., Zhai, R.G., Schulze, K.L., Verstreken, P.,
227 Jianglan Liu THE ROLE OF THE EXOCYST IN EXOCYTOSIS AND CELL MIGRATION References Cao, Y., Zhou, Y., Tepass, U., Crair, M.C., and Bellen, H.J. (2005). Mutations in
Drosophila sec15 reveal a function in neuronal targeting for a subset of exocyst
components. Neuron 46, 219-232.
Merrifield, C.J., Qualmann, B., Kessels, M.M., and Almers, W. (2004). Neural
Wiskott Aldrich Syndrome Protein (N-WASP) and the Arp2/3 complex are
recruited to sites of clathrin-mediated endocytosis in cultured fibroblasts. Eur J
Cell Biol 83, 13-18.
Mizutani, K., Miki, H., He, H., Maruta, H., and Takenawa, T. (2002). Essential role
of neural Wiskott-Aldrich syndrome protein in podosome formation and
degradation of extracellular matrix in src-transformed fibroblasts. Cancer Res 62,
669-674.
Mondesert, G., Clarke, D.J., and Reed, S.I. (1997). Identification of genes controlling
growth polarity in the budding yeast Saccharomyces cerevisiae: a possible role of
N-glycosylation and involvement of the exocyst complex. Genetics 147, 421-434.
Monsky, W.L., Kelly, T., Lin, C.Y., Yeh, Y., Stetler-Stevenson, W.G., Mueller, S.C.,
and Chen, W.T. (1993). Binding and localization of M(r) 72,000 matrix
metalloproteinase at cell surface invadopodia. Cancer Res 53, 3159-3164.
Moore, B.A., Robinson, H.H., and Xu, Z. (2007). The crystal structure of mouse
Exo70 reveals unique features of the mammalian exocyst. J Mol Biol 371,
228 Jianglan Liu THE ROLE OF THE EXOCYST IN EXOCYTOSIS AND CELL MIGRATION References 410-421.
Morales M, Colicos MA, Goda Y. (2000). Actin-dependent regulation of
neurotransmitter release at central synapses. Neuron. 27(3): 539-50.
Moskalenko, S., Henry, D.O., Rosse, C., Mirey, G., Camonis, J.H., and White, M.A.
(2002). The exocyst is a Ral effector complex. Nat Cell Biol 4, 66-72.
Moskalenko, S., Tong, C., Rosse, C., Mirey, G., Formstecher, E., Daviet, L., Camonis,
J., and White, M.A. (2003). Ral GTPases regulate exocyst assembly through dual
subunit interactions. J Biol Chem 278, 51743-51748.
Mott, H.R., Nietlispach, D., Hopkins, L.J., Mirey, G., Camonis, J.H., and Owen, D.
(2003). Structure of the GTPase-binding domain of Sec5 and elucidation of its
Ral binding site. J Biol Chem 278, 17053-17059.
Mott, J.D., and Werb, Z. (2004). Regulation of matrix biology by matrix
metalloproteinases. Curr Opin Cell Biol 16, 558-564.
Mueller, S.C., Ghersi, G., Akiyama, S.K., Sang, Q.X., Howard, L., Pineiro-Sanchez,
M., Nakahara, H., Yeh, Y., and Chen, W.T. (1999). A novel protease-docking
function of integrin at invadopodia. J Biol Chem 274, 24947-24952.
Munson, M., and Novick, P. (2006). The exocyst defrocked, a framework of rods
revealed. Nat Struct Mol Biol 13, 577-581.
Murray, J.T., Panaretou, C., Stenmark, H., Miaczynska, M., and Backer, J.M. (2002).
229 Jianglan Liu THE ROLE OF THE EXOCYST IN EXOCYTOSIS AND CELL MIGRATION References Role of Rab5 in the recruitment of hVps34/p150 to the early endosome. Traffic 3,
416-427.
Murthy, M., Garza, D., Scheller, R.H., and Schwarz, T.L. (2003). Mutations in the
exocyst component Sec5 disrupt neuronal membrane traffic, but neurotransmitter
release persists. Neuron 37, 433-447.
Murthy, M., and Schwarz, T.L. (2004). The exocyst component Sec5 is required for
membrane traffic and polarity in the Drosophila ovary. Development 131,
377-388.
Nachury, M.V., Loktev, A.V., Zhang, Q., Westlake, C.J., Peranen, J., Merdes, A.,
Slusarski, D.C., Scheller, R.H., Bazan, J.F., Sheffield, V.C., and Jackson, P.K.
(2007). A core complex of BBS proteins cooperates with the GTPase Rab8 to
promote ciliary membrane biogenesis. Cell 129, 1201-1213.
Nakahara, H., Howard, L., Thompson, E.W., Sato, H., Seiki, M., Yeh, Y., and Chen,
W.T. (1997). Transmembrane/cytoplasmic domain-mediated membrane type
1-matrix metalloprotease docking to invadopodia is required for cell invasion.
Proc Natl Acad Sci U S A 94, 7959-7964.
Nakahara, H., Mueller, S.C., Nomizu, M., Yamada, Y., Yeh, Y., and Chen, W.T.
(1998). Activation of beta1 integrin signaling stimulates tyrosine phosphorylation
of p190RhoGAP and membrane-protrusive activities at invadopodia. J Biol Chem
230 Jianglan Liu THE ROLE OF THE EXOCYST IN EXOCYTOSIS AND CELL MIGRATION References 273, 9-12.
Nakahara, H., Nomizu, M., Akiyama, S.K., Yamada, Y., Yeh, Y., and Chen, W.T.
(1996). A mechanism for regulation of melanoma invasion. Ligation of
alpha6beta1 integrin by laminin G peptides. J Biol Chem 271, 27221-27224.
Nakahara, H., Otani, T., Sasaki, T., Miura, Y., Takai, Y., and Kogo, M. (2003).
Involvement of Cdc42 and Rac small G proteins in invadopodia formation of
RPMI7951 cells. Genes Cells 8, 1019-1027.
Ngsee, J.K., Elferink, L.A., and Scheller, R.H. (1991). A family of ras-like
GTP-binding proteins expressed in electromotor neurons. J Biol Chem 266,
2675-2680.
Novick, P., and Brennwald, P. (1993). Friends and family: the role of the Rab
GTPases in vesicular traffic. Cell 75, 597-601.
Novick, P., Ferro, S., and Schekman, R. (1981). Order of events in the yeast secretory
pathway. Cell 25, 461-469.
Novick, P., Field, C., and Schekman, R. (1980). Identification of 23 complementation
groups required for post-translational events in the yeast secretory pathway. Cell
21, 205-215.
Novick, P., and Guo, W. (2002). Ras family therapy: Rab, Rho and Ral talk to the
exocyst. Trends Cell Biol 12, 247-249.
231 Jianglan Liu THE ROLE OF THE EXOCYST IN EXOCYTOSIS AND CELL MIGRATION References Novick, P., and Schekman, R. (1979). Secretion and cell-surface growth are blocked
in a temperature-sensitive mutant of Saccharomyces cerevisiae. Proc Natl Acad
Sci U S A 76, 1858-1862.
Ohnishi H, Yamamori S, Ono K, Aoyagi K, Kondo S, Takahashi M. (2001). A src
family tyrosine kinase inhibits neurotransmitter release from neuronal cells. Proc
Natl Acad Sci U S A. 98(19):10930-5.
Oikawa, T., Yamaguchi, H., Itoh, T., Kato, M., Ijuin, T., Yamazaki, D., Suetsugu, S.,
and Takenawa, T. (2004). PtdIns(3,4,5)P3 binding is necessary for
WAVE2-induced formation of lamellipodia. Nat Cell Biol 6, 420-426.
Olencki, T., Peereboom, D., Wood, L., Budd, G.T., Novick, A., Finke, J., McLain, D.,
Elson, P., and Bukowski, R.M. (2001). Phase I and II trials of subcutaneously
administered rIL-2, interferon alfa-2a, and fluorouracil in patients with metastatic
renal carcinoma. J Cancer Res Clin Oncol 127, 319-324.
Owen, D.J., Collins, B.M., and Evans, P.R. (2004). Adaptors for clathrin coats:
structure and function. Annu Rev Cell Dev Biol 20, 153-191.
Oztan, A., Silvis, M., Weisz, O.A., Bradbury, N.A., Hsu, S.C., Goldenring, J.R.,
Yeaman, C., and Apodaca, G. (2007). Exocyst requirement for endocytic traffic
directed toward the apical and basolateral poles of polarized MDCK cells. Mol
Biol Cell 18, 3978-3992.
232 Jianglan Liu THE ROLE OF THE EXOCYST IN EXOCYTOSIS AND CELL MIGRATION References Palmer, R.E., Lee, S.B., Wong, J.C., Reynolds, P.A., Zhang, H., Truong, V., Oliner,
J.D., Gerald, W.L., and Haber, D.A. (2002). Induction of BAIAP3 by the
EWS-WT1 chimeric fusion implicates regulated exocytosis in tumorigenesis.
Cancer Cell 2, 497-505.
Papayannopoulos, V., Co, C., Prehoda, K.E., Snapper, S., Taunton, J., and Lim, W.A.
(2005). A polybasic motif allows N-WASP to act as a sensor of PIP(2) density.
Mol Cell 17, 181-191.
Park, H.O., and Bi, E. (2007). Central roles of small GTPases in the development of
cell polarity in yeast and beyond. Microbiol Mol Biol Rev 71, 48-96.
Pfeffer, S.R. (1999). Transport-vesicle targeting: tethers before SNAREs. Nat Cell
Biol 1, E17-22.
Pfeffer, S.R. (2001). Rab GTPases: specifying and deciphering organelle identity and
function. Trends Cell Biol 11, 487-491.
Pollard, T.D., and Borisy, G.G. (2003). Cellular motility driven by assembly and
disassembly of actin filaments. Cell 112, 453-465.
Polzin, A., Shipitsin, M., Goi, T., Feig, L.A., and Turner, T.J. (2002). Ral-GTPase
influences the regulation of the readily releasable pool of synaptic vesicles. Mol
Cell Biol 22, 1714-1722.
Pommereit, D., and Wouters, F.S. (2007). An NGF-induced Exo70-TC10 complex
233 Jianglan Liu THE ROLE OF THE EXOCYST IN EXOCYTOSIS AND CELL MIGRATION References locally antagonises Cdc42-mediated activation of N-WASP to modulate neurite
outgrowth. J Cell Sci 120, 2694-2705.
Prehoda, K.E., Scott, J.A., Mullins, R.D., and Lim, W.A. (2000). Integration of
multiple signals through cooperative regulation of the N-WASP-Arp2/3 complex.
Science 290, 801-806.
Prigent, M., Dubois, T., Raposo, G., Derrien, V., Tenza, D., Rosse, C., Camonis, J.,
and Chavrier, P. (2003). ARF6 controls post-endocytic recycling through its
downstream exocyst complex effector. J Cell Biol 163, 1111-1121.
Rea, S., Martin, L.B., McIntosh, S., Macaulay, S.L., Ramsdale, T., Baldini, G., and
James, D.E. (1998). Syndet, an adipocyte target SNARE involved in the
insulin-induced translocation of GLUT4 to the cell surface. J Biol Chem 273,
18784-18792.
Redondo-Munoz, J., Escobar-Diaz, E., Samaniego, R., Terol, M.J., Garcia-Marco,
J.A., and Garcia-Pardo, A. (2006). MMP-9 in B-cell chronic lymphocytic
leukemia is up-regulated by alpha4beta1 integrin or CXCR4 engagement via
distinct signaling pathways, localizes to podosomes, and is involved in cell
invasion and migration. Blood 108, 3143-3151.
Ren, M., Xu, G., Zeng, J., De Lemos-Chiarandini, C., Adesnik, M., and Sabatini, D.D.
(1998). Hydrolysis of GTP on rab11 is required for the direct delivery of
234 Jianglan Liu THE ROLE OF THE EXOCYST IN EXOCYTOSIS AND CELL MIGRATION References transferrin from the pericentriolar recycling compartment to the cell surface but
not from sorting endosomes. Proc Natl Acad Sci U S A 95, 6187-6192.
Ridley, A.J., Schwartz, M.A., Burridge, K., Firtel, R.A., Ginsberg, M.H., Borisy, G.,
Parsons, J.T., and Horwitz, A.R. (2003). Cell migration: integrating signals from
front to back. Science 302, 1704-1709.
Riefler, G.M., Balasingam, G., Lucas, K.G., Wang, S., Hsu, S.C., and Firestein, B.L.
(2003). Exocyst complex subunit sec8 binds to postsynaptic density protein-95
(PSD-95): a novel interaction regulated by cypin (cytosolic PSD-95 interactor).
Biochem J 373, 49-55.
Robinson, N.G., Guo, L., Imai, J., Toh, E.A., Matsui, Y., and Tamanoi, F. (1999).
Rho3 of Saccharomyces cerevisiae, which regulates the actin cytoskeleton and
exocytosis, is a GTPase which interacts with Myo2 and Exo70. Mol Cell Biol 19,
3580-3587.
Rogers, K.K., Wilson, P.D., Snyder, R.W., Zhang, X., Guo, W., Burrow, C.R., and
Lipschutz, J.H. (2004). The exocyst localizes to the primary cilium in MDCK
cells. Biochem Biophys Res Commun 319, 138-143.
Rohatgi, R., Ho, H.Y., and Kirschner, M.W. (2000). Mechanism of N-WASP
activation by CDC42 and phosphatidylinositol 4, 5-bisphosphate. J Cell Biol 150,
1299-1310.
235 Jianglan Liu THE ROLE OF THE EXOCYST IN EXOCYTOSIS AND CELL MIGRATION References Rosse, C., Formstecher, E., Boeckeler, K., Zhao, Y., Kremerskothen, J., White, M.D.,
Camonis, J.H., and Parker, P.J. (2009). An aPKC-exocyst complex controls
paxillin phosphorylation and migration through localised JNK1 activation. PLoS
Biol 7, e1000235.
Rosse, C., Hatzoglou, A., Parrini, M.C., White, M.A., Chavrier, P., and Camonis, J.
(2006). RalB mobilizes the exocyst to drive cell migration. Mol Cell Biol 26,
727-734.
Roth, D., Guo, W., and Novick, P. (1998). Dominant negative alleles of SEC10
reveal distinct domains involved in secretion and morphogenesis in yeast. Mol
Biol Cell 9, 1725-1739.
Rouiller, I., Xu, X.P., Amann, K.J., Egile, C., Nickell, S., Nicastro, D., Li, R., Pollard,
T.D., Volkmann, N., and Hanein, D. (2008). The structural basis of actin filament
branching by the Arp2/3 complex. J Cell Biol 180, 887-895.
Roumanie, O., Wu, H., Molk, J.N., Rossi, G., Bloom, K., and Brennwald, P. (2005).
Rho GTPase regulation of exocytosis in yeast is independent of GTP hydrolysis
and polarization of the exocyst complex. J Cell Biol 170, 583-594.
Rykx, A., De Kimpe, L., Mikhalap, S., Vantus, T., Seufferlein, T., Vandenheede, J.R.,
and Van Lint, J. (2003). Protein kinase D: a family affair. FEBS Lett 546, 81-86.
Saarikangas, J., Zhao, H., Pykalainen, A., Laurinmaki, P., Mattila, P.K., Kinnunen,
236 Jianglan Liu THE ROLE OF THE EXOCYST IN EXOCYTOSIS AND CELL MIGRATION References P.K., Butcher, S.J., and Lappalainen, P. (2009). Molecular mechanisms of
membrane deformation by I-BAR domain proteins. Curr Biol 19, 95-107.
Sakurai-Yageta, M., Recchi, C., Le Dez, G., Sibarita, J.B., Daviet, L., Camonis, J.,
D'Souza-Schorey, C., and Chavrier, P. (2008). The interaction of IQGAP1 with
the exocyst complex is required for tumor cell invasion downstream of Cdc42 and
RhoA. J Cell Biol 181, 985-998.
Salminen, A., and Novick, P.J. (1987). A ras-like protein is required for a post-Golgi
event in yeast secretion. Cell 49, 527-538.
Salminen, A., and Novick, P.J. (1989). The Sec15 protein responds to the function of
the GTP binding protein, Sec4, to control vesicular traffic in yeast. J Cell Biol 109,
1023-1036.
Sans, N., Prybylowski, K., Petralia, R.S., Chang, K., Wang, Y.X., Racca, C., Vicini,
S., and Wenthold, R.J. (2003). NMDA receptor trafficking through an interaction
between PDZ proteins and the exocyst complex. Nat Cell Biol 5, 520-530.
Seals, D.F., Azucena, E.F., Jr., Pass, I., Tesfay, L., Gordon, R., Woodrow, M., Resau,
J.H., and Courtneidge, S.A. (2005). The adaptor protein Tks5/Fish is required for
podosome formation and function, and for the protease-driven invasion of cancer
cells. Cancer Cell 7, 155-165.
Sells, M.A., Boyd, J.T., and Chernoff, J. (1999). p21-activated kinase 1 (Pak1)
237 Jianglan Liu THE ROLE OF THE EXOCYST IN EXOCYTOSIS AND CELL MIGRATION References regulates cell motility in mammalian fibroblasts. J Cell Biol 145, 837-849.
Sells, M.A., Knaus, U.G., Bagrodia, S., Ambrose, D.M., Bokoch, G.M., and Chernoff,
J. (1997). Human p21-activated kinase (Pak1) regulates actin organization in
mammalian cells. Curr Biol 7, 202-210.
Sheetz, M.P., Felsenfeld, D., Galbraith, C.G., and Choquet, D. (1999). Cell migration
as a five-step cycle. Biochem Soc Symp 65, 233-243.
Shipitsin, M., and Feig, L.A. (2004). RalA but not RalB enhances polarized delivery
of membrane proteins to the basolateral surface of epithelial cells. Mol Cell Biol
24, 5746-5756.
Singh, S., Powell, D.W., Rane, M.J., Millard, T.H., Trent, J.O., Pierce, W.M., Klein,
J.B., Machesky, L.M., and McLeish, K.R. (2003). Identification of the p16-Arc
subunit of the Arp 2/3 complex as a substrate of MAPK-activated protein kinase 2
by proteomic analysis. J Biol Chem 278, 36410-36417.
Sivaram, M.V., Furgason, M.L., Brewer, D.N., and Munson, M. (2006). The structure
of the exocyst subunit Sec6p defines a conserved architecture with diverse roles.
Nat Struct Mol Biol 13, 555-556.
Sivaram, M.V., Saporita, J.A., Furgason, M.L., Boettcher, A.J., and Munson, M.
(2005). Dimerization of the exocyst protein Sec6p and its interaction with the
t-SNARE Sec9p. Biochemistry 44, 6302-6311.
238 Jianglan Liu THE ROLE OF THE EXOCYST IN EXOCYTOSIS AND CELL MIGRATION References Slack-Davis, J.K., Eblen, S.T., Zecevic, M., Boerner, S.A., Tarcsafalvi, A., Diaz,
H.B., Marshall, M.S., Weber, M.J., Parsons, J.T., and Catling, A.D. (2003). PAK1
phosphorylation of MEK1 regulates fibronectin-stimulated MAPK activation. J
Cell Biol 162, 281-291.
Sommer, B., Oprins, A., Rabouille, C., and Munro, S. (2005). The exocyst
component Sec5 is present on endocytic vesicles in the oocyte of Drosophila
melanogaster. J Cell Biol 169, 953-963.
Songer, J.A., and Munson, M. (2009). Sec6p anchors the assembled exocyst complex
at sites of secretion. Mol Biol Cell 20, 973-982.
Spiczka, K.S., and Yeaman, C. (2008). Ral-regulated interaction between Sec5 and
paxillin targets Exocyst to focal complexes during cell migration. J Cell Sci 121,
2880-2891.
Spiliotis, E.T., and Nelson, W.J. (2006). Here come the septins: novel polymers that
coordinate intracellular functions and organization. J Cell Sci 119, 4-10.
Steffen, A., Le Dez, G., Poincloux, R., Recchi, C., Nassoy, P., Rottner, K., Galli, T.,
and Chavrier, P. (2008). MT1-MMP-dependent invasion is regulated by
TI-VAMP/VAMP7. Curr Biol 18, 926-931.
Stroupe, C., Collins, K.M., Fratti, R.A., and Wickner, W. (2006). Purification of
active HOPS complex reveals its affinities for phosphoinositides and the SNARE
239 Jianglan Liu THE ROLE OF THE EXOCYST IN EXOCYTOSIS AND CELL MIGRATION References Vam7p. Embo J 25, 1579-1589.
Suetsugu, S., Yamazaki, D., Kurisu, S., and Takenawa, T. (2003). Differential roles
of WAVE1 and WAVE2 in dorsal and peripheral ruffle formation for fibroblast
cell migration. Dev Cell 5, 595-609.
Sugihara, K., Asano, S., Tanaka, K., Iwamatsu, A., Okawa, K., and Ohta, Y. (2002).
The exocyst complex binds the small GTPase RalA to mediate filopodia
formation. Nat Cell Biol 4, 73-78.
Tague, S.E., Muralidharan, V., and D'Souza-Schorey, C. (2004). ADP-ribosylation
factor 6 regulates tumor cell invasion through the activation of the MEK/ERK
signaling pathway. Proc Natl Acad Sci U S A 101, 9671-9676.
Takaya, A., Ohba, Y., Kurokawa, K., and Matsuda, M. (2004). RalA activation at
nascent lamellipodia of epidermal growth factor-stimulated Cos7 cells and
migrating Madin-Darby canine kidney cells. Mol Biol Cell 15, 2549-2557.
Takenawa, T., and Suetsugu, S. (2007). The WASP-WAVE protein network:
connecting the membrane to the cytoskeleton. Nat Rev Mol Cell Biol 8, 37-48.
Terbush, D.R., Guo, W., Dunkelbarger, S., and Novick, P. (2001). Purification and
characterization of yeast exocyst complex. Methods Enzymol 329, 100-110.
TerBush, D.R., Maurice, T., Roth, D., and Novick, P. (1996). The Exocyst is a
multiprotein complex required for exocytosis in Saccharomyces cerevisiae. Embo
240 Jianglan Liu THE ROLE OF THE EXOCYST IN EXOCYTOSIS AND CELL MIGRATION References J 15, 6483-6494.
TerBush, D.R., and Novick, P. (1995). Sec6, Sec8, and Sec15 are components of a
multisubunit complex which localizes to small bud tips in Saccharomyces
cerevisiae. J Cell Biol 130, 299-312.
Ting, A.E., Hazuka, C.D., Hsu, S.C., Kirk, M.D., Bean, A.J., and Scheller, R.H.
(1995). rSec6 and rSec8, mammalian homologs of yeast proteins essential for
secretion. Proc Natl Acad Sci U S A 92, 9613-9617.
Tsuboi, T., Ravier, M.A., Xie, H., Ewart, M.A., Gould, G.W., Baldwin, S.A., and
Rutter, G.A. (2005). Mammalian exocyst complex is required for the docking step
of insulin vesicle exocytosis. J Biol Chem 280, 25565-25570.
Uemura, T., Oda, H., Kraut, R., Hayashi, S., Kotaoka, Y., and Takeichi, M. (1996).
Zygotic Drosophila E-cadherin expression is required for processes of dynamic
epithelial cell rearrangement in the Drosophila embryo. Genes Dev 10, 659-671.
Ullrich, O., Reinsch, S., Urbe, S., Zerial, M., and Parton, R.G. (1996). Rab11
regulates recycling through the pericentriolar recycling endosome. J Cell Biol 135,
913-924.
Vadlamudi, R.K., Li, F., Barnes, C.J., Bagheri-Yarmand, R., and Kumar, R. (2004).
p41-Arc subunit of human Arp2/3 complex is a p21-activated kinase-1-interacting
substrate. EMBO Rep 5, 154-160.
241 Jianglan Liu THE ROLE OF THE EXOCYST IN EXOCYTOSIS AND CELL MIGRATION References Van den Steen, P.E., Dubois, B., Nelissen, I., Rudd, P.M., Dwek, R.A., and
Opdenakker, G. (2002). Biochemistry and molecular biology of gelatinase B or
matrix metalloproteinase-9 (MMP-9). Crit Rev Biochem Mol Biol 37, 375-536.
Vega, I.E., and Hsu, S.C. (2001). The exocyst complex associates with microtubules
to mediate vesicle targeting and neurite outgrowth. J Neurosci 21, 3839-3848.
Vega, I.E., and Hsu, S.C. (2003). The septin protein Nedd5 associates with both the
exocyst complex and microtubules and disruption of its GTPase activity promotes
aberrant neurite sprouting in PC12 cells. Neuroreport 14, 31-37.
VerPlank, L., and Li, R. (2005). Cell cycle-regulated trafficking of Chs2 controls
actomyosin ring stability during cytokinesis. Mol Biol Cell 16, 2529-2543.
Walch-Solimena, C., Collins, R.N., and Novick, P.J. (1997). Sec2p mediates
nucleotide exchange on Sec4p and is involved in polarized delivery of post-Golgi
vesicles. J Cell Biol 137, 1495-1509.
Wang, H., Tang, X., Liu, J., Trautmann, S., Balasundaram, D., McCollum, D., and
Balasubramanian, M.K. (2002). The multiprotein exocyst complex is essential for
cell separation in Schizosaccharomyces pombe. Mol Biol Cell 13, 515-529.
Wang, H.S., Hung, Y., Su, C.H., Peng, S.T., Guo, Y.J., Lai, M.C., Liu, C.Y., and Hsu,
J.W. (2005). CD44 cross-linking induces integrin-mediated adhesion and
transendothelial migration in breast cancer cell line by up-regulation of LFA-1
242 Jianglan Liu THE ROLE OF THE EXOCYST IN EXOCYTOSIS AND CELL MIGRATION References (alpha L beta2) and VLA-4 (alpha4beta1). Exp Cell Res 304, 116-126.
Wang, H.Y., Zhang, F.C., Gao, J.J., Fan, J.B., Liu, P., Zheng, Z.J., Xi, H., Sun, Y.,
Gao, X.C., Huang, T.Z., Ke, Z.J., Guo, G.R., Feng, G.Y., Breen, G., Clair, D.S.,
and He, L. (2000). Apolipoprotein E is a genetic risk factor for fetal iodine
deficiency disorder in China. Mol Psychiatry 5, 363-368.
Wang, L., Li, G., and Sugita, S. (2004). RalA-exocyst interaction mediates
GTP-dependent exocytosis. J Biol Chem 279, 19875-19881.
Wang, Q.J. (2006). PKD at the crossroads of DAG and PKC signaling. Trends
Pharmacol Sci 27, 317-323.
Wang, S., and Hsu, S.C. (2006). The molecular mechanisms of the mammalian
exocyst complex in exocytosis. Biochem Soc Trans 34, 687-690.
Wang, S., Liu, Y., Adamson, C.L., Valdez, G., Guo, W., and Hsu, S.C. (2004). The
mammalian exocyst, a complex required for exocytosis, inhibits tubulin
polymerization. J Biol Chem 279, 35958-35966.
Wang, S.J., Fuh, J.L., Huang, S.Y., Yang, S.S., Wu, Z.A., Hsu, C.H., Wang, C.H., Yu,
H.Y., and Wang, P.J. (2008). Diagnosis and development of screening items for
migraine in neurological practice in Taiwan. J Formos Med Assoc 107, 485-494.
Waters, M.G., and Hughson, F.M. (2000). Membrane tethering and fusion in the
secretory and endocytic pathways. Traffic 1, 588-597.
243 Jianglan Liu THE ROLE OF THE EXOCYST IN EXOCYTOSIS AND CELL MIGRATION References Weber, T. M., Joost, H., Simpson, I. A., and Cushman, S. W. (1988) Receptor
Biochemistry and Methodology, ed. R. C. Kahn and L. C. Harrison, New York:
Alan R. Liss, 171–187.
Wedlich-Soldner, R., Altschuler, S., Wu, L., and Li, R. (2003). Spontaneous cell
polarization through actomyosin-based delivery of the Cdc42 GTPase. Science
299, 1231-1235.
Wedlich-Soldner, R., Wai, S.C., Schmidt, T., and Li, R. (2004). Robust cell polarity
is a dynamic state established by coupling transport and GTPase signaling. J Cell
Biol 166, 889-900.
Wenk, M.R., and De Camilli, P. (2004). Protein-lipid interactions and
phosphoinositide metabolism in membrane traffic: insights from vesicle recycling
in nerve terminals. Proc Natl Acad Sci U S A 101, 8262-8269.
Whyte, J.R., and Munro, S. (2001). The Sec34/35 Golgi transport complex is related
to the exocyst, defining a family of complexes involved in multiple steps of
membrane traffic. Dev Cell 1, 527-537.
Whyte, J.R., and Munro, S. (2002). Vesicle tethering complexes in membrane traffic.
J Cell Sci 115, 2627-2637.
Wu, H., Turner, C., Gardner, J., Temple, B., and Brennwald, P. (2010). The Exo70
subunit of the exocyst is an effector for both Cdc42 and Rho3 function in
244 Jianglan Liu THE ROLE OF THE EXOCYST IN EXOCYTOSIS AND CELL MIGRATION References polarized exocytosis. Mol Biol Cell 21, 430-442.
Wu, S., Mehta, S.Q., Pichaud, F., Bellen, H.J., and Quiocho, F.A. (2005). Sec15
interacts with Rab11 via a novel domain and affects Rab11 localization in vivo.
Nat Struct Mol Biol 12, 879-885.
Xi, X., Guo, Y., Chen, H., Xu, C., Zhang, H., Hu, H., Cui, L., Ba, D., and He, W.
(2009). Antigen specificity of gammadelta T cells depends primarily on the
flanking sequences of CDR3delta. J Biol Chem 284, 27449-27455.
Xu, K.F., Shen, X., Li, H., Pacheco-Rodriguez, G., Moss, J., and Vaughan, M. (2005).
Interaction of BIG2, a brefeldin A-inhibited guanine nucleotide-exchange protein,
with exocyst protein Exo70. Proc Natl Acad Sci U S A 102, 2784-2789.
Yamaguchi, H., Lorenz, M., Kempiak, S., Sarmiento, C., Coniglio, S., Symons, M.,
Segall, J., Eddy, R., Miki, H., Takenawa, T., and Condeelis, J. (2005). Molecular
mechanisms of invadopodium formation: the role of the N-WASP-Arp2/3
complex pathway and cofilin. J Cell Biol 168, 441-452.
Yamashita, M., Kurokawa, K., Sato, Y., Yamagata, A., Mimura, H., Yoshikawa, A.,
Sato, K., Nakano, A., and Fukai, S. (2010). Structural basis for the Rho- and
phosphoinositide-dependent localization of the exocyst subunit Sec3. Nat Struct
Mol Biol 17, 180-186.
Yamazaki, D., Fujiwara, T., Suetsugu, S., and Takenawa, T. (2005). A novel function
245 Jianglan Liu THE ROLE OF THE EXOCYST IN EXOCYTOSIS AND CELL MIGRATION References of WAVE in lamellipodia: WAVE1 is required for stabilization of lamellipodial
protrusions during cell spreading. Genes Cells 10, 381-392.
Yamazaki, D., Suetsugu, S., Miki, H., Kataoka, Y., Nishikawa, S., Fujiwara, T.,
Yoshida, N., and Takenawa, T. (2003). WAVE2 is required for directed cell
migration and cardiovascular development. Nature 424, 452-456.
Yanagawa, R., Furukawa, Y., Tsunoda, T., Kitahara, O., Kameyama, M., Murata, K.,
Ishikawa, O., and Nakamura, Y. (2001). Genome-wide screening of genes
showing altered expression in liver metastases of human colorectal cancers by
cDNA microarray. Neoplasia 3, 395-401.
Yeaman, C., Grindstaff, K.K., Hansen, M.D., and Nelson, W.J. (1999). Cell polarity:
Versatile scaffolds keep things in place. Curr Biol 9, R515-517.
Yeaman, C., Grindstaff, K.K., and Nelson, W.J. (1999). New perspectives on
mechanisms involved in generating epithelial cell polarity. Physiol Rev 79, 73-98.
Yeaman, C., Grindstaff, K.K., and Nelson, W.J. (2004). Mechanism of recruiting
Sec6/8 (exocyst) complex to the apical junctional complex during polarization of
epithelial cells. J Cell Sci 117, 559-570.
Yeaman, C., Grindstaff, K.K., Wright, J.R., and Nelson, W.J. (2001). Sec6/8
complexes on trans-Golgi network and plasma membrane regulate late stages of
exocytosis in mammalian cells. J Cell Biol 155, 593-604.
246 Jianglan Liu THE ROLE OF THE EXOCYST IN EXOCYTOSIS AND CELL MIGRATION References Yin, H.L., and Janmey, P.A. (2003). Phosphoinositide regulation of the actin
cytoskeleton. Annu Rev Physiol 65, 761-789.
Yoshimura, S., Egerer, J., Fuchs, E., Haas, A.K., and Barr, F.A. (2007). Functional
dissection of Rab GTPases involved in primary cilium formation. J Cell Biol 178,
363-369.
Yoshizaki, H., Mochizuki, N., Gotoh, Y., and Matsuda, M. (2007). Akt-PDK1
complex mediates epidermal growth factor-induced membrane protrusion through
Ral activation. Mol Biol Cell 18, 119-128.
Zajac, A., Sun, X., Zhang, J., and Guo, W. (2005). Cyclical regulation of the exocyst
and cell polarity determinants for polarized cell growth. Mol Biol Cell 16,
1500-1512.
Zhang, G., Xi, J., Wang, X., Guo, J., Zhang, H., Yang, Y., Qiao, S., Wang, L., Zhang,
H., He, L., and Zhu, Y. (2008). Efficient recovery of a functional extracellular
domain of bovine IgG2 Fc receptor (boFcgamma2R) from inclusion bodies by a
rapid dilution refolding system. J Immunol Methods 334, 21-28.
Zhang, X., Bi, E., Novick, P., Du, L., Kozminski, K.G., Lipschutz, J.H., and Guo, W.
(2001). Cdc42 interacts with the exocyst and regulates polarized secretion. J Biol
Chem 276, 46745-46750.
Zhang, X., Orlando, K., He, B., Xi, F., Zhang, J., Zajac, A., and Guo, W. (2008).
247 Jianglan Liu THE ROLE OF THE EXOCYST IN EXOCYTOSIS AND CELL MIGRATION References Membrane association and functional regulation of Sec3 by phospholipids and
Cdc42. J Cell Biol 180, 145-158.
Zhang, X., Zajac, A., Zhang, J., Wang, P., Li, M., Murray, J., TerBush, D., and Guo,
W. (2005). The critical role of Exo84p in the organization and polarized
localization of the exocyst complex. J Biol Chem 280, 20356-20364.
Zhang, X.M., Ellis, S., Sriratana, A., Mitchell, C.A., and Rowe, T. (2004). Sec15 is
an effector for the Rab11 GTPase in mammalian cells. J Biol Chem 279,
43027-43034.
Zhao, Z.S., Manser, E., Chen, X.Q., Chong, C., Leung, T., and Lim, L. (1998). A
conserved negative regulatory region in alphaPAK: inhibition of PAK kinases
reveals their morphological roles downstream of Cdc42 and Rac1. Mol Cell Biol
18, 2153-2163.
Zuo, X., Guo, W., and Lipschutz, J.H. (2009). The exocyst protein Sec10 is necessary
for primary ciliogenesis and cystogenesis in vitro. Mol Biol Cell 20, 2522-2529.
Zuo, X., Zhang, J., Zhang, Y., Hsu, S.C., Zhou, D., and Guo, W. (2006). Exo70
interacts with the Arp2/3 complex and regulates cell migration. Nat Cell Biol 8,
1383-1388.
248