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Rho/ROCK dependent mRNA translocation to tumor cell pseudopodia Stuart, Heather Christine 2006

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RHO/ROCK DEPENDENT M R N A TRANSLOCATION TO T U M O R C E L L PSEUDOPODIA by H E A T H E R CHRISTINE STUART B.Sc , The University of Western Ontario, 2003 A THESIS SUBMITTED IN PARTIAL F U L F I L L M E N T OF THE REQUIREMENTS FOR THE DEGREE OF M A S T E R OF SCIENCE i n THE F A C U L T Y OF G R A D U A T E STUDIES (Anatomy) THE UNIVERSITY OF BRITISH C O L U M B I A July 2006 © Heather Christine Stuart, 2006 Abstract The psuedopodial fraction of MS V - M D C K - I N V cells was isolated and proteomic analysis by LS-MS/MS revealed the enrichment of a variety of cytoskeletal and adhesion proteins, glycolytic enzymes, proteins required for translation, ubiquitin/proteasome associated proteins, R N A binding proteins and cell signaling proteins. The selective enrichment of these proteins indicated the potential existence of mRNA in the pseudopodial domain. Propidium iodide labeling demonstrated the presence of mRNA in the pseudopodia. The localization of mRNA in the pseudopodia could be inhibited by treatment with Rho-kinase inhibitor, Y27632. Further investigation of the involvement of Rho/ROCK signaling in the pseudopodia demonstrated local activation of RhoA. This activation occurred preferentially to the activation of another RhoGTPase family member, Racl . However, Racl activation could be reversed with Y27632 treatment indicating communication between signaling components in the pseudopodia. Photobleaching of actin and mRNA in the pseudopodia in the presence of Y27632 showed a decreased recovery time for mRNA but not actin to the bleached regions. This supports a role for Rho-kinase in pseudopodial mRNA translocation. Microarray analysis of the pseudopodial fraction disclosed the presence of a number of upregulated mRNAs. In situ hybridization with anti-sense oligonucleotides to P-actin, RhoA, Shp-2, m-ras and Arp2/3 p41 subunit confirmed the presence of these mRNA in the pseudopodia. Active transport of mRNA via the cytoskeleton to intracellular domains has been documented in a number of cell types. It was shown here by treatment with nocodazole that mRNA localization to M S V - M D C K - I N V cell pseudopodia occurs independently of microtubules. Therefore the M S V - M D C K - I N V tumor cell model displaying distinct i i actin rich pseudopodial domains is characterized by distinct protein and mRNA complements. The regulation of these domains is mediated by local activation of a Rho/ROCK signaling pathway that contributes to mRNA localization in a microtubule independent manner. Table of Contents Abstract i i Table of Contents • iv List of Figures v List of Illustrations vi List of Abbreviations • vii Acknowledgements ix Dedication x CHAPTER 1 Introduction 1 1.1 Tumor cell motility and invasion 1 1.2 Actin microfilaments and microtubules as a driving force in cell motility. ..11 1.3 RhoGTPases as mediators of cell protrusion and motility 27 1.4 mRNA localization: a component of cell polarity 43 1.5 Summary ; 53 CHAPTER 2 Results 54 2.1 Materials and Methods 54 2.2 Results 60 CHAPTER 3 Discussion 80 Bibliography 97 iv List of Figures Figure 1. Tumor cell pseudopodia grow through 1 pm pore filters allowing for their isolation and characterization.. 61 Figure 2. Immunofluorescence labeling of proteins identified in actin-rich pseudopodia of M S V - M D C K - I N V cells 63 Figure 3. mRNA localizes to M S V - M D C K - I N V cell pseudopodia in a ROCK-dependent manner 65 Figure 4. RhoA is selectively activated in the pseudopodia of M S V - M D C K - I N V cells 69 Figure 5. Actin turnover in M S V - M D C K - I N V cell pseudopodia is R O C K independent 71 Figure 6. mRNA turnover in M S V - M D C K - I N V cell pseudopodia is R O C K dependent. 72 Figure 7. M S V - M D C K - I N V cell pseudopodia contain a distinct mRNA complement 74 Figure 8. mRNA translocation to the pseudopodia is microtubule independent in M S V -MDCK-INV cells 78 List of Illustrations Illustration 1. Organization of actin and microtubule cytoskeleton in cellular protrusions 8 Illustration 2. (3-Actin-rich pseudopodia of M S V - M D C K - I N V cells are highly blebbed as visualized by scanning electron microscopy 9 Illustration 3. The Rho GTPase cycle 28 Illustration 4. RhoGTPase-regulated pathways affect actin filament organization 31 Illustration 5. RhoGTPases regulate microtubules 38 Illustration 6. Basic structure of the GFP-based FRET probe for Rho GTPases (Raichu-Rho) 67 Illustration 7. Acceptor photobleaching FRET. Bleaching of the acceptor yields increased emission from the donor 68 vi List of abbreviations +TIPS - Positive end associated microtubule associated proteins y-TuRC - y tubulin ring complex ABP - Actin binding protein ACP - Actin crosslinking proteins ADF - Actin depolymerizing factor ADP - Adenosine diphosphate AP-1 - Activator protein APC - Adenomatous Polyposis Coli Arp - Actin related protein ATP - Adenosine triphosphate Cc - Critical concentration CFP - Cyan fluorescent protein CLIP-170 - Cytoplasmic linker protein dhMotC - Dihydromotoporamine DIG - digoxigenin DLC1 - Deleted in liver cancer DRP - Diaphanous related formin dsRBD - Double stranded R N A binding domain EB1 - End binding EBS - Elongation factor binding site E C M - Extracellular matrix E F l a - Elongation factor EGFP - Enhanced green fluorescent protein EMT - Epithelial mesenchymal transition Ena/Vasp - Enabled/Vasodilator-stimulated phosphoprotein ER - Endoplasmic reticulum E R M - Ezrin, Radixin, Moesin F-actin - Filamentous actin F A K - Focal adhesion kinase FH - formin homology FRAP - Fluorescence recovery after photobleach FRET - Fluorescence resonance energy transfer G-actin - Globular actin GAP - GTPase activating factor GDI - Guanine nucleotide dissociation factor GDP - Guanine diphosphate GEF - Guanine nucleotide exchange factor GFP - Green fluorescent protein GTP - Guanine triphosphate HGFR - Hepatocyte growth factor receptor INV - Invasive IQGAP - named for its homology to GTPase activating proteins (GAPs) and its IQ (calmodulin binding) motifs IRS-58 - insulin-receptor-substrate-58 K L C - Kinesin light chain L I M K - Lin-1 1, Isl-1 and Mec-3 kinase L P A - Lysophosphatidic acid M A P - Microtubule associated protein MBP- Myelin basic protein M D C K - Madin-Darby canine kidney mDia - mammalian diaphanous related protein M f - Mobile fraction MLC2 - Myosin light chain M L C P - Myosin light chain phosphatase M M P - Matrix metalloprotease M R C K - Myotonic dystrophy kinase-related cdc42 binding kinase mRNA - Messenger ribonucleic acid M S V - Maloney sarcoma virus MTOC - Microtubule organizing centre NPC - Nucleoporin complex N-WASP - Wiskott-Aldrich-syndrome-protein P A K - p21 activated kinase Pi - Phosphate PI - Phosphoinositide PIP2 - Phosphatidylinositol 4, 5-bisphosphate RNP - Ribonucleoprotein R O C K - Rho kinase rRNA - Ribosomal ribonucleic acid SCAR - Suppressor of cAMP receptor TIMP - Tissue inhibitors of metalloproteases UTR - Untranslated region W A V E - WASP-family verprolin homology protein YFP - Yellow fluorescent protein ZBP - Zipcode binding protein Acknowledgements I would like to acknowledge my supervisor, Dr. Ivan. R Nabi, for providing me the opportunity to work in his lab and for his constant guidance and encouragement throughout my research experience. As well, Zongjian Jia, for his instruction and patience while I continued to learn. I would like to thank the past and present members of the Nabi lab: Thao Dang, Jacky Goetz, Barhat Joshi, Liliana Kojic, Patrick Lajoie, Pascal St. Pierre and Nathalie Y for their humor, advice and friendship. I would like to acknowledge my committee members: Dr. Aly Karsan, Dr. Calvin Roskelley, Dr. Linda Matsucchi and Dr. Tim O'Connor for their advice and encouragement. Dedication I would like to dedicate this thesis to my family. My parents and brothers have always had faith in me. Their patience and encouragement has been a constant source of support. Thanks guys! x Chapter 1 Introduction 1.1 Tumor cell motility and invasion Cancer on a global scale The effects of cancer are felt worldwide. Uncontrolled proliferation and metastasis are the denning characteristics of cancer and it is these traits that are accredited with the increasing number of cancer associated deaths reported each year. In Canada alone in 2005, 149 000 new incident cases were diagnosed and 69 500 deaths were recorded. It is estimated that approximately 1 in 4 Canadians will eventually die of cancer (NCIC, 2005) . The severity of the disease increases with the diversity of cancers that arise. Lung cancer remains the leading cause of cancer associated deaths and when combined with prostate/breast (in male and female populations respectively) and colorectal cancer these cancers account for 50% of cancer cases in Canada. The severity of different types of cancer can be measured crudely be dividing the number of deaths by the number of diagnosed cases. Using these criteria some of the most fatal cancers include pancreatic, lung and stomach cancer. Fortunately, pancreatic and stomach cancer comprise only 2.2 and 1.9% of cancer in Canada, although lung cancer accounts for 29%. The differences between sites of origin for cancer do not stop with their incidence and severity; there are many factors affecting to cancer growth and development, starting from small changes in cellular gene expression and progressing dynamic interactions between a tumor and its individual host cell environment. The variation in severity amongst different cancers, can be attributed to the ability of a tumor cell to migrate to sites of the body separate from the site of origin and proliferate. Cancers with high mortality rates accomplish this most quickly. Therefore, 1 different cancers can be sorted according to their differential metastatic potentials or their ability to migrate to and grow at sites remote from to the original tumor. The mechanisms governing the motility of tumor cells that are highly metastatic are under intense investigation in order to better understand how metastasis occur and how they can be prevented. Modes of tumor cell motility The distinguishing factor between benign and metastatic tumors is the ability of cancer cells to become displaced from their site of origin and proliferate in a new environment. Therefore the study of how tumor cells move and the players regulating movement is very important. This research is complicated because there are several methods of movement employed by cancer cells and the plasticity of tumor cell migration allows cells to change their mode in response to environmental cues. The complexity of studying tumor cell migration arises from the multiple modes of cell motility that are each regulated by distinct pathways and affected uniquely by the external environment. The following will outline several modes of motility used by tumor cells during migration including: mesenchymal (directed), amoeboid (non-directed and protease independent) and collective (cell contacts maintained) movement (Sahai, 2005). These varying modes of migration are dependent on the development of specific cellular structures, such as protrusions, that promote movement by interacting with the environment. Protrusions are unique and often cell type specific and include filopodia, lamellipodia, pseudopodia and ruffles (Buccione et al., 2004). 2 A basic outline of the steps involved in migration include: polarization and elongation of the cell, development of leading edge protrusion, attachment to the extracellular environment or matrix, contraction of the cytoskeleton and development of tractile force in the cell body and finally retraction of the lagging edge (Abercrombie et al., 1977; Friedl and Brocker, 2000; Lauffenburger and Horwitz, 1996). There are, however, a number of factors that influence each step in migration and lead to the characterization of several distinct modes of motility. Integrins are transmembrane proteins that work to attach the extracellular matrix (ECM) to the outside of the cell and are essential for generating contractile force over the cell body. They can form clusters within the cell membrane called focal complexes, and when further stabilized these complexes develop into focal contacts (Burridge and Chrzanowska-Wodnicka, 1996; Hynes, 2002; Zamir et al., 2000). Integrins contribute to migration in several ways. Firstly, the integrin expression in the cell has an affect on the adhesive properties of the cell. For example, certain types of cancer, such as human lung carcinomas express low levels of some integrins, specifically pi and P3 (Falcioni et al., 1994), which facilitates an amoeboid type of cell migration. Secondly, integrins are present as heterodimers (Hynes et al., 1989), and as such require a set of subunits to comprise the complete protein. There are 18 a and 8 P subunits that can be paired to form integrins with distinct properties (Hynes, 2002). For example, a5pi binds selectively to fibronectin substrates (Cukierman et al., 2001), whereas a2p2 binds with specificity to collagen (Maaser et al., 1999). Therefore, tumor cell motility is affected by (1) the presence of integrins and (2) the type of integrins present. 3 Another factor that affects cancer cell motility is the presence or absence of extracellular proteases. Once activated, integrins can recruit proteases to aid in the degradation of the E C M , which facilitates the cell's movement through extracellular space. For example, a2pi binds to matrix metalloprotease-1 (MMP- l ) and confines it to points of cell contact with the E C M (Dumin et al., 2001). However, proteases are not necessary for all types of migration and as such become a defining characteristic of certain modes of motility. Other factors that contribute to a distinct mode of motility are the type of cell and the cell morphology. The following describes the established types of migration and how differential regulation of each may facilitate movement in select environments. The best known mode of motility is developed when cells transform from an epithelial to a mesenchymal phenotype and is therefore termed mesenchymal migration. This method is significant as 10-40% of carcinomas undergo epithelial-mesenchymal transition (EMT) (Thiery, 2002). However, most cells that utilize mesenchymal migration arise from connective tissue tumors, such fibrosarcomas (Wolf et al., 2003) or gliomas (Paulus et al., 1996). Cell morphology is elongated and highly polarized (Friedl and Wolf, 2003). This is a consequence of PIP3 induced Rac activation at the leading edge that stimulates actin polymerization and cell protrusion (Ridley et al., 2003). Focal contacts form at the tip of the protrusions and attach the cell to the E C M in an integrin dependent manner. As a result, cell motility and invasion requires degradation of the E C M by secreted proteases such as MMPs (Friedl and Wolf, 2003). Typically, Rho activation is limited to the lagging egde where it is involved in membrane retraction (Ridley et a l , 2003). 4 Mesenchymal cell motility is relatively slow, at speeds of approximately 0.1-1 pm/min (Zhang and Vande Woude, 2003). A second type of motility as described by Sahai (2005) is amoeboid motility. This type of movement has been liken to the Rho/ROCK dependent movement of tumor cells described by Sahai and Marshall (2003). Cells have a rounded morphology, but external stimulation leads actin polymerization at the leading edge and pseudopod formation. Studies in the mammary adenocarcinoma cell line, MTLn3, show that this process is dependent on cofilin, but Rho/ROCK signaling is likely involved in actin remodeling (Ghosh et al., 2004). Amoeboid motility is not blocked by integrin inhibition (Hegerfeldt et al., 2002) and is independent of protease secretion (Wolf et al., 2003). In contrast to mesenchymal motility, the speed of amoeboid motility is moderately fast, as approximately 4um/min. (Wyckoff et al., 2000). Finally, collective motility is characterized by clusters or sheets of tumor cells moving together. This form of motility has not been studied to the same extent as the mesenchymal and amoeboid modes of motility, but it has been described in lobular breast carcinomas and some ovarian carcinomas (Pitts et al., 1991; Sood et al., 2001). This mode of motility would be similar to a collective E M T in which adherence junctions are maintained and cells do not become isolated (Friedl, 2004). Therefore, there are a number of methods using by migrating cells to invade. Each method is assoicated with a particular signaling pathway that can optimize cell migration by modulating cell morphology and reponse to external stimuli. The problem facing scientists today is that tumor cells can switch their mode of motility by changing the dominant signaling pathway. For example, cells can switch between Rho and Rac 5 mediated signaling (Sander et al., 1999). This is an adaptive response that facilitates cell movement in a range of microenvironments (Sahai and Marshall, 2003; Wolf et al., 2003). Tumor cell protrusions A prerequisite for all moving cells is that they develop polarity, meaning distinct functionally specific regions, usually caused by the asymmetric distribution of proteins and mRNA within the cell. There are a number of membrane protrusions that form as a result of polarization and are associated with tumor cell motility. The following text provides a description of four types of protrusions, their functional roles and their regulatory mechanisms. Lamellipodia are large, broad membrane protrusions containing dense actin meshworks that are found on the leading edge of migrating cells (Illustration 1). The meshworks are formed by the coordinated efforts of a number of actin binding proteins that allow for filament branching rather than bundling. Lamellipodia; are characterized by having a width of approximately 1 -5 pm and may contain radiating actin filaments that protrude both within the lamella and beyond it. For purposes of definition, F-actin bundles within lamellipodia are referred to as microspikes, while those protruding past the cell edge are termed filopodia (Small, 1988). Lamellipodia are considered to be a primary site for actin polymerization (Glacy, 1983) and play a significant role in developing the polarity necessary for cell motility. They are also involved in substrate adhesion and, when they are associated with ruffling, lamellipodia contribute to nutrient uptake through macropinocytosis and phagocytosis (Bar-Sagi and Feramisco, 1986). 6 Another category of protrusive structures is termed filopodia (Illustration 1), which exhibit long, slender, finger-like protrusions. They are composed of bundled actin filaments, which form structures 0.1 pm wide and normally 5-35 pm long, however, extensions can reach up to 70 pm. Filopodia are extremely dynamic and can protrude and retract at a rate of 10 prn/min or faster during intense activity (Gustafson and Wolpert, 1961). They are found at the leading edge of migrating cells and are often linked by a webbing of lamellipodial sheets (Mitchison and Cramer, 1996). In addition to sensing the external environment of the E C M and neighboring cells, filopodia function as important adhesion components (Vasioukhin et al., 2000). Both a-actinin and E-cadherin are recruited to the tips of filopodial protrusions and are necessary for the formation of adherens junctions. The formation of adherens junctions between cells can be stimulated by calcium induced filopodia that penetrate into neighboring cells. Proteins such as a-actinin, catenins and E-cadherin are recruited to the tips of filopodia where they are necessary for solidifying cell-cell contact (Vasioukhin et al., 2000). Among the array of membrane protrusions is the well documented, but largely unexplained phenomenon of cellular blebbing. Blebs are characterized as round, knob-like structures that form transiently on the membranes of cells (Figure 2). This occurs when a cell has become round in an attempt to engage in a specific cellular process. For example, blebbing has been observed; in cultured cells during mitosis at the beginning of cytokinesis (Boss, 1955), during both necrotic and apoptotic cell death (Kerr et al., 1972; Trump and Berezesky, 1995) and at the leading edge of cells during locomotion (Trinkaus, 1973). Although blebs have been described in conjunction with a number of 7 lame Hi podia ruff les actin-rich region f i l o pod i a a c t i n bundles actin meshwcrk cenuceome MTOC) focal adhesion stress fibers *l p r o t r u s i o n Illustration 1. Organization of actin and microtubule cytoskeleton in cellular protrusions. (Etienne-Manneville, 2004). cellular phenomena, there is little known about their functional significance or the signaling mechanisms regulating their appearance. It has been determined that the amount of F-actin is inversely proportional to the number of blebs (Cunningham, 1995) and that bleb formation is caused by changes in intracellular hydrostatic pressure (Fedier and Keller, 1997). As such, pressure variations can in part be attributed to the state of actin, microtubules and intermediate filaments within the cell (Hagmann et al., 1999). More recently, a Rho/ROCK/p38MAPK dependent bleb pathway has been observed. The inhibition of each of these signaling molecules leads to a decrease in bleb formation 8 (Jia et al., 2006; Pfeiffer et al., 2004). The transient nature of blebs makes this type of protrusion a difficult target to study. However, the use of live cell imaging will help in elucidating the cellular significance of such structures. Illustration 2. p-Actin rich pseudopodia of MSV-MDCK-INV cells are highly blebbed as visualized by scanning electron microscopy. Scale bar 1 um (Nguyen et al., 2000) The term 'pseudopodia' means 'fake feet' and with this definition many membrane protrusions can be classified as pseudopodia. However, most distinct protrusive domains have specified names that distinguish them (eg. lamellipodia, filopodia, invadopodia, etc.). Membrane protrusions that are dissimilar to other previously described protrusions are often referred to generally as pseudopodia, even i f they exhibit distinctive characteristics. Thus, pseudopodia has been used loosely to describe a variety of membrane protrusions (Cardone et al., 2005), with no defined characteristics associated with this name. Therefore, the term pseudopodia, for the purposes of this thesis, will refer to Rho-dependent protrusions that are characterized by actin rich densities at the tip of membrane extensions. The following continues to define pseudopodia as they apply to our cell model. Pseudopodia are temporary protrusions that extend and retract from the membrane during cell motility. They have been previously characterized in an invasive variant of transformed canine kidney cells (MDCK) which demonstrate multiple pseudopodial domains (Nguyen et al., 2000). The pseudopodia are finger-like extensions of the cell membrane that display distinctive membrane blebs (Illustration 2). The formation of pseudopodia is regulated by a c-Met/HGFR (Hepatocyte growth factor receptor) feedback loop that is constitutively active via an autocrine stimulation pathway (Vadnais et al., 2002). Effectors of c-Met involved in pseuodpodial regulation include both Rho, of the Rho family of small GTPases and its downstream kinase, ROCK. (Jia et al., 2005; Jia et al., 2006). The Rho/ROCK pathway has been shown to regulate the localization of mRNA to the pseudopodia (Jia et al., 2005) and this phenomenon will be a major cellular event explored in this thesis. 10 1.2 Actin and Microtubules as the driving force behind cell motility Actin Composition and Polarization Actin plays a vital role in cell motility and is the most abundant protein found in eukaryotic cells. It comprises 14% (Yates and Greaser, 1983) of muscle, 1-5% of non-muscle by weight and is found at an intracellular concentration of approximately 0.5 mM (Pollard and Cooper, 1986). Actin contains 375 residues and is part of a large and highly conserved gene family (Vandekerckhove and Weber, 1978b), demonstrated by the observation that actin genes from amoeba and animals are 80% homologous. Humans carry six genes encoding various isoforms of actin (Vandekerckhove and Weber, 1978a; Vandekerckhove and Weber, 1978c), including four a actin isoforms found in muscle (Vandekerckhove and Weber, 1978b) and P and y isoforms found in non-muscle cells (Vandekerckhove and Weber, 1978c). a-actin is important in contractile structures (Pollard, 1986), while P-actin pools are found at the front of the cell where actin polymerization occurs (Otey et al., 1988). A closely related family of actin-like proteins is the Arps (Actin-related proteins). They are 50% homologous to actin and function primarily to stimulate actin assembly (Arp2/3) (Mullins et al., 1998), but have been shown to associate with microtubule motor proteins (ie. Arpl) (Schroer et al., 1996). There are two types of actin, G-actin (globular or monomeric) and F-actin (filamentous). G-actin subunits are free actin monomers that, when combined into a linear molecule, form F-actin. The combination of F and G actin can exist in four states, F-actin-ATP, F-actin-ADP, G-actin-ATP, and G-actin-ADP (Asakura, 1961). The most 11 common states are G-actin-ATP and F-actin-ADP, as these facilitate either actin polymerization or depolymerization respectively. The G-actin subunit contains two lobes that form a cleft between them that can bind a nucleotide and M g 2 + in the ATPase fold (Korn, 1982). The nucleotide that is bound (ie, ATP or ADP) affects the conformation of the subunit and subsequently either promotes or hinders the likelihood of becoming incorporated into F-actin. It is interesting to note that the addition of ions, specifically M g 2 + , K + or Na + , to a G-actin solution promotes the formation of F-actin (Rouayrenc and Travers, 1981). Conversely, diluting the same solution reverts the actin pool to primarily a G-actin state. This observation indicates that actin polymerization and depolymerization can occur independently of supplementary signaling molecules. Studies involving the different forms of actin have uncovered several substances that have proven useful in the determination of cytoskeletal form and function. For example, cytochalasin D blocks the addition of G-actin monomers at the positive end of a filament (Schliwa, 1982). Latrunculin blocks the addition of G-actin by binding to the monomer itself (Coue et al., 1987). Another commonly used substance is phalloidin, which binds the interface between F-actin subunits and prevents depolymerization (Lengsfeld et al., 1974). Since phalloidin only binds to F-actin, fluorescently-tagged phalloidin is very useful in labeling experiments aimed at identifying polymerized actin filaments. F-actin displays a characteristic polarity; meaning that all the subunits are oriented in the same direction. This allows for the assignment of positive and negative ends of the filament. The development of these poles (ie, through the process of actin polymerization) involves three steps; a lag period, followed by elongation and finally achievement of a steady state (Frieden and Goddette, 1983). The lag period involves the 12 aggregation of G-actin into short, unstable oligomers; an event called nucleation. This process is be time consuming, however, once 3-4 subunits have successfully joined together they can act as a stable nucleus for further polymerization. Once elongation occurs, the length of the filament grows quickly as monomers are added to both ends. Eventually filament growth reaches a steady state in which the rate of polymerization equals that of depolymerization. Therefore, there is no net increase or decrease in the length of the actin filament. The concentration of G-actin in the surrounding cytosol when one end of the filament has reached growth equilibrium is referred to as the critical concentration (Cc) (Kasai et al., 1962). A different Cc is usually assigned to the positive and negative ends of the filament as the positive end allows polymerization to occur at a faster rate than the negative pole (Wegner, 1976). Elongation can occur with both forms of G-actin (ie. ATP or ADP associated), however, the rate is increased in the case of G-actin-ATP. Additionally, although not essential for polymerization, ATP is usually hydrolyzed to ADP + Pi when G-actin-ATP is added to a filament. When steady state equilibrium has been reached, the concentration of G-actin in the cellular environment is usually between the Cc of the positive end and the Cc of the negative end. Therefore, subunits are constantly added to the positive end and subtracted from the negative end, a phenomena known as treadmilling (Kirschner, 1980). Treadmilling is often observed in motility structures such as lamellipodia. Using FRAP (fluorescence recovery after photo-bleaching) techniques researchers have observed the constant recycling of GFP-tagged actin to filament tips (Tyska and Mooseker, 2002). 13 Actin dynamics Once actin filaments have formed they can develop into two different structural arrangements within the cell, namely, bundles or networks (Edds, 1979). Bundles occur when filaments are tightly packed together and oriented in parallel (Buckley et al., 1981; Byers and Fujiwara, 1982; Wong et al., 1983). Many types of protrusions are formed from bundled filaments. These are often characterized according to the number of filaments per bundle, which corresponds to the width of the protrusion, and the length of the filaments. Such identifying properties are typically correlated with the number and type of actin binding proteins associated with the filaments. Networks are composed of loosely packed filaments that form crisscrossed arrays (Wessells et al., 1971), often at angles perpendicular to each other. Branch points that are at an angle of 70° or less are called Y junctions, whereas angles of >70° are called X junctions (Schliwa and van Blerkom, 1981). The filaments are commonly shorter than those found in bundles, but can exist tens of nanometers apart from others in the same network. As mentioned in the description of actin bundles, many filament interactions are dictated by the actin binding proteins (ABPs) they are associated with. For example, the presence of actin-related protein (ARP) 2/3 is frequently observed at Y junctions (Svitkina and Borisy, 1999), while filamin A often functions as a stabilizing protein in the X junctions of lamellipodia (Flanagan and al., 2001). There are two types of sub-networks, generally referred to as cortical and cytosolic actin networks (Ponti et al., 2004). The cortex is a narrow zone juxtaposed to the plasma membrane that houses a planar, web-like actin network. Cortical networks differ substantially between cell types, for exarnple, between erythrocytes (Lux, 1979), 14 platelets (White, 1984), epithelial (Asch et al., 1979) and muscle cells (Jockusch, 1983). The cytoplasmic actin networks show more homogeneity between cells and appears similar to a three dimensional gel. As previously mentioned the filaments in any of the cytoskeletal arrangements are held together by actin crosslinking proteins (ACPs) or ABPs (Dubreuil, 1991). The length and flexibility of these proteins determines whether actin filaments form into bundles or networks. For example, short proteins hold the filaments together closely and form bundles, whereas longer proteins allow for the looser, more random organization seen in networks. Actin binding proteins There are an extremely large number of proteins involved in the cytoskeletal dynamics that lead to cell motility. A small number of them will be described below, and many of those remaining can be classified based on functional generalizations made regarding protein families. Many ABPs, such as fimbrin or spectrin, belong to the Calponin homology domain (CH-domain) family. The members of this family all contain an actin binding domain that is homologous to calponin, a muscle protein. Fimbrin (Bretscher, 1981; Glenney et al., 1981a) and a-actinin (Maruyama and Sakai, 1981) of the C H domain family are examples of short ABPs while filamin, spectrin and dystrophin are examples of longer ABPs. In addition to enabling networks of actin filaments, proteins such as filamin (Pavalko et al., 1989) or dystrophin (Ervasti and Campbell, 1993a) can function to crosslink actin networks in the cytosol to the plasma membrane through interactions with various membrane proteins. Another protein family that can carry out similar 15 functions is the ERJVI (Ezrin, Radixin, Moesin) family, which, upon activation by phosphorylation, can crosslink microfilaments to membranes (Berryman et al., 1993). Not only does this allow actin to interact with cellular membranes, but filaments can be directly or indirectly connected to the extracellular matrix (ECM) through interactions with transmembrane proteins. Interestingly, the distribution of E R M proteins has been found to differ between tissues, for example between epithelial and endothelial cells (Berryman et al., 1993), indicating that they carry a tissue specific functionality. Gelsolin is unique in its ability to engage in both an actin capping and severing events (Janmey et al., 1985; Yin and Stossel, 1979). Upon calcium binding, an actin binding site is exposed that allows gelsolin to bind to actin filaments. This interaction between gelsolin and actin, weakens the association between actin and filamin, and allows breaking or severing of the filaments. Gelsolin maintains its binding partnership with actin following a break and forms a cap which discourages further elongation of the filament and facilitates pointed end depolymerization (McGough et al., 2003). Filamin, however, is not completely released from the actin filament until it can interact with signaling molecules such as, phosphatidylinositol 4,5-bisphosphate (PIP2) or lysophosphatidic acid (LPA) (Janmey and Stossel, 1987; Yamamoto et al., 2001). The dissolution of actin filaments increases actin turnover and is thought to be an initial step in changing cell morphology. A n example of how the above interactions take place in functional environment is described in the following sequence. In quiescent cells, gelsolin is bound to PIP2 at the plasma membrane. Upon stimulation by an external stimuli, this association is severed followed by an increase in the cytosolic calcium concentration (Chou et al., 2002). Following its release, gelsolin is able to interact with 16 actin and promote cell motility by increasing actin turnover (Chellaiah et al., 2000; Witke etal., 1995). Vil l in (Glenney et al., 1981b) is another ABP found in the same family as gelsolin, which also possesses capping and severing capabilities. The intriguing feature regarding such dual functionality is that switching between capping and severing is regulated by the intracellular calcium concentration. For example at high calcium concentrations villin functions as a severing protein and conversely at low concentrations as a capping protein (Bretscher and Weber, 1980). The phosphorylation of villin, however, has veto power over its capping function, as even at low calcium concentrations villin acts as a severing protein (Kumar et al., 2004). The presence of PIP2 inhibits both the severing and capping abilities of villin and facilitates the bundling of actin filaments (Kumar and Khurana, 2004) The development of different types of protrusions has been the focus of many studies. For example, it is thought that filopodia arise from a pre-existing lamellipodial actin network where extending filaments are prevented from capping. This can be facilitated by such protein families as the Ena/Vasp (Enabled/Vasodilator-stimulated phosphoprotein) family, which binds barbed ends and prevents them from being capped (Bear et al., 2002). Therefore, as the filaments elongate they collide and are bundled together (Svitkina et al., 2003) by proteins such as fascin. The bundling of actin at this stage leads to a polarization of the filaments, because a-actinin is found at the rear of the bundle while fascin is found along its length. Such polar properties contribute to the strength and stiffness of the bundle (Yamashiro et al., 1998). 17 In summary, actin dynamics are regulated by a number of cellular proteins. Actin filament treadmilling events implicating the simultaneous polymerization and depolymerization of actin filaments are responsible for the development of membrane protrusions involved in cell motility. Therefore research into the temporal and spatial regulation of actin microfilaments is important in studying tumor cell migration. Microtubules Composition and Polarization Another major component of the cytoskeleton is microtubules. In combination with actin, microtubules are vital to cellular processes such as; cell division, migration, vesicle transport and cellular polarization. The full role for microtubules in cell motility is still being elucidated. They are necessary to provide a protrusive force on the membrane, however, they are not observed in motile structures, such as filopodia and only minimally in lamellipodia. The following text provides an outline of microtubule structure and localized regulation, while attempting to reference the global mechanisms coupling microtubule dynamics to cell motility. A microtubule is a cylindrical structure, approximated 24 nm in diameter, which is composed of repeating subunits and capable of elongating and shortening in response to intracellular cues. The building blocks of microtubules include three tubulin monomers, a, p, and y, which are encoded by separate genes. Dimers composed of a and P monomers are the main subunit of microtubules, while y monomers are involved in nucleation events and are preferentially located in the microtubule organizing centre. The ap-tubulin dimer contains two GTP binding sites. One found on a tubulin, which binds 18 GTP irreversibly and the other on P tubulin, which binds GTP reversibly and is capable of hydrolyzing it from GTP to GDP. Following hydrolysis, GDP can be replaced on the P tubulin binding site by GTP (Erickson, 1998). At any point during cellular lifespan there are at least two pools of microtubules. An unstable form involved in dynamic instability, and having a half life between 5-10 min (Saxton et al., 1984; Schulze and Kirschner, 1986), and the stable form associated with microtubule stabilization, having a half life of several hours (Webster et al., 1987). The stabilized form helps initiate polarization by orientating the cellular polarity axis with respect to the direction of migration (Bulinski and Gundersen, 1991). There are a number of post translational modifications that occur on tubulin subunits that contribute to various aspects of microtubule stability and confirmation. The most common alterations include; detyrosination (Argarana et al., 1978), acetylation (L'Hernault and Rosenbaum, 1985a), phosphorylation (Eipper, 1972), palmitoylation (Caron, 1997), polyglutamylation (Edde et al., 1990) and polyglycylation (Redeker et al., 1994). Detyrosination involves the a-tubulin subunit becoming detyrosinated to produce a form of tubulin termed Glu-tubulin. This results in the reversible loss of the C-terminal tyrosine residue, which exposes a glutamate residue that acts as the new C-terminal residue (Idriss, 2000). This novel end residue confers an added stability to the protein and provides a selective advantage for certain interactions. For example, the motor protein, kinesin, preferentially binds to the Glu form of tubulin (Liao and Gundersen, 1998). It is important to note that the presence of Glu-tubulin does not mean 19 that microtubules are stable, but rather that stable microtubules recruit the Glu-tubulin form (Cook et al., 1998; Khawaja et al., 1988). The addition of an acetyl group, acetylation, to amino acid residues is now recognized in a variety of proteins (Yang, 2005). With regards to tubulin, specifically a tubulin, acetylation has been found to occur in the 8 amino group of internal lysine residues (L'Hernault and Rosenbaum, 1985a). Aceytylation has been described in motility structures such as flagella. It was demonstrated that acetylation of tubulin occurs in the flagellar matrix during motility and deacetylation in the cell body following reabsorption of the flagella (L'Hernault and Rosenbaum, 1985b). However, the exact role of this modification has yet to be determined, apart from correlation studies showing that stable microtubules tend to accumulate a population of acetylated a tubulins (Palazzo et al.,2003). Post-translational phosphorylation of proteins has been long established as a key regulatory mechanism within cells (Eipper, 1972). Tubulin is not an exception to this mechanism. The addition of phosphate groups to amino acid residues is facilitated by a large number of protein kinases including cAMP dependent kinases (Goodman et al., 1970), Ca 2 +/Calmodulin kinases (Burke and DeLorenzo, 1981), casein 1/2 kinases (Serrano et al., 1987; Singh et a l , 1984) and tyrosine kinases (Wandosell et al., 1987). Although both subunits act as phosphate recipients, serine residues on the P tubulin subunit are preferentially phosphorylated (Piras and Piras, 1974). While there is considerable evidence supporting the occurrence of tubulin phosphorylation events, information pertaining to the function of such a process needs to be expanded upon. However, phosphorylation most likely contributes to tubulin regulation in a similar 20 manner to its regulation of other proteins, for example, mediating binding interactions, complex assembly and protein localization. Palmitoylation is the covalent attachment of the long chain fatty acid, palmitate, to a protein of interest. The reaction is reversible and occurs preferentially at cysteine residues (Schlesinger et al., 1993). It occurs primarily to link cellular proteins to lipid membranes, including the plasma membrane, membrane bound vesicles and organelles (Cole and Lippincott-Schwartz, 1995). Since there are no substantial hydrophobic regions in tubulin (Cleveland and Sullivan, 1985) that would otherwise facilitate membrane interactions palmitoylation serves to promote this interaction. The reaction occurs on the a tubulin subunit and appears to be important in providing a link between microtubules and membrane structures that are necessary for cellular trafficking. Polyglutamylation, the progressive addition of glutamyl units to a protein, has been demonstrated in studies done in neuronal tissue. Specifically, glutamylation occurs on a glutamate residue located in the carboxy terminal domain of a tubulin. Although specific consequences of glutamylation have not been elucidated, glutamylated a tubulin comprises 40-50% of the total a tubulin in brain tissue, indicating a significant motive for this process. The glutamylated residue is located in a domain that is necessary for the binding of microtubule associated proteins and calcium ions (Littauer et al., 1986; Maccioni et al., 1988; Serrano et al., 1986), therefore glutamylation could be required for the regulation of these interactions. Initial assembly of microtubules occurs by the longitudinal addition of aP-tubulin dimmers in a head-to-tail arrangement to form protofilaments. Thirteen protofilaments then link together laterally to form a sheet that, upon circularization, becomes a 21 microtubule. Subsequent elongation occurs, however elongation of individual protofilaments can occur at different rates. For the most part microtubules exist as singlets, that is single cylindrical structures, however they have been found as doublets or triplets in cilia or flagella and centrioles or basal bodies, respectively. Like actin filaments, microtubules are polar structures and have designated positive and negative ends. The positive end is associated with fast polymerization, forming a microtubule that terminates with a B subunit (Baas, 1996). The negative end polymerizes very slowly and the terminating a subunit, in mammalian cells, is often anchored at the centrosome or MTOC (microtubule organizing centre). In a migrating cell, the negative ends are found in close association with the MTOC while the positive ends are oriented towards the cell periphery in the direction of cell migration. The MTOC is essential, not only for microtubule nucleation and organization, but for organization of microtubule associated structures such as mitochondria, the Golgi and the endoplasmic reticulum. The MTOC is perinuclear and in mammals, is composed of a lattice of microtubule associated proteins (MAPs) and a pair of centrioles. A centriole consists of a pinwheel array of triplet microtubules that are surrounded by proteins such as y tubulin and pericentrin (Zimmerman et al., 1999). The y tubulin monomers can combine to form a ring shaped structure termed the y tubulin ring complex (y-TuRC). This functions as a nucleation site for microtubule assembly and this process is so efficient that it can facilitate polymerization at ap-tubulin concentrations below the C c (C c has the same definition as when describing actin concentration). Centrioles are usually centered in the MTOC, but do not come in contact with the negative poles of cytosolic microtubules (Vaughn and Harper, 1998). 22 Microtubule dynamics A phenomenon, known as 'dynamic instability', occurs in microtubules that involves endless cycles of growth and shrinkage. While such an event can occur at both ends of a polarized microtubule, it preferentially occurs at the positive end for two reasons. First of all, the C c is lower at the positive pole enabling polymerization and depolymerization to occur at ap-tubulin concentrations less than that at the negative pole. This helps to ensure that, in the absence of external stabilizing factors, that the positive end is almost always engaging in dynamic cycling of aP-tubulins. Second of all, the P monomer is the terminating tubulin at the positive end, allowing the exposure of the exchangeable GDP-GTP binding site. When GTP is bound, stabilization or polymerization is favored, whereas when GDP is bound depolymerization is favored. The presence of a GDP bound tubulin subunit usually occurs for one of two reasons; (1) rapid depolymerization exposes a GDP subunit that was previously buried within the tubule or (2) the tubule is growing so slowly that GTP is hydrolyzed to GDP before another subunit can be added. The terms, 'rescue' and 'catastrophe', are used to describe the addition of a GTP bound tubulin subunit to the positive end to prevent depolymerization and the loss of a GTP bound tubulin subunit at the positive end to facilitate rapid depolymerization, respectively (Desai and Mitchison, 1997). This process of dynamic instability has been shown to occur as microtubules explore cellular compartments in response the signaling events (Kirschner and Mitchison, 1986). During migration, signals from growth factors and the extracellular matrix induce stabilization of the positive end of microtubules in specific locations within the cell. Stabilization can occur through various mechanisms including, the presence of a GTP cap or the binding of 23 microtubule stabilizing proteins. Such stabilization allows for the appropriate orientation of the MTOC and initial determination of the direction of movement. Therefore, dynamic instability and positive end stabilization are necessary for cell migration as they enable linkage of microtubules to the cell cortex and initiate cellular polarization in response to environmental triggers (Watanabe et al., 2005). Having introduced cytoskeletal dynamics with regards to actin filaments and microtubules, it is interesting to note the similarities and differences between these two structures. Microtubule polymerization is similar to that of actin filaments in several instances. Firstly, polymerization in both cases is dependent on the C c of the subunits involved. That is, at concentrations of actin and aP-tubulin above the C c , polymerization is facilitated, while depolymerization is favored at concentrations below C c . At concentrations similar to the C c there is a cycling of polymerization and depolymerization that does not result in any net change in filament/tubules length. Secondly, for both filamentous actin and microtubules, the C c is lower at the positive end. This still allows for polymerization and depolymerization at both poles, but enables both processes to occur at a faster rate at the positive end. Thirdly, nucleation can be facilitated in both cases from subfragments of previous filaments and tubules. For example, following a severing event, a small remaining aggregate of actin or tubulin subunits can serve as a nucleation site for future polymerization. The primary difference between actin filaments and microtubules with regards to their structural dynamics is their assembly process. Filament elongation occurs simply by the joining of G-actin-ATP monomers to a pre-existing filament. In comparison, aP-tubulin must combine longitudinally to form protofilaments, before thirteen protofilaments can 24 come together to form a cylindrical unit. It is only upon microtubule formation that elongation can occur. Microtubule associated proteins Microtubule associated proteins (MAPs) are proteins that interact directly with microtubules and contribute to their assembly, function and regulation. One main category is the assembly MAPs, which are responsible for cross-linking microtubules in the cytoplasm. Typically assembly MAPs are characterized by two important domains; a basic microtubule-binding domain and an acidic projection domain. The projection domain has been shown to be important in linking microtubules to other cellular structures, such as membranes or intermediate filaments, and regulating the spacing between interactions (Chen et al., 1992). Two types of assembly MAPs have been identified: Type 1 and Type 2. Type 1 MAPs, including M A P I A and M A P IB, are large filamentous proteins that have a lys-lys-glu-X amino acid repeat sequence that allows them to bind negatively charged tubulin (Noble et al., 1989; Vaillant et al., 1998). Such an interaction functions to neutralize repulsive forces between tubulin subunits and stabilize intra-microtubule associations. Type 2 MAPs are characterized by 3-4 repeats of an 18 amino acid sequence that functions as a microtubule binding domain (Doll et al., 1993). Included in Type 2 MAPs is MAP2 which is found mainly in dendrites and functions to facilitate crossbridge formation between microtubules and could potentially link microtubules to intermediate filaments (Johnson and Jope, 1992). Another Type 2 M A P is MAP4, which is found ubiquitously and serves to stabilize microtubules during mitosis (Shiina and Tsukita, 1999). Lastly 25 Tau, which consists of several isoforms stemming from splice variants, acts to crosslink microtubules into bundles mainly in neurons (Hirokawa, 1994). Although it will be discussed in more detail later on, it is of importance to remember that the regulation of MAPs is a potent factor in the regulation of microtubules. For example, upon phosphorylation some MAPs lose the ability to bind microtubules, leading to a loss of stabilization and subsequent depolymerization (Maccioni and Cambiazo, 1995). Another set of MAPs are localized to a specific area of microtubule structure, specifically the positive end and are therefore termed +TIPS. While not all of these will be discussed in detail the +TIPS include; CLIP-170, CLASPs, EB1, APC, dynein, dynactin and L i s l (Galjart, 2005; Galjart and Perez, 2003; Schuyler and Pellman, 2001; Xiang, 2006). One major function of these proteins is to sense and react to stimuli in localized cortical sites. In summary, microtubules are an important component of moving cells. Their dynamics not only promote the acquisition of asymmetric cell shape, but are part of a number of up and downstream regulatory pathways. Their ability to interact with microtubule associated proteins, become post-translationally modified and/or stabilized can impact many factors contributing to cell motility. Thus, a better understanding of microtubule dynamics will increase our knowledge about how tumor cells migrate. 26 1.3 Rho family GTPases as mediators of cellular protrusion and motility RhoGTPases and their effectors Rho GTPases The RhoGTPase family contributes significantly to the regulation of actin filaments and microtubules. It is a large family composed of several subsets of proteins. However, RhoA, Racl and cdc42 are the most clearly defined in regulating motility and will be the main focus of the Rho family discussion. RhoGTPases are small signaling molecules that cycle through activation and inactivation by the association with either GTP or GDP, respectively. They are regulated spatially and temporally by a number of proteins including, GEFs (guanine exchange factors), GDIs (guanine dissociation factors) and GAPs (GTPase activating proteins). Briefly, GEFs promote the conversion of RhoGDP to RhoGTP, GDIs inhibit the conversion of RhoGDP to RhoGTP and GAPs enhance the intrinsic hydrolyzing activity of GTPases (ie, conversion of RhoGTP to RhoGDP) (Illustration 3). In resting cells, Rho proteins are found mainly in the GDP bound form and in complexes with GDIs in the cytosol. Upon appropriate signaling, Rho can dissociate from GDI and relocate to the membrane via a C-terminal prenyl group. • Activation most likely occurs at or near the membrane through the help of GEFs. 27 G T P Illustration 3. The Rho GTPase cycle. (Raftopoulou and Hall, 2004) Once activated, RhoGTPases have the ability to regulate a number of proteins depending on their cellular localization. For example, RhoA plays a role in actin dynamics via actin effector molecules, leading to the assembly of contractile actin and myosin complexes and allowing for the contraction of the rear of the cell during migration. In resting cells, inactive RhoA is found mainly in the cytoplasm, however, some evidence suggests its presence in stress fibres (Katoh et al., 2001). RhoA is targeted to the membrane for activation in interphase cells, but its localization pattern in migrating cells has yet to be elucidated (Yoshizaki et al., 2003). In cells engaging in directional motility, Racl and cdc42 localize at the leading edge where they contribute to the polymerization of actin that results in lamellipodia and filopodia formation, respectively. It is of interest that, the largest concentration of cdc42 occurs immediately along the tip of the leading edge, whereas Rac concentration peaks just back from this (Itoh et al., 2002) and continues to decrease according to a gradient as 28 it nears the cell body (Kraynov et al., 2000). Cdc42 also localizes to the Golgi complex, possibly to help regulate secretory transport involved in mediating polarization. The leading edge of lamellipodia has been recognized as site where many proteins involved in actin polymerization are localized. This regional accumulation lends itself to the rapid development of membrane protrusion (Frischknecht and Way, 2001). Lamellipodial protrusion is associated with activation of the small GTPase, Racl . For example, stimulation by growth factors or integrin receptors (Hall, 1998) can induce post-translational modifications in Racl that allow it to translocate to the membrane (Michaelson et al., 2001) where it can become activated by upstream effector molecules (eg. GEFs) (Kranewitter et al., 2001). From here, the insulin-receptor-substrate-58 (IRS-58) protein can link activated Racl to S C A R / W A V E . S C A R / W A V E proteins are members of the WASP family of scaffolding proteins and have been shown to localize along the tips of lamellipodial protrusions (Hahne et al., 2001; Nakagawa et al., 2001). Additionally S C A R / W A V E is known to activate Arp2/3 (Machesky and Insall, 1998; Miki et al., 1998), which is a potent promoter of actin polymerization. The end result is the formation of lamellipodia (Miki et al., 2000) which facilitates motility in response to the original extracellular signal. A brief synopsis of the known signaling events involved in filopodia formation begins with the small GTPase, cdc42. The cdc42 protein, along with PIP2, binds to N -WASP (Wiskott-Aldrich-syndrome-protein) which activates Arp2/3. Actin-related protein (Arp2/3) facilitates nucleation of actin monomers and promotes polymerization of filaments found in filopodia (Takenawa and Mik i , 2001; Wear et al., 2000). This mechanism is known to modulate filopodia formation, however, functional N-WASP is 29 not required for their generation as noted in N-WASP knockout models (Lommel et al., 2001; Snapper et al., 2001). Interestingly, the protein, IRS-58, with a previously described function in lamellipodia, is also found in filopodia and may interact with cdc42 to regulate their formation in an Arp2/3 independent manner (Govind et al., 2001). This offers a possible alternative pathway for filopodia formation and could explain the normal phenotype of the N-WASP knockouts. Rho effectors Growth factor stimulation and adhesion complex stabilization are important large scale regulating events upstream of RhoGTPases. However, effector molecules are directly responsible for regulating RhoGTPases. Effectors of Rho include, but are not limited to: Rho kinase (ROCK), mDia and rhotekin (Illustration 4). Effectors of Rac include: P A K and IQGAP1; effectors of cdc42 include: P A K , WASP and IQGAP1 (Illustration 4). Experiments conducted to identify the above effectors include the use of lysophosphatidic acid (LP A) to induce the stabilization of microtubules. It was determined that RhoA, while not interacting directly with microtubules (Best et al., 1996), is necessary and sufficient for their stabilization (Cook et al., 1998; Nagasaki and Gundersen, 1996). The question was proposed as to what linked RhoA to microtubules during stabilization. The answer that became apparent was the effector molecule, mDia2. mDial and mDia2 are part of the DRE family (diaphanous-related formins) found originally in mice. Therefore, mDia was recognized as an effector of RhoA and was credited with inducing the stabilization of microtubules, possibly through a capping mechanism (Infante et al., 2000; Palazzo et al., 2004). 30 Rho p160ROCK LIMK M L C _ p h o s p h a t a s e cofllin F-uctin stabilization MLC phosphorylation uctimmyosin crossllnking mDia a c n n pol> n iL-ri/ . i l in i i S c a r / W A V E A r p 2 / 3 Rac ptfSPAK • LIMK • cofilin Cdc42 W A S p / N - W A S p + A r p 2 / 3 Illustration 4. RhoGTPase-regulated pathways affect actin filament organization. (Raftopoulou and Hall, 2004) Rac and cdc42 effectors Racl and cdc42 have effectors of their own that work independently and in association with effectors from other pathways. P A K is a specific effector of Racl and cdc42 that is activated in response to signals for cell migration. It can phosphorylate a serine residue on a protein called opl8/stathmin and deactivate it. The active form of opl8/stathmin contributes to microtubule destabilization (Daub et al., 2001; Wittmann et al., 2004). Therefore its inhibition leads to microtubule stabilization. This pathway is advantageous at the leading edge of migrating cells, where stabilization of pioneer microtubules decreases the frequency of catastrophe and increases the time allowed for growth of the tubule (Waterman-Storer and Salmon, 1997; Wittmann et al., 2003). 31 Therefore, Racl and cdc42 can function to initialize a P A K dependent pathway that can facilitate microtubule stabilization at the leading edge. Another effector molecule that is common to both Racl and cdc42 is IQGAP1. In addition to being a known actin-binding protein, IQGAP1 interacts with the +TIP protein, CLIP-170 in vitro (Zumbrunn et al., 2001). IQGAP1 localizes with actin at the leading edge of polarized cells, which is near to where CLIP-170 associated microtubules are localized. The interaction between IQGAP1 and CLIP-170 is enhanced by the activation of Racl and cdc42. This serves to provide directed docking sites for microtubule positive ends that are in close proximity to actin and the cell cortex (Zumbrunn et al., 2001). IQGAP1 also associates with APC, another +TIP, and functions to link actin and microtubules in specific cortical regions of the leading edge (Watanabe et al., 2004). The blockade of this interaction results in the disruption of actin networks, the hindering of the microtubule immobilization process and the prevention of M T O C polarization. Additionally, A P C interacts with and activates Asef, a protein with intrinsic GEF activity that is capable of recruiting and activating Racl (Kawasaki et al., 2000). Therefore, the Racl and cdc42 pathways that direct microtubule stabilization and localization, involve, but are not limited to the specific interactions that occur between M A P +TIPs; APC, CLIP-170 and IQGAP, the latter of which also binds to actin in similar locations. The amphipathic binding qualities of IQGAP 1 allow it to act as an adaptor between actin and tubulin association and regulation, with the potential for localized feedback loops specific for migratory patterns in the cell. The importance of positive end stabilization of microtubules has been discussed. However, the negative ends play a significant role in orientation and polarization of the 32 MTOC. This process is essential for cell migration and is largely regulated in a cdc42 dependent manner (Etienne-Manneville and Hall, 2003; Stowers et al., 1 9 9 5 ; Tzima et al., 2003). Dynein and dynactin, which are proteins associated with the positive end of tubules, are also involved in the reorientation of the negative ends of microtubules at the MTOC following signals for cell migration (Etienne-Manneville and Hall, 2003; Palazzo et al., 2001b). These two effectors can form a motor protein complex that directs the negative ends by generating a mechanical force. The force is generated at the positive end and is transferred to the negative end were it is used to pull the microtubules into place. This cdc42 dependent pathway has shown to enhance the polarization of the MTOC, however, the exact mechanisms by which this occurs is still to be determined. Regulation of cytoskeletal dynamics by Rho GTPases The manner in which the cytoskeleton is organized and arranged is most evident as cellular morphology, however the end result does not reflect the vast array of signaling events that are necessary for actin and tubulin dynamics. Signaling pathways involve the interaction of a large number of proteins, signaling molecules and other cellular material, each fulfilling a specific role such as phosphorylation, cleavage, or stabilization. The complexity of these pathways is amplified by factors like crosstalk and redundancy, which enhance efficiency and survival of a cell respectively, but leave a convoluted trail for scientists attempting to elucidate cellular mechanisms. The following text attempts to provide a summary of some of the major elements involved in cytoskeletal arrangement and how their interactions may lead to cellular changes. However, the vastness of this 33 field and the speed with which knowledge is being accumulated limits a complete and comprehensive account of cytoskeletal dynamics. Actin dynamics Actin binding proteins (ABPs) play an important role in the regulation of actin, however, it is the regulation of ABPs themselves that is the most complex. Post-translation modification, ion-binding and cofactor association are some of the methods involved in A B P regulation. Alterations to protein structure may result in a conformational change that reveals a site necessary for a specific binding interaction or possibly a change in energy state that allows for further activation or phosphorylation events. The complexity of signaling pathways is generated by a vast number of signaling molecules, the diversity of their actions and the redundancy that each can adopt in various cellular circumstances. The following describes a select number of signaling proteins, their proposed role in the cell and the consequences they render with regards to cell motility. p21-activated kinase (PAK) proteins are a family of serine/threonine kinases that have been identified as important targets for the RhoGTPases, Rac and cdc42. This relationship has been shown to be crucial in establishing directionality in cell movement (Sells et al., 1999), laying down cell adhesions and enhancing contractility within a cell (Kiosses et al., 1999). PAKs are localized to actin rich structures, such as pseudopodia and membrane ruffles, and the activated form, which is bound to active Rac or cdc42, can be found along the leading edge of migrating cells. An important target for P A K proteins is the L I M kinase family (Stanyon and Bernard, 1999). Once activated, P A K can 34 phosphorylate the L I M kinases, which can in turn phosphorylate ADF/cofilin. This latter phosphorylation event leads to the inhibition of ADF/cofilin (Carlier et al., 1999) and subsequently its severing ability of F-actin. Therefore, PAKs have been associated with elongation of F-actin filaments. Filamin has already been discussed as an actin binding protein that crosslinks filaments and can bind to additional proteins, such as transmembrane receptors (Hjalm et al., 2001) and small GTPases (Stossel et al., 2001). Filamin has been identified as a binding partner of the P A K family protein, PAK1. PAK1 is found downstream of Rho family proteins such as Rac and cdc42 and the induction of cellular polarization and cytoskeletal rearrangement has been associated with PAK1 activation (Sells et al., 1999). The interaction between filamin and P A K , following P A K activation, could function to (1) activate filamin through phosphorylation and (2) couple P A K signaling to actin filaments (Vadlamudi et al., 2002). The ADF/cofilin family plays a role in mediating actin dynamics and modulating the protrusions associated with cellular motility (Ghosh et al., 2004). Cofilin is activated by dephosphorylation of an internal serine residue, which leads to an increase in free barbed ends on actin filaments, F-actin content and in general, cellular locomotion (Ghosh et al., 2004). Following activation, cofilin is thought to contribute to the generation of lamellipodia and the determination of the direction of cell movement (Zebda et al., 2000). Traditionally the predicted mode of action of ADF/cofilin was as a depolymerization factor (Carlier et al., 1997), however, recently it has been linked to filament polymerization (Condeelis, 2001). While still controversial, evidence suggests a dynamic role for ADF/cofilin involving both polymerizing and depolymerizing 35 properties. Inactivation of ADF/cofilin occurs when it is phosphorylated by members of the L I M Kinase family. Interestingly, both of these proteins can function downstream of the Rho GTPase family of signaling molecules (Arber et al., 1998) which have been linked to lamellipodial protrusion through the stabilization of actin filaments (Sumi et al., 2001; Yang et al., 1998). Phosphoinositides (Pis) have been shown to localize to the leading edge of polarized cells (Funamoto et al., 2002). In this position they can directly contribute to membrane protrusion events by influencing actin binding proteins. In several cases they are associated with the inhibition of cell motility. For example phosphatidylinositol-4,5-bisphosphate (PIP2) can bind to and inhibit the severing/capping function of ADF/ cofilin (Ojala et al., 2001; Yonezawa et al., 1990). This minimizes actin filament dynamics thus limiting cell motility. Another example where Pis restrict cell movement is the dissociation of profilin from G-actin monomers in the presence of PIP 2. This slows the exchange of ADP for ATP on the actin monomers and the subsequent delivery to free filament ends (Goldschmidt-Clermont et a l , 1992). The result is a decrease in the rate of filament elongation and potential membrane protrusions (Lassing and Lindberg, 1985). Microtubule dynamics The assembly and disassembly of microtubules are important processes in the rearrangement of the cytoskeleton and the production of intracellular force. However, the development of stable microtubules holds similar importance to its dynamic counterpart. There are many factors that play a role in microtubule stabilization and the following text 36 attempts to outline the key determinants. A comprehensive review of all the components would exhaust the realms of information covered in this thesis. It has been observed that the positive ends of stabilized microtubules are oriented in the direction of cell movement along the leading edge, but the localization of mDia2 and RhoA is not limited to this area. Further investigation revealed that microtubule stabilization also appeared to be affected by substrate adhesion. It was determined that integrin mediated activation of F A K (focal adhesion kinase) was at least in part responsible for the association of mDia2 and RhoA. This association was observed in localized lipid raft domains in the plasma membrane and lead to the formation of Glu-microtubules (Palazzo et al., 2001a). mDial and mDia2 were previously known to associate with the +TIP proteins, APC (adenomatous polyposis coli) and EB1 (end-binding l)(Wen et al., 2004), thereby providing a continuum in which the regulation of Glu-microtubules can occur. The signaling cascade begins With integrin mediated F A K activation which leads to mDia and RhoA localization in lipid rafts. This facilitates the interaction of mDia with MAPs, APC and EB1, and the stabilization of microtubules; providing an appealing outline of some the events that occur during microtubule mediated cell migration (Illustration 5). Members of the DRF family have also been found to play a role in actin filament nucleation which would provide a link between actin filament and microtubule regulation downstream of RhoA during cytoskeletal rearrangement. 37 m i c r o t u b u l e s Illustration 5. RhoGTPases regulate microtubules. (Raftopoulou and Hall, 2004) Microtubule dynamics are not strictly limited to responding to upstream regulation. Rather they themselves can influence migratory signaling cascades that modify important proteins, such as those in the RhoGTPase family. For example, following the removal of nocodazole, a known microtubule depolymerizing agent, membrane ruffling is induced leading to the activation of Racl (Waterman-Storer et al., 1999). During the ruffling process, microtubules extend towards the membrane of these protrusions and activate Racl in manner that facilitates cell migration. Additionally, RhoA has been found to engage in cyclical feedback loops with microtubules. Following a depolymerization event, intracellular signals govern the development of contractile actin bundles and focal adhesions. These structures lead to the activation of RhoA (Ren et al., 1999), though the mechanism is largely unknown. The interaction between microtubules, effector molecules and regulatory proteins appears in part to be self-governing. Regulation occurs by engaging in positive and negative feedback loops in response to each other and environmental stimuli. 38 Rho GTPases and Invasion (Role for Rho/ROCK pathway) Rho GTPases have long been known to play an important role in tumor cell invasion. Traditionally, the cdc42 group of GTPases was responsible for filopodia formation which functioned to sense tactic environmental signals and guide cell directionality (Ridley, 2001). Rac GTPases contributed largely to the development of lamellipodia as a result of actin polymerization at the leading edge of polarized cells (Ridley et al., 1992). Rho GTPases, through the indirect phosphorylation of myosin light chains were able to induce in cells, acto-myosin contractility that is necessary for movement (Kimura et al., 1996). Traditionally RhoGTPase was thought to localize to the rear of migrating cells and help to regulate lagging edge retraction (Burridge and Wennerberg, 2004; Raftopoulou and Hall, 2004). However more recently its activated form has been shown to form a sharp band at the edge of protrusions and to localize only sporadically to the lagging edge (Pertz et al., 2006). Complementary data on myosin II shows differential localization of myosin IIA in protrusive regions and myosin IIB in the retracting tails of moving cells (Kolega, 2003). Our knowledge about the roles of RhoGTPases has expanded and it is evident that they contribute to tumor cell motility and invasion in many diverse ways that vary in spatial and temporal regulation and between cell lines. For example, while cdc42 has been associated primarily with regulating cell polarity, a study by Wilkinson et al. (2005) shows that it plays a role in facilitating invasion via downstream activation of M R C K (myotonic dystrophy kinase-related cdc42 binding kinase). M R C K activation functions in two ways, firstly, by phosphorylating and activating M L C 2 (myosin light chain) and secondly, by phosphorylating and deactivating 39 M L C P (myosin light chain phosphatase). Both of these effects lead to increased contractility and subsequent invasion in BE colorectal carcinoma cells. It is of note that the action of M R C K acts redundantly to that of R O C K (effector of RhoA) indicating a convergence between Rho and cdc42 signaling (Wilkinson et al., 2005). There have been discrepancies associated with Racl signaling and its effects on invasion. For example, it is known that activation of Rho GTPases can mediate the expression of genes necessary for invasion, but the effects of Racl have been shown to both promote and inhibit this consequence. The activation of Racl and Racl GEF, Tiaml, in renal carcinoma cells is associated with the upregulation of TIMP1 and TIMP2 (tissue inhibitors of metalloproteases) (Engers et al., 2001). These inhibitors of extracellular proteases slow the degradation of the basement membrane and subsequently tumor cell invasion. Conversely, the activation of Racl in fibroblasts, fibrosarcomas and lung cancer cell lines, has been shown to increase MMP1 (matrix metalloprotease) and MT1-MMP expression (Kheradmand et al., 1998; Soon et al., 2003; Zhuge and Xu, 2001). Thus, the increase in protease expression promotes the breakdown of the extracellular matrix and facilitates tumor cell invasion. Similarly, the activation of RhoA has been shown to correlate with both increased and decreased tumor cell invasion. The small molecule inhibitor dhMotC (dihydromotoporamine) inhibits tumor cell invasion in MDA-MB-231 cells (McHardy et al., 2004). This inhibition however, leads to increased RhoA activation, increased stress fibre and focal adhesion formation and an increase in the ROCK-dependent activation of the Na+/H+ exchanger. This evidence portrays RhoA as a factor associated with decreasing tumor cell motility. In other studies, the expression of RhoA and its activation 40 promote invasion and motility in rat MM1 hepatoma cells (Yoshioka et al., 1999). The explanation of this mechanism is such that the high RhoA expression facilitates protein translocation to the membrane, where its activation leads to local R O C K activation and acto-myosin contractility facilitating membrane protrusion and invasion. Therefore, the role of RhoGTPases in invasion is not clean cut, but the following presents a possible approach for categorizing the regulation of the Rho/ROCK pathway and correlating activation with differential invasive phenotypes. A study by Sahai and Marshall (2003) shows that differing modes of tumor cell invasion are differentially regulated by a Rho/ROCK signaling pathway. This paper suggests that there are two morphologies associated with tumor cell invasion. The first displays a rounded and blebbed phenotype that depends on Rho/ROCK signaling to promote motility (A375m2 melanoma and LS174T colon carcinoma). The second shows an elongated phenotype that invades independent of Rho/ROCK signaling (BE colon carcinoma and SW962 squamous cell carcinoma). A series of experiments demonstrated an increased Rho activation in blebbed cells compared to elongated cells and an observed decrease in motility when Rho was activated in elongated cells. When combined with in vivo studies that show decreased tumor cell invasion in the presence of R O C K inhibitor (Itoh et al., 1999), a strong argument is presented for differential regulation of Rho/ROCK signaling in distinct tumor cell phenotypes. An effort is, therefore, being made to categorize specific signaling pathways and correlate them with certain cell types and modes of motility. This may provide an advantage when determining appropriate treatment approaches for patients' diagnosed with cancer. However, the issue is complicated further by the ability of tumor cells to 41 switch between modes of motility (Sahai, 2005). Natural processes such as the migration of epithelial cells during development involve epithelial-mesenchymal transition (Ciruna and Rossant, 2001). Therefore, it is reasonable to tumor cells behave similarly and when faced with environmental stress can alter their mode of motility to best support their survival. This is demonstrated in instances where anti-cancer drugs targeting certain pathways have induced changes in cell motility that render the cell drug resistant (Wolf et al., 2003). This effect is a major obstacle in cancer therapy. However, by continuing to increase knowledge about distinct signaling pathways, we are able to more accurately treat and diagnose this disease. 42 1.4 mRNA localization: a component of cell polarity Importance of mRNA localization The first part of this thesis has focused on the structure and regulation of cellular proteins. However, as expressed in the central dogma, all proteins are translated from messenger RNA (mRNA) molecules in the cytoplasm. The intracellular protein distribution is not uniform and cell functionality relies on the localization of proteins required for specific functions. For example, P-actin mRNA is localized to the leading edge of fibroblasts to support the large requirement for actin polymerization in motile cells (Lawrence and Singer, 1986). A morphogen gradient is established by the localization of bicoid mRNA in drosophila oocytes and is necessary for the development of antero-posterior axis upon translation in the embryo (St Johnston et al., 1989). Therefore, the question remains as to whether proteins are translated prior to transport to their respective functional destinations or if mRNA is distributed to targeted sites for local translation. The answer most likely reflects a combination of both protein and mRNA localization. The importance of mRNA localization is evident in that it is a driving force for cell polarization and key mechanism in post transcriptional gene regulation (Bashirullah et a l , 1998). Therefore, the next paragraph describes what is known about mRNA translocation. Knowledge is limited to specific mechanisms as there is great diversity in form and function of R N A localization between species, cell types and mRNA sequences. A great deal of what we currently understand about mRNA localization has been developed in non-mammalian systems such as yeast (Long et al., 1997), drosophila and xenopus oocytes (Serano and Cohen, 1995; Yisraeli et al., 1990). However, more recent studies in chicken fibroblasts and neurons have helped to further characterize additional 43 RNA transport pathways (Gu et al., 2002). While mechanical and regulatory differences do exist, there are some common underlying components that undoubtedly form a basis for mRNA transport. It is therefore appropriate to describe some of the mechanisms and how they are regulated with a final focus on the role of Rho family of GTPases in regulation mRNA transport to tumor cell protrusions. Regulating factors Cis-acting factors There are a number of mechanisms that a cell can use to localize mRNA including, active transport via the cytoskeleton (Yisraeli et al., 1990), coordinated degradation and stabilization of mRNA molecules (Bashirullah et al., 1999) and/or diffusion through the cytoplasm in conjunction with site specific trapping (Glotzer et al., 1997). The largest bodies of evidence exist for the former two examples and this dialogue will focus primarily on transport methods that rely on cytoskeletal components. Upon transcription in the nucleus messenger R N A is shuttled into the cytoplasm through nuclear pores. Its subsequent localization is often dictated by a cis-acting regulatory element in the 3'UTR termed the zipcode sequence (Kislauskis and Singer, 1992). This zipcode facilitates the binding of a trans-acting factor, usually a protein that can induce a conformational change in the mRNA molecule and render it accessible to additional regulatory factors. The zipcode has not been identified in all localized mRNAs and those where it has been identified show varying levels of consensus, indicating the specificity of R N A targeting. For example, in P-actin mRNA the zipcode is a 54 nucleotide sequence just 3' to the stop codon that is essential for correct mRNA 44 localization (Kislauskis et al., 1994). The zipcodes for a- and y-actin isoforms are still found in the 3'UTR but contain limited sequence homology to their P-actin counterpart (Kislauskis et al., 1993). This finding, however, is not surprising as the protein isoforms localize differentially in the cell, with a-actin and y-actin found primarily in perinuclear regions and P-actin situated mainly at cell periphery (Hoock et al., 1991; Otey et al., 1988). Alternatively, nanos mRNA from drosophila oocytes contains four regions in the 3'UTR involved in localization. These regions act synergistically to target nanos to the posterior pole of the developing egg (Bergsten and Gavis, 1999; Crucs et a l , 2000). The primary sequence of mRNA targeting elements is important in distinguishing between specific RNAs. However, the secondary structure of the molecule also contributes to the ability of the R N A to become localized. In yeast, ASH1 mRNA contains a region in its 3 'UTR that forms a stem-loop structure (Chartrand et al., 1999). This structure is not only necessary, but essential for ASH1 mRNA localization. Another example of this is found in the drosophila oocyte where the presence of stem loop secondary structure in the 3'UTR of bicoid (bed) mRNA is more important for targeting than the primary sequence (Macdonald and Kerr, 1998). Trans-acting factors The zipcodes sequences, as previously mentioned, facilitate the binding of trans-acting factors to the mRNA molecule. It is the trans-acting factors that direct mobility and the translation of this mRNA. Together the mRNA and trans-acting factors form a complex called a ribonucleoprotein (RNP) and that has been identified as vesicle for mRNA transport through the cytoplasm and anchoring upon arrival at a destination 45 (Heasman et al., 2001; Kloc and Etkin, 1994). The RNPs appear as granules in the cell (Ainger et al., 1993) and contain many components necessary for protein translation; such as arginyl-tRNA synthetase, elongation factor 1-a and ribosomal R N A (rRNA) (Barbarese et al., 1995). As a reference point, R N A granules containing myelin basic protein (MBP) were reported to have mean radius 0.6-0.81 pm (Barbarese et al., 1995). There are a number of proteins or trans-acting factors that bind R N A for different purposes. However, the following text will describe mRNA binding proteins that facilitate the mobilization and localization of mRNA. One of the best characterized trans-acting factors is named staufen. Staufen is required in many cases for RNP formation as well as mRNA localization. Staufen is composed of five double stranded RNA binding domains (dsRBD), the second of which (dsRBD2) is split by a short insertion (Micklem et al., 2000). A l l five of these domains are required for correct binding and localization. It is postulated that different regions are required specifically for actin versus microtubule based transport (Micklem et al., 2000). For example, staufen colocalizes with oskar and bicoid mRNA in drosophila oocytes and is necessary for their localization at the posterior pole and anchoring at the anterior pole, respectively (Ferrandon et al., 1994; St Johnston et al., 1991). The localization of both oskar and bicoid has been shown to be microtubule dependent and reliant on the integrity of the dsRBD2 domain of staufen (Clark et al., 1994; Ferrandon et al., 1994; Micklem et al., 2000). Conversely, the staufen dependent targeting of prospero mRNA during embryonic neuroblast division in drosophila requires the presence of intact actin microfilaments and the functional dsRBD5 domain of staufen (Broadus and Doe, 1997; Broadus et a l , 1998). 46 Zipcode binding protein 1 (ZBP1) is another previously described trans-acting factor (Ross et al., 1997). This 68 kDa protein binds to a sequence (ACACCC) in the 3'UTR of p-actin mRNA in many species and is necessary for the localization of P-actin mRNA within the cell (Oleynikov and Singer, 2003). Interestingly, dominant-negative mutants of ZBP1 inhibit cell polarity and motility in fibroblasts and the development of filopodia and filopodial synapses in dendrites (Eom et al., 2003). In addition to regulating mRNA transport, ZBP1 also contributes to the translational regulation of P-actin mRNA (Huttelmaier et al., 2005). Studies have shown that binding of ZBP1 to the zipcode region of P-actin mRNA prevents translation until the targeting location has been reached, at which point ZBP1 is phosphorylated by src enabling the release of the mRNA molecule which facilitates translation (Huttelmaier et al., 2005). A third example of a trans-acting factor is elongation factor-a (EFla). This protein catalyzes amino-acyl tRNA binding to ribosomes during translation in a GTP-dependent manner (Sprinzl, 1994). E F l a is highly conserved among eukaryotic species (>75%) and is commonly found in RNPs (Sprinzl, 1994). In addition to facilitating protein translation, E F l a , provides a link between mRNA localization and the cytoskeleton as it has been shown by electron microscopy to bind to actin filaments and co-localized with mRNA (Bassell et al., 1994a; Liu et al., 1996a). mRNA transport mechanisms Cytoskeletal Transport The transport of mRNA along the cytoskeleton has been demonstrated in many models systems, such as in somatic cells (Bashirullah et al., 1998), yeast (Jansen, 2001) 47 and drosophila (Palacios and St Johnston, 2001). Actin filaments and microtubules are the primary components of the cytoskeletal transport system; in general facilitating short and long distance mRNA shuttling, respectively. Actin-based transport has been characterized in a number of examples such as the localization of P-actin mRNA to leading edge in fibroblasts (Bassell et al., 1994a; Bassell et al., 1994b) and ASH1 mRNA targeting to the daughter bud tip to repress mating type switching in yeast (Chartrand et al., 2001). Examples of microtubule dependent transport include transport of MBP mRNA in oligodendrocytes (Worboys, 1994) or the targeting or ZBP1 associated P-actin mRNA from the cell body to growth cones in developing neurons (Zhang et al., 2001). The mechanism of mRNA transport along actin microfilaments or microtubules is largely unknown. However, research continues to suggest that active transport along the cytoskeleton via motor proteins is a main mechanism. The following studies describe some examples of the motor proteins involved in R N A transport, such as myosin (Bohl et al., 2000), kinesin (Januschke et al., 2002) and dynein (Wilkie and Davis, 2001), as well as the circumstances in which they are important. The transport of P-actin mRNA along actin filaments to the leading edge of fibroblasts has been well characterized (Sundell and Singer, 1991). The likelihood that this pathway is driven by myosin motor proteins is strong (Latham et al., 2001). The localization of P-actin mRNA is potentiated by an increase in the formation of actin stress fibres and decreased by treatment with myosin inhibitors (Latham et al., 2001). Additionally, studies using fluorescently tagged ZBP1, that binds P-actin mRNA, shows fluorescent particles moving toward developing protrusions at speeds (0.6 urn/sec) 48 consistent with what would be expected from myosin driven transport (Oleynikov and Singer, 2003). Transport via kinesin and dynein occurs along microtubules and is separated into (+) positive end and (-) negative end transport, respectively. This can be interpreted that mRNA is being moved toward either the positive or negative end of the microtubule. Both methods have been observed in a variety of species with a combination of kinesin and dynein driven transport sometimes occurring in the same cell (Duncan and Warrior, 2002). Kinesin (or positive end) based transport is observed during the movement of MBP mRNA in oligodendrocytes (Carson et al., 1997). Fluorescently tagged M B P mRNAs are seen moving in the cytoplasm of the cell, most oscillating back and forth, but some traveling in a directed manner toward peripheral processes (Ainger et al., 1993). In this study mRNA containing particles moved away from the cell body at a speed of 0.2 um/sec. In a separate study, this peripheral localization was blocked by incubation with antisense R N A to kinesin light chain (KLC), indicating the likely dependence on kinesin for M B P mRNA transport to processes in oligodendrocytes (Carson et al., 1997). A specific example of dynein (negative end) based transport occurs during oogenesis in drosophila (Januschke et al., 2002; Schnorrer et al., 2000). As the oocyte develops, it is surrounded by 'nurse' cells that provide organelles and cytoplasm necessary for growth. The transfer of cellular material occurs through cytoplasmic bridges called ring canals, through which microtubules extend (Swan et al., 1999). Although many mRNAs travel along these microtubules to the oocyte, bicoid mRNA, which localizes anteriorly in the developing egg, uses dynein as motor to direct its movement. This is evident in that upon overexpression of components of the dynactin 49 complex (of which dynein binding is essential for motor protein function) prevents the correct localization of bed in the oocyte. That is, bed mRNA enters the egg, but is not properly localized to the anterior pole. This effect is not observed with other mRNAs such as oskar, which are also transported from the 'nurse' cell to the oocyte (Schnorrer et al., 2000). Regulatory pathways affecting mRNA transport While there is still much to learn regarding the factors and mechanisms involved in mRNA transport, there is even less known about the regulation of this occurrence. However, there is an increasing body of evidence to support the involvement of certain signaling molecules such as the Rho family of small GTPases (Latham et al., 2001). The following will attempt to outline a potential role for the Rho GTPases as well as some of the associating factors that may contribute to the localization of specific mRNAs. The diaphanous-related formin family is a group of proteins that contain conserved formin homology (FH) domains and function to regulate signaling with certain kinases and actin binding proteins (Edmonds et al., 1995; Wasserman, 1998). This family has been shown to work downstream of RhoA and cdc42 in yeast (Bnilp) (Imamura et al., 1997), mammals (mDial-3) (Watanabe et al., 1997) and drosophila (Diaphanous) (Peng et al., 2003). Diaphanous-related formins (DREs) are associated with mRNA translocation because they contain an E F l a binding site (EBS) (Umikawa et a l , 1998) and the relationship between E F l a and P-actin mRNA has been described previously. It has been shown in yeast that the EBS, a short amino acid sequence located between the FH1 and FH2 domains of DRFs, can prevent binding of E F l a to actin 50 filaments and block F-actin bundling in vitro (Liu et al., 2002; Umikawa et al., 1998). In mammalian cells EBS deletion mutants of mDia3 prevent F-actin bundling in the cell cortex and filopodia, but have no effect on stress fibre formation (Peng et al., 2003). Studies showing the effect of RhoA on P-actin mRNA localization (Latham et al., 1994; Latham et al., 2001) indicate that its activation promotes mRNA targeting. This, in conjunction with the role of mDia proteins as downstream effectors of RhoA, presents a model in which activation of RhoA and subsequently mDia affects the interaction of E F l a with actin filaments (Condeelis and Singer, 2005); the EFla-actin complex formation being necessary for the association of p-actin mRNA and its localization to the leading edge of motile cells. This hypothesis is supported by evidence that GFP-tagged to the amino acid sequence of the EBS fails to localize P-actin mRNA appropriately (Liu et al., 2002) and that recombinant EBS prevents the association between E F l a and F-actin and subsequently of P-actin mRNA with the EFla-F-Actin complex (Liu et al., 2002). mRNA localization in metastasis A study by Shestakova et al. (1999) investigated the relationship between metastasis in cancer and the cellular localization of P-actin mRNA. When comparing highly metastatic breast adenocarcinoma (MTLn3) cells to cells with low metastatic potential (MTC), cell polarity was identified as factor differentiating the two (Shestakova et al., 1999; Wang et al., 2002). That is, as cells became more invasive they lose the characteristic polarity associated with directed motility. The explanation for this phenotype described the polar cell as having to turn itself around in order to allowing the 51 leading edge to face a chemoattractant, whereas a non-polar and invasive cell could react to its environment and polarize spontaneously in a given direction (Condeelis and Segall, 2003; Condeelis, 2003; Wang et al., 2002). Another feature associated with polarity in these cells was the ability to asymmetrically localize mRNA, specifically P-actin mRNA (Condeelis and Segall, 2003; Shestakova et al., 1999). This observation fits well with evidence that P-actin mRNA localization is required for the development of a stable leading edge and that this localization is dependent on ZBP1 (Wang et al., 2002). Additionally, the levels of ZBP1 were found to be up to lOx lower in cells isolated from a primary tumor that invasive tumor cells from the same progenitor cells (Wyckoff et al., 2000). Therefore as cells develop metastatic potential they lose static polarization and develop a dynamic membrane that can react readily to chemoattractants from any direction. With the loss of cell polarity there is a decrease in stably localized mRNA and the enhancement of mRNA transport mechanisms that efficiently translocate mRNA to rapidly polarizing regions. Thus metastatic cells become hypersensitive to their environment through their ability to spontaneously polarize in the direction of stimuli rather than maintaining a fixed leading edge. The Rho-dependent pseudopodia of the M S V - M D C K - I N V cells display similar characteristics as these domains are transient and non-directional, but consistently ensure pseudopodial mRNA localization. 52 1.5 Summary In summary, several aspects of tumor cell biology, specifically focusing on the cellular mechanisms involved in metastasis, have been described. Tumor cells use different modes of motility and protrusive structures to facilitate movement in their environment. The dynamic nature of tumor cells is dependent on components of the cytoskeleton including actin microfilaments and microtubules. Rho family GTPases, RhoA, Racl and cdc42, play a role in regulating actin filaments and microtubules. Membrane protrusions that develop as a result of cytoskeleton dynamics are polarized structures that require localized signaling molecules. Messenger R N A is recruited to many cellular protrusions as part of this polarization process. Therefore, investigation of the mechanisms of mRNA localization that facilitate polarization in tumor cells is important. A better understanding of the polarized protrusions that are necessary for tumor cell migration will enhance our ability to treat and diagnosis metastatic cancer. The purpose of this study is to further characterize Rho-dependent pseudopodia as a polarized signaling domain using the M S V - M D C K - I N V cell line as a tumor cell model. Specifically, the role of RhoGTPases in regulating mRNA localization to the pseudopodia will be investigated. A variety of techniques will be used including FRET, FRAP and in situ hybridization to explore the composition and regulation of Rho-dependent pseudopodia. 53 Chapter 2 Results 2.1 Materials and Methods Antibodies, Plasmids and Reagents Antibodies to P-actin, Bip/GRP78, calnexin and talin were purchased from Sigma-Aldrich (Ontario, Canada). Anti-FAK, anti-phospho-tyrosine (p-Tyr; PY-99), anti-phospho-RBl (SY80), anti-HSC70, anti-lamin A/C were obtained from Santa Cruz Biotechnology (California, USA). Biosource International (California, USA) provided the antibody to anti-phospho-FAK Y397. Anti-p97 (VCP), anti-HSP90, anti-EFla and anti-nuclear pore complex proteins (NPC, Mab414) were purchased from Abeam Inc. (Massachusetts, USA), Stressgen (Michigan, USA), Upstate (Virginia, USA) and Covance Research Products (Quebec, Canada), respectively. Calbiochem (Ontario, Canada) supplied antibodies to calpain 2 and paxillin. Antibodies to vinculin and a5pi integrin were purchased from Chemicon Inc (California, USA). Secondary antibodies conjugated to Alexa 488, 588 and 633 as well as fluorescent dyes; propidium iodide, Alexa Phalloidins 488 and 568, Sytox-green, Sytol4 and Syto RNASelect were purchased from Molecular probes (Ontario, Canada). The nuclear stain, Hoechst, was supplied by Sigma-Aldrich. pEGFP and pEGFP-Actin plasmids were from BD Bioscience (Ontario, Canada). Raichu FRET probes for activated RhoA and Racl were provided by Dr. Michiyuki Matsuda (Department of Tumor Virology, Osaka University, Japan). The dominant active Racl plasmid was obtained from Nathalie Lamarche (McGill University, Montreal, Quebec). R O C K inhibitor, Y27632, was supplied by Calbiochem and Nocodazole by Sigma-Aldrich. 54 Cell culture and pseudopod purification M S V - M D C K - I N V cells were cultured in high glucose D M E M supplemented with 1% non-essential amino acids, 1% glutamine, 1% vitamins 1% penicillin/streptomycin (Invitrogen, Ontario, Canada) and 10% fetal bovine serum (Medicorp, Quebec, Canada) as described by Le et al. (1998). Cells were maintained in a 37°C incubator with 5% C02/air. Transfections were carried out using Effectene transfection reagent (Qiagen, Ontario, Canada). Pseudopodia were purified using 100mm diameter filters containing 1 um pores, as previously described by Nguyen et al. (2000). Immunofluorescence labeling M S V - M D C K - I N V cells were plated on coverslips in 35 mm tissue culture dishes (Becton Dickinson, New Jersey, USA) or 1 pm pore filter cell culture inserts in multi-well companion plates (Becton Dickinson) as described previously, Nguyen et al. (2000) for 24 hrs. Nocodazole (2 pM) (Sigma) and Y27632 (20 uM) (Calbiochem) treatments were performed for the times indicated, before fixation with 3% paraformaldehyde (PFA) or precooled (-80°C) methanol/acetone (80%/20%) (the latter used when immunofluorescence labeling involves either anti-NPC or anti-Lamin A/C). Following PFA fixation the cells were permeabilized with 0.2% Triton-X-100 (Fisher Scientific, Ontario, Canada). Incubation in 30% bovine serum albumin (BSA, Fisher Scientific) prior to labeling provided a blocking step and when applicable, cells were treated with 50 ng/ul RNAse at 37°C for 30 min. R N A was labeled using either 1 ng/pl propidium idodide (Molecular Probes) or 400 n M SytoRNASelect (Molecular Probes). Cells were incubated with indicated primary antibodies for Ihr prior to 30 min with appropriate 55 fluorescent secondary antibodies. Coverslips were washed and mounted on slides with gelvatol or filters were mounted between a slide and a coverslip. Cells were imaged on a FV1000 Olympus confocal microscope using 60x or lOOx planapochromat objectives. Quantification of protein expression in actin rich cellular domains was performed using ImagePro software (Media Cybernetics, Maryland, USA). A thresholding function was used to devise a mask of either phalloidin or actin-GFP labeled regions of the cell. Expression of proteins of interest in the pseudopodia was determined in masked regions and these intensity/area values were compared with intensity/area values of the cell outside the masked regions. Quantification represents two experiments that evaluated 30 cells each. Oligonucleotides The following oligonucleotides were designed to complementary regions of sense mRNA strands. Actin sense, 5' A T T G C G G C G C T A G T T G T A G A T A A C G G C T C C G G T A T G T G C A A G G C G 3'; Actin, C G C C T T G C A C A T A C C G G A G C C G T T A T C T A C A A C T A G C G C C G C A A T ; Fibronectin, G T G G C C A G G A A T G G T G G C T T C C T T C C A A C G G C C T G G A G A G T T T T T A G G ; Shp-2, A C T G G G C C T C G C C A A A A A A C T G C C A T C A A C T C C T C T T G T C A A C A G C ; m-ras, G C A T C A G A T C G A C C T T G T T G G C C A C G A G G A T C A T C G G G A A T G A C T C T C T G ; RhoA, G T C T G G A T A G G A G A G A G G C C T C A A G T G A T C A T G A T C T T C C C G C C C A G C T G ; Arp2/3 p41, G C A A T C T G G G T A C G A T C C C T G T T C C A G G C A T G A C A G G T G A T T G G C T C T A G T ; syntrophin, CCTGTCTTCTTC A A G A C C T G C A C C G C C T C A T C A T G G G T A G C A G A G G A G A G . Oligonucleotides 56 were with digoxigenin-dUTP tailed using a DIG Oligonucleotide Tailing Kit (Roche, Ontario, Canada). In situ hybridization Approximately 20000 cells were plated per well in 8 well Lab-Tek chamber slides (Nalge Nunc International, New York, USA) and incubated at 37°C (5% C0 2) for 24 hrs. Cells were fixed with 3% paraformaldehyde (PFA) for 30 min at room temperature and permeabilized in 0.2% Triton-X-100 for 10 min at room temperature. Post fixation was performed with 3% (PFA) for 5 min at room temperature before washing twice with freshly prepared 0.1% DEPC (diethylpyrocarbonate) treated l x PBS (phosphate buffered saline). Hydrogen peroxide (1%) treatment for 1 hr was used to quench endogenous peroxidase. Prehybridization took place at 37°C for 2hrs in solution containing 10% dextran sulphate (Sigma), 2 m M R V C (ribonucleoside vanadyl complexes, Sigma), 0.1% Blocking reagent (Roche), 1 ug/ul E. coli M R E 600 tRNA (Roche), 2x SSC (0.3 M sodium chloride, 0.03 M sodium citrate) 50% formamide (Sigma), 0.25 ug/ul sheared salmon sperm D N A (Eppendorf, Germany). Oligonucleotides were added to the prehybridization solution at a final concentration of 0.05 pmol/ul and were allowed to hybridize for 40 hrs at 37°C. Following hybridization were consecutive triple washes with 2x SSC, 50% formamide, 0.1% SDS (sodium dodecyl sulphate, E M D Chemicals Inc. New Jersey, USA), and l x SSC, 50% formamide, 0.1% SDS. 57 Developing DIG (digoxigenin-dUTP) -labeled oligonucleotides Cells were incubated with blocking solution (2x SSC, 8% formamide, 2 m M R V C , 1% Blocking reagent) at 37°C for 30 min before the addition of the anti-DIG-AP (digoxigenin-dUTP alkaline phosphatase, Roche) at a dilution of 1:5000 for 2 hrs at 37°C. Slides were washed in triplicate with 2x SSC, 8% formamide and left to equilibrate in AP (alkaline phosphatase) detection solution (100 mM Tris (Calbiochem), 100 m M NaCl (EMD Chemicals Inc.), 50 m M M g C l 2 (BDH Inc., Quebec, Canada)) for 10 min. Developing occurred by incubation with staining solution (NBT/BCIP, Roche) tablet in 10 ml H2O) for 48 hrs. Following developing, cells were washed with 95% Ethanol and mounted using gelvatol. Fluorescence recovery after photobleach (FRAP) M S V - M D C K - I N V cells stably expressing EGFP-Actin (selected for using G418, Invitrogen) or untransfected M S V - M D C K - I N V cells were plated in 4-well Lab-Tek II chambered coverglasses (Nalge Nunc International) for 24 hrs. Untransfected cells were stained with 400 n M Sytol4 (Molecular Probes) dye for Ihr. Subsequently cells were incubated with 20 u M Y27632 for 1 hr in phenol red-free D M E M . pEGFP-Actin transfected cells were imaged for 90 s following bleaching of a selected actin-rich region in a pseudopod using a 488 nm laser at 100%. Cells stained with Sytol4 were imaged for 30 s following bleaching of a R N A labeled pseudopod. Mobile fractions and recovery half times were calculated using Graphpad Prism statistical software. 58 Fluorescence resonance energy transfer (FRET) FRET experiments were carried out using single molecule Rho (Raichul298) and Rac (Raichul026) activity sensing FRET plasmids (Michiuki Matsuda, Osaka University). Cells were transfected with either Rho 1298 or Rac 1026, or dually transfected with Rho 1298 and a dominant active Rac plasmid in 4-well Lab-Tek II chambered coverglasses (Nalge Nunc International) for 24 hrs. Treatment with 20 u M Y27632 in phenol-red free D M E M for 1 hr occurred prior imaging. Transfected cells were initially imaged in both CFP and YFP channels on an Olympus FV1000 following excitation with a 405 nm laser. Selected regions were bleached using a 515 nm laser and subsequent images of both CFP and YFP channels were taken. Olympus Fluoview Ver. 1.4a software was used to calculate the average pixel intensity of a selected region before and after bleaching. FRET efficiency was calculated in the CFP channel using Olympus Fluoview Ver. 1.4a software taking into account background pixel intensity for each image. The percent bleach value was obtained by subtracting the average pixel intensity following bleaching from the average pixel intensity prior to bleaching. 59 2.2 Results Isolation and characterization of proteins in the pseudopodia The ability of the pseudopodia of M S V - M D C K - I N V cells to pass through 1 um pores when plated on filters has been previously characterized (Nguyen et al., 2000). Briefly, following culture in serum-enriched media, the pseudopodia pass through the pores, leaving the nucleus and cell body fraction on the upper side of the filter. This is evident in Fig. 1 (A-D) with the nuclei labeled in green (Sytox green) (Fig. 1 A) on the upper side of the filter and the pseudopodia labeled for F-actin (phalloidin) (Fig. 1 B) and phosphorylated tyrosine residues (Fig. 1 C) on the underside of the filter. The pseudopodia of well characterized invasive tumor cell lines, HT1080 (Fig 1. E-H) and MDA-MB-231 (Fig 1.1-L) also pass through the filter pores. In these cases the nuclei were labeled with Hoechst (blue) (Fig. 1 E, I) and the pseudopodia labeled with phalloidin (green) (Fig. 1 F, J) and an antibody to phospho-tyrosine residues (red) (Fig. 1 G, K). This validates the use of 1 pm pore filter units as method of separating pseudopodial and cell body fractions. The invasion of pseudopodia through the filters allows for the selective isolation of this domain by scraping the underside of the filter (Jia et al., 2005). The pseudopodial fraction was analyzed for protein content by liquid chromatography tandem mass spectroscopy and 167 proteins were identified (Jia et al., 2005). Once serum related proteins were subtracted a number of proteins falling into the following categories were identified; cytoskeletal (28), adhesion (5), glycolytic enzymes (13), chaperones (10), translation-associated (23), RNA-binding (14), ubiquitin/proteasome-associated (11), signaling (16), membrane trafficking (6), organelle-associated (13) and 28 unclassified 60 merge 11_| merge I merge IWItf proteins per class # peptides per class a Cytoskeleton (28) • Glycolysis (13) . Chaperones (10) • Translation (23) • RNA binding protein (14) • Ubiquitin/proteasome (11) • Signaling (16) • Organelles (13) • Trafficking (6) • Adhesion (5) • Others (28) m Cytoskeleton (656) • Glycolysis (353) • Chaperones (315) • Translation (238) • RNA binding protein (92) 0 Ubiquitin/proteasome (75) • Signaling (76) D Organelles (67) • Trafficking (58) • Adhesion (42) • Others (157) Figure 1. Tumor cell pseudopodia grow through 1 fi in pore filters allowing for their isolation and characterization. M S V - M D C K -INV (A-D) (Jia et al., 2005), HT1080 (E-H), and MDA-MB-231 (I-L) are plated on 1 pm pore filters. The upper side of the filter is shown above the white dotted line and the pseudopodia growing through the filter are labeled underneath the dotted line. The nuclei are labeled on the upper side of the filter with Sytox-green (A) or Hoechst (E, I). F-actin is labeled with Texas Red phalloidin (B) or Alexa 568 phalloidin (F, G) and a-phospho-tyrosine-99 labeling is detected with Alexa 633 (C) or 647 (G, K ) conjugated to a secondary antibody. Merged images of the filters are shown in (D), (H) and (L). M S V - M D C K -INV cell pseudopodia were isolated from a filter and their lysate was analyzed by L C - M S / M S . Classification of the proteomic data is shown in (M). The pie chart on the left represents the number of proteins identified in each class and the chart on the right shows the relative abundance (number of peptides per protein) of proteins (Jia et al., 2005). 61 proteins. Figure 1 M shows a pie chart representation of the fraction of categorized proteins identified and the relative abundance of peptides per category. A selection of highly expressed proteins was chosen for validation by immunofluorescence labeling (Fig. 2 A-P) (Jia et al., 2005). Although there is a diffuse cytoplasmic labeling for a number of the proteins, enrichment in pseudopodial labeling is still evident. Quantification of these results was achieved by thresholding the actin rich regions of the pseudopodia. Thresholding was accomplished by selecting an actin pixel density value and using computer software to identify regions in the cell with a pixel density greater than or equal to the chosen value. Application of equivalent thresholding parameters to all cells allowed objective identification of actin rich pseudopodia. A mask was made of these regions in multiple cells. The average pixel density for proteins of interest labeled within these regions was determined (Fig. 2 Q). The control M S V -MDCK- IN V cells that were transfected with pEGFP show that enrichment in the pseudopodia was not due to a volume effect or the diffusion of cytoplasmic proteins (Fig. 2 A-B). Therefore, quantification shows significance based on comparing the ratio of pseudopodia to cell body fraction of the enriched proteins to pEGFP in the transfected cell. The expression of a selection of cytoskeleton-associated and adhesion proteins as well the translation-associated protein, E F l a (Fig. 2 M-N), and the cell cycle regulator, phospho-Rbl were noticeably enhanced in the pseudopodia. This supports the concept of a localized signaling domain capable of forming adhesions and facilitating cytoskeletal induced membrane protrusions. Phospho-Rbl was originally identified as upregulated in the pseudopodia from a Kinetworks KPSS1.3 phospho-site screen (Jia et al., 2005) and 62 Figure 2. Immunofluorescence labeling of proteins identified in actin-rich pseudopodia of MSV-MDCK-INV cells. Cells were transfected with a pEGFP plasmid (A and B) and the colocalization of GFP (A) and phalloidin (B) was used as a control. Other untransfected cells were labeled with antibodies to HSP90 (C), calpain2 (E), vinculin (G), calnexin (I), BiP (K), and E F l a (M) and detected with Alexa-conjugated secondary antibodies. Additionally, F-actin was labeled with Alexa phalloidin (B,D,F,H,J,L,and N). M S V - M D C K - I N V cells stably transfected with pEGFP-Actin (O, P) were fixed with methanol/acetone and labeled for the nucleoporin complex (O). The degree to which proteins labeling colocalized with actin was quantified (Q) and significance was calculated with respective to GFP labeling. (± S.E.; * , p<0.05, **, p<0.001). Scalebar 20 urn. 63 although its functionality in this domain is unclear the immunofluorescence labeling supports this initial detection. The ER-associated protein, BiP (Fig. 2 K-L) was detected in the pseudopodia by proteomic analysis and confirmed by blot and immunofluorescence. This demonstrates the presence, not the upregulation of BiP in the pseudopodia. However, BiP does appear more highly expressed in the pseudopodia than calnexin (another ER marker) which was not identified in the proteomic screen. As well, BiP has been identified as being selectively expressed in metastatic tumor cells (Arap et al., 2004), which would support its presence in the pseudopodia. Proteins involved in nucleocytoplasmic shuttling such as lamin A / C and nucleoporin complex (NPC) (Fig. 2 O-P) are traditionally localized to the nuclear envelope region, and while the strong labeling of NPC by immunofluorescence validates it presence in the pseudopodia, it does not clarify its functionality. mRNA localizes to the pseudopodia in a ROCK dependent manner A number of translation-associated proteins and RNA-binding proteins were identified in the proteomic screen. This relates to studies linking E F l a (identified as highly expressed in the pseudopodia) to mRNA granules (Sprinzl, 1994) and showing its importance in mRNA trafficking and anchoring (Liu et al., 2002). Therefore, interpretation of the results from the proteomic analysis predicted the presence of mRNA in the pseudopodia. This is in accordance with studies by Latham et al. (2001) showing Rho/ROCK dependent localization of P-actin mRNA to the leading edge of crawling cells. Staining M S V - M D C K - I N V cells with propidium iodide showed that mRNA was localized to most actin rich pseudopodial domains (Fig. 3 A) and that this localization 64 Phalloidin/PI GFP/Phalloidin Figure 3. mRNA localizes to MSV-MDCK-INV cell pseudopodia in a ROCK-dependent manner. (A, C, E) Cells were labeled with propidium iodide (red) and phalloidin (green). (B, D) Alternatively pEGFP transfected cells (green) were labeled with phalloidin (red). Cells in (C) and (D) were treated with Y27632 for Ihr prior to fixation. The cytosolic mRNA specificity of propidium iodide is shown by treating cells with RNAse for 30min prior to fixation (E). The colocalization of propidium iodide or GFP with phalloidin in the pseudopodia was quantified and treatment with Y27632 significantly inhibits propidium iodide labeling in the pseudopodia. (±S.E.; *, p<0.005). Scalebar 10 urn. 65 was prevented following treatment with the R O C K inhibitor, Y27632 (Fig. 3 C). The integrity of mRNA labeling by propidium iodide was confirmed by an RNAse treatment control that restricted fluorescence to the nucleus and degraded all cytoplasmic R N A (Fig. 3 E). Once again, pEGFP transfected cells were used as a volume control, showing no effect on GFP localization following treatment with the R O C K inhibitor (Fig. 3 B, D). These results were quantified by making a mask of the actin rich regions of the pseudopodia and determining the average pixel density of the propidium iodide within this area (Fig. 3 F). The cells treated with R O C K inhibitor show a significant decrease in mRNA content in the pseudopodia. This confirmed our prediction and implicated a Rho/ROCK dependent in regulating mRNA translocation. Rho activation is increased in the pseudopodia compared to the cell body It has been shown that R O C K plays a role in regulating mRNA transport to the pseudopodia (Jia et al., 2005). Therefore was of interest to investigate the upstream activation of the R O C K pathway in this domain. A single molecule FRET (fluorescence resonance energy transfer) construct was obtained that signals Rho activation by differential emission of CFP (cyan fluorescent protein) and YFP (yellow fluorescent protein) fluorophores. The GTP binding region of RhoGTPase and the Rho binding domain (RBD) of Rho effector, P K N , are sandwiched between the CFP (energy donor) and YFP proteins (energy acceptor). Upon GTP binding to Rho, a conformational change occurs in the protein that allows RBD to bind to the active Rho and decreases the proximity of YFP and CFP to each other. Subsequently when CFP is excited by a 66 405 rim laser, the energy is transferred directly to YFP and emitted at a wavelength of 530 nm, which is specific to YFP fluorescence. Thus making detectable the regions of the cell in which Rho activation is occurring. A similar construct was used to determine Rac GTPase activation, with a GTP binding domain specific to Rac that interacts with the Rac binding domain of an effector protein. 433 nm 475 nm Illustration 6. Basic structure of the GFP-based FRET probe for Rho GTPases (Raichu-Rho). Adapted from (Nakamura et al., 2005). The FRET method that was used was acceptor photobleaching FRET. This method photobleaches the YFP acceptor rendering it incapable of accepting energy transfer from the CFP donor. Therefore, when the CFP donor is excited it will emit energy at 475 nm, a wavelength specific to CFP fluorescence. Thus, when FRET is occurring, an increase in CFP emission is observed following photobleaching of YFP 67 F R E T before Acceptor Bleaching Bleaching alter Acteptc* Bteachsng I l l u s t r a t i o n 7. Acceptor photobleaching FRET. Bleaching of the acceptor yields increased emission from the donor, ( w w w . z e i s s . c o m ) U s i n g t h i s t e c h n i q u e w e i n v e s t i g a t e d the d i f f e r e n t i a l a c t i v a t i o n o f R h o a n d R a c i n the p s e u d o p o d i a a n d c e l l b o d y o f M S V - M D C K - I N V c e l l s . T h e r e i s a n i n c r e a s e i n R h o a c t i v a t i o n i n the p s e u d o p o d i a o v e r the c e l l b o d y as s h o w n b y the i n c r e a s e d C F P e m i s s i o n f o l l o w i n g p h o t o b l e a c h i n g o f Y F P ( F i g . 4 A - D ) . T h i s ag rees w i t h the c o n c e p t tha t p s e u d o p o d i a a re s i g n a l i n g d o m a i n s that are r e g u l a t e d i n pa r t t h r o u g h l o c a l R h o a c t i v a t i o n . I n t e r e s t i n g l y w h e n the c e l l s w e r e t r ea ted f o r I h r w i t h Y 2 7 6 3 2 the re i s a s i g n i f i c a n t d e c r e a s e i n R h o a c t i v a t i o n i n the p s e u d o p o d i a , b r i n g i n g i t to a l e v e l that i s s i m i l a r to that o f the c e l l b o d y ( F i g . 4 J). T h e s a m e r esu l t i s o b t a i n e d w i t h the c e l l s w e r e t r a n s f e c t e d f o r 2 4 h r s w i t h a d o m i n a n t - a c t i v e R a c c o n s t r u c t . T h i s w o u l d i n d i c a t e that i n h i b i t i o n o f R O C K a n d a c t i v a t i o n o f R a c l e a d to a d e c r e a s e i n R h o a c t i v a t i o n . W h i l e there i s n o d i f f e r e n c e i n R a c a c t i v a t i o n b e t w e e n the p s e u d o p o d i a a n d c e l l b o d y o f un t r ea t ed c e l l s ( F i g . 4 E-H) , the i n h i b i t i o n o f R O C K l e a d s t o a n i n c r e a s e i n R a c a c t i v a t i o n i n t he p s e u d o p o d i a ( F i g . 4 J). T h i s f u r t h e r s u p p o r t s the i d e a tha t R O C K a n d R a c are i n v o l v e d i n f e e d b a c k l o o p s that a f f e c t the a c t i v a t i o n o f R h o i n l o c a l i z e d d o m a i n s . 68 255 Transfect ion F R E T e f f ic iency 026 0.17 0 19 0.19 0 19 0.17 025 0.18 0 18 0 15 0 02 001 002 001 0 02 001 0 02 001 R h o R h o + Y R h o + D A - R a c R a c R a c + Y Figure 4. RhoA is selectively activated in the pseudopodia of MSV-MDCK-INV cells. Cells were transfected with CFP/YFP FRET plasmids specific for active RhoA (A-D) or active Racl (E-H). Fluorescence images of the CFP channel are taken before (A, C, E, G) and after (B, D, F, H) photobleaching with a 515 nm laser. Regions of the pseudopodia (A, B, E, F) and cell body (C, D, G, H) were selected for testing RhoGTPase activation, which was measured by increases in average pixel density of CFP emission in the bleached region. Increases in intensity can be seen to the in pseudo-colored images of the bleached region (to the right of each image). Regions of high intensity are colored in red and low intensity in blue. (I) Shows a table indicating the region of the cell that was bleached, the donor and acceptor fluorphores, the number of cells, the percent bleach of the Y F P channel and the FRET efficiency of the CFP channel ± standard error. Cells were transfected either with active RhoA or active Racl FRET plasmid and treated with Y27632 for Ihr prior to imaging. Alternatively, cells were dually transfected with an active RhoA FRET plasmid and a dominant-active Racl plasmid. (J) The differences in FRET efficiency between the pseudopodia (black) and the cell body (blue) were quantified under the indicated conditions. (±S.E.; *, p<0.01). Scalebar20 pm. 69 mRNA turnover but not Actin-GFP turnover in the pseudopodia is ROCK dependent A technique termed fluorescence recovery after photobleaching (FRAP) was used to investigate the turnover of actin-GFP protein or mRNA in the pseudopodia of M S V -M D C K - I N V cells. The time required for fluorescently labeled protein or mRNA to return to a photobleached region is an indication of the turnover rate of that molecule and is represented as Ty2 (half time of recovery). As well, the percent of fluorescently tagged molecule that returns to the site is represented as the mobile fraction (Mf). The results shown in Fig. 5 and Fig. 6 demonstrate the differential effect of R O C K inhibition on the Ti/ 2 and M f of actin-GFP protein and Sytol4 labeled mRNA. Figure 5 (D-F) shows that R O C K inhibition has no significant effect on actin turnover in cells stably expressing actin-GFP. The Ty2 (Fig. 5 H) and mobile fractions (Fig. 5 I) of untreated and Y27632 treated cells reflect that there is no difference between the two. The actin-GFP protein contains only the coding region of actin and not the 5'or 3'UTRs that may encode mRNA localization signals. Therefore this result reflects the ability of actin to diffuse to the pseudopodia and does not indicate the use of active transport for actin localization. It does however demonstrate the highly dynamic, ROCK-independent nature of actin turnover in the pseudopodia. Similar experiments using Sytol4, a dye labeling cytoplasmic mRNA (Knowles and Kosik, 1997), show that R O C K inhibition slows mRNA turnover in actin rich protrusions. The treatment of MS V - M D C K - I N V cells with Y27632 results in the development of larger and more spread actin rich protrusions that are still classified as pseudopodia, but are distinct from the slender finger-like protrusions seen in untreated cells. Although it was previously shown (Fig. 3) that inhibition of R O C K prevents 70 prebleach bleach postbleach A 3 jg^jjbiii . > C { f J D t F I t -1 1 • Control •Y27632 I r i 1 1 1 1 i i i 0 10 20 30 40 50 60 70 80 90 100 Time (a) 2 J H a l f t i m e o f r e c o v e r y (T%) M o b i l e f r a c t i o n (Mf ) c o n t r o l Y27632 c o n t r o l Y27632 Figure 5. A c t i n t u r n o v e r i n M S V - M D C K - I N V c e l l p s e u d o p o d i a is R O C K i n d e p e n d e n t . Fluorescence recovery after photobleaching (FRAP) was used to determine the effect of R O C K inhibition on actin turnover in the pseudopodia. Cells stably expressing pEGFP-actin were imaged prior to bleaching (A, D), immediately after bleaching (B, E) and 90 s after bleaching (C, F). Cells in panel (D-F) were treated with Y27632 for 1 hr prior to imaging. The white square outlines the region that was bleached and the inset of each picture shows a pseudo-color, zoomed image of this region. (G) A representative graph of three experiments shows the fluorescence intensity of the bleached region over time following bleaching at t = Osec. Untreated cells are shown in blue and Y27632 treated cells in pink. Values have been normalized to the pre-bleach intensity. (H) Comparison of the half time of recovery between untreated and Y27632 treated cells shows no significant difference. (I) The mobile fraction represents the percent of fluorescence recovered in the bleached region. The bar graph shows no significant difference between the mobile fractions of untreated and Y37632 treated cells. Scalebar 20 um. 71 prebleach bleach postbleach • C P W'm a Control +Y27632 1 0 1 5 Time (s) 1 f, Half time of recovery (T% p , Mobile Fraction (Mf) .o.e Control Y27632 Control Y27632 Figure 6. mRNA turnover in MSV-MDCK-INV cell pseudopodia is ROCK dependent. Fluorescence recovery after photobleaching (FRAP) was used to determine the effect of R O C K inhibition on mRNA turnover in the pseudopodia. Cells were labeled with cell permeant dye Sytol4 to identify nucleic acids in the cytoplasm. Images were taken prior to bleaching (A, D), immediately after bleaching (B, E) and 30s after bleaching (C, F). Cells in panel (D-F) were treated with Y27632 for Ihr prior to imaging. The white square outlines the region that was bleached and the inset of each picture shows a pseudo-color, zoomed image of this region. (G) A representative graph of three experiments shows the fluorescence intensity of the bleached region over time following bleaching at t = Osec. Untreated cells are shown in blue and Y27632 treated cells in pink. Values have been normalized to the pre-bleach intensity. (H) Comparison of the half time of recovery between untreated and Y27632 treated cells. Cells treated with Y27632 show a significantly increased time for fluorescence to return to the bleached area. (I) The mobile fraction represents the percent of fluorescence recovered in the bleached region. The bar graph shows no significant difference between the mobile fractions of untreated and Y27632 treated cells. (±S.E.; *, pO.Ol ) . Scalebar 20 pm mRNA localization to the pseudopodia, this blockade is hot complete in all cells and small levels of mRNA can be observed in the pseudopodial domains. Therefore, the FRAP experiments in Fig. 6 show the effect of Y27632 treatment on the turnover of mRNA in cells still displaying some pseudopodial localization (Fig. 6 D-F). The result indicated that inhibition of R O C K decreases the turnover of mRNA in the pseudopodia as demonstrated with an increased T>/2 compared to control cells (Fig. 6 H). The difference in M f between treated and untreated cells shows no significant differences however, the observed trend shows a decreased M f in the presence of Y27632 (Fig. 6 I). This evidence supports the initial result that R O C K is important in regulating mRNA transport and turnover in the pseudopodia. Pseudopodia contain a distinct spectrum of mRNA The pseudopodia contain a distinct protein complement as made evident in the proteomic screen prepared on this fraction. In addition to the observed presence of mRNA in the pseudopodial domain to it would be intuitive to assume that the pseudopodia may also contain a distinct selection of mRNAs. The pseudopodial fraction was isolated as before and the lysate was analyzed by Affymetrix microarray analysis. When compared to the cell body fraction there were a number of differences in gene expression as shown in Fig. 7 (A), where the red bars indicated upregulated genes and the blue bars downregulated genes. The genes encoding proteins that were upregulated in the pseudopodia could be classified into the following categories; phosphatases, phospholipases, ras-related, signaling, R N A related, ubiquitination-associated, involved in cell cycle, cytoskeletal, ribosomal, translation or transcription-associated, transport and 73 1 , 2 3 1 2 3 8? B 4 -1£ 0 *1.5 +3 C P s e u d . R N A / T o t a l R N A 0 10 20 30 40 SO 60 70 Syntrophin Actin sense w A*. Actin • Fiforanectin Shp2 i n ••:V. m-ras Figure 7. M S V - M D C K - I N V cell pseudopodia contain a distinct mRNA complement. Pseudopodia were isolated and mRNA content was analyzed. (A) Results from an Affymetrix gene array of mRNA from 3 preparations of cell bodies and pseudopodia from M S V - M D C K - I N V cells. Upregulated genes are in red and downregulated genes in blue. 23 913 genes were identified. (B) Brightfield (left column) and differential interference contrast (right column) images of M S V -M D C K - I N V cells. Images are taking following in situ hybridization with oligonucleotides to the sense strand of actin and the anti-sense strands of actin, fibronectin, Shp-2 and m-ras mRNAs. Dark spots indicate the presence of DIG (digoxigenin) tailed oligonucleotide bound to its complementary mRNA molecule. Images of cells hybridized with actin sense oligonucleotides shown high background labeling but no labeling characteristic of mRNA. Labeling for actin, Shp-2 and m-ras is apparent in the pseudopodia, but fibronectin is primarily in the cell body. (C) The amount of mRNA in the pseudopodia is quantified as a fraction of the total mRNA in the cell. mRNA labeling was documented manually by counting the number of spots in the pseudopodia and cell body. The pseudopodia are defined as anything in an extension beyond where it narrows away from the cell body. Results taken from two independent experiments. Scalebar 20 um. 74 metabolic. The existence of mRNAs in the pseudopodia encoding translation associated proteins provides further evidence for local translation in this domain. Altogether, the combined presence of these genes supports the hypothesis that pseudopodia are localized signaling domains that are actively driven by a dynamic cytoskeleton. A selection of the mRNAs that were identified as highly upregulated in the pseudopodia was chosen to have their localization validated by in situ hybridization (Fig. 7 B). These mRNAs included actin, Shp2 (Src homology 2 phosphotyrosyl phosphatase, a nonreceptor-type phosphatase), M-ras (Ras small GTPase), RhoA (Rho small GTPase), p41 (subunit of Arp2/3 complex), and syntrophin (peripheral membrane protein with unique signaling properties) (Oak et al., 2001). P-actin and Arp2/3 p41 subunit mRNA have been previously demonstrated to localize at or near membrane protrusions (Latham et al., 2001; Mingle et al., 2005). As well, overexpression of RhoA mRNA has been associated with tumor progression (Horiuchi et al., 2003; Kamai et al., 2001). Both M -ras and Shp-2 have been linked to HGF induced signaling (Duan et al., 2006; Zhang et al., 2004). Therefore, as MSV-MDCK-rNV cells are regulated via an HGF signaling pathway (Vadnais et al., 2002), it was of interest to investigate the localization of M-ras and Shp-2 mRNA. Lastly, syntrophin has been shown to be upregulated in M S V -M D C K - I N V cells (unpublished data). Fibronectin mRNA, identified as being largely downregulated in the pseudopodia, was used as a control. Oligonucleotides ranging in length from 45-55 base pairs were prepared for each mRNA taking into account primary and secondary structure properties that might.prevent optimal binding. The oligonucleotides were DIG tailed and allowed to hybridize to mRNA in individual M S V -M D C K - I N V cells. Following hybridization the bound oligonucleotides were identified 75 using an alkaline phosphatase detection system that allowed for visualization through a light microscope. Differential phase contrast and brightfield images (Fig. 7 B) were acquired of the labeled cells and the amount of mRNA in the pseudopodia was quantified (Fig. 7 C). Hybridization with an oligonucleotide specific for the sense strand of P-actin mRNA was used as a negative control and while the image shows a strong background labeling the typical punctuated labeling seen when an oligonucleotide is bound to mRNA is not observed. Quantification was done manually by counting the number of visible mRNA granules in the pseudopodia and cell body. The pseudopodia were defined as the region beyond the cell body where narrowing begins to occur. Pseudopodial mRNA is presented as a fraction of the total mRNA in the cell. . The results shown in the Fig. 7 (C) confirm the results from the microarray data in that M-ras, RhoA, p41, Shp2 and syntrophin are all present in the pseudopodia at levels similar to that of P-actin. Conversely, fibronectin mRNA is decreased in the pseudopodia by in situ hybridization. The in situ hybridization results confirm the presence of specific mRNAs in the pseudopodial regions, but not the upregulation that was indicated in the microarray analysis. This is reasonable and to be expected as the concentration of mRNA in the samples submitted for microarray analysis was the same for pseudopodial and cell body fractions. Alternatively, the concentration of total mRNA in the pseudopodia in situ is substantially less than that of the cell body. Therefore, it is difficult to show enrichment of specific mRNA in the pseudopodia by in situ hybridization. 76 Localization of mRNA to the pseudopodia is microtubule independent The previous results demonstrate that the pseudopodia are home to a unique spectrum of mRNAs different from the cell body. Due to the transient nature of pseudopodia it is unlikely that R N A can diffuse quickly enough through the cytoplasm to reach the tip of the protrusion in time to have the necessary effects. Therefore, a method of active transport, most likely via actin microfilaments or microtubules and similar to what has been observed in fibroblasts (Latham et al., 2001) or oligodendrocytes (Carson et al., 1997), respectively, is predicted. It has previously been shown that treatment of M S V - M D C K - I N V cells with the microtubule depolymerizing agent, nocodazole, for 30 min (Fig. 8 E) results in the majority of cells becoming round and exhibiting dynamic membrane blebs (Jia et al., 2006). The remainder of cells become spread and develop actin stress fibres. However, following a 90 min nocodazole M S V - M D C K - I N V cells show recovery in that stress fibres diminish and actin rich pseudopodia reform in the absence of microtubules (Fig. 8 B). It was this result that raised the question, upon reformation of pseudopodial domains what happens to mRNA localization? Figure 8 demonstrates the effects of four conditions on mRNA localization in M S V - M D C K - I N V cells. Experimental conditions included; nocodazole treatment for 90 min (Fig. 8B), nocodazole treatment for 30 min followed by a 60 min wash in normal media (Fig. 8 C) and nocodazole treatment for 30 min followed by a 60 min wash in the presence of Y37632 (Fig. 8 D). Cells were fixed and labeled for nuclei (Hoechst), RNA (SytoRNA select) and F-actin (Alexa phalloidin 647). The lower panel of each image shows M S V - M D C K - I N V cells labeled for tubulin. 77 Control Noc90 Noc30+WO60 Noc30+WO60+Y / _ 1 4 B I C n 1 "x mm T u b u l i n Jf Noc30 Control Noc90 Noc30 Noc30+ +WO60 (WO60 +Y) Figure 8. mRNA translocation to the pseudopodia is microtubule independent in M S V - M D C K -INV cells. Following treatment with nocodazole for 90 min, mRNA localizes to the pseudopodia. Immunofluorescence images of cells labeled with a nuclear dye (Hoechst, blue), an mRNA dye (Syto RNASelect, green) and an antibody to (3-actin (detection with a secondary antibody conjugated to Alexa 647, red) on the right and cells labeled with anti-tubulin (detection with a secondary antibody conjugated to Alexa647, green). Tubulin labeling shows the presence or absence of intact microtubules. (A) Untreated cells, (E) cells treated with Nocodazole for 30 min show characteristic rounded and blebbed phenotype. (D) Cells treated with Nocodazole for 30 min, then washed out (WO) in media with Y27632 for 60 min display a decrease in mRNA in the pseudopodia. (C) Cells treated with Nocodazole for 30 min, then washed out in media with for 60 min show a return of mRNA to pseudopodial domains. (F) When treated with Nocodazole for 90 min mRNA is localized to newly formed pseudopodia. (F) Amount of mRNA in pseudopodia is quantified by determining the colocalization of Syto RNASelect labeling with p-actin labeling. Pseudopodial mRNA localization compared with control cells is significantly decreased in cells washed out in the presence of Y27632, but not following 90min Nocodazole treatment. (±S.E.; *, p<0.05). Scalebar 10 pm. 78 Tubulin labeling demonstrates the presence of intact microtubules (Fig 8. A , C, D) or depolymerized microtubules (Fig. 8 B) following each treatment. The amount of mRNA in the pseudopodia was quantified as before in that a mask of actin regions was prepared that was used as an outline to determine the average pixel density of mRNA labeling in this region (Fig. 8 F). The presence of Y27632 restricted mRNA translocation to the pseudopodia as expected from previous results, while washing with regular media allowed mRNA to relocate to newly formed pseudopodia. Treatment with nocodazole for 90 min resulted in not only a redistribution of mRNA to the pseudopodia, but a slight (if non-significant) increase in R N A labeling in this area. Based on this evidence it can be concluded that mRNA localization to pseudopodial domains is not reliant on intact microtubules in M S V - M D C K - I N V cells. 79 Chapter 3 Discussion The Rho-dependent pseudopodia of MSV-MDCK-INV cells During cell polarization, M S V - M D C K - I N V cells develop multiple actin-rich pseudopodia that are characterized by their transient nature and Rho-dependence. This type of pseudopodia is both similar and dissimilar to pseudopodia in other cell types. For example the pseudopodia of MDA-MB-231 are also regulated by a Rho/ROCK signaling pathway (Cardone et al., 2005). Alternatively, Rac and cdc42 have been shown to regulate lamellipodial and filopodial protrusions, respectively (Burridge and Wennerberg, 2004; Raftopoulou and Hall, 2004). In this study we show that these pseudopodia are localized signaling domains with distinct protein and mRNA complements different from those existing in the cell body. This result was obtained by proteomic and microarray analysis and validated by immunofluorescence labeling and in situ hybridization for a selection of proteins and mRNA, respectively. These domains are locally regulated by a Rho/ROCK signaling pathway that is involved in regulating the translocation of mRNA to the pseudopodia. The localization of mRNA occurs in a microtubule independent manner, as the effect of long term nocodazole treatment shows small increases in mRNA delivery. An increase in the levels of GTP-bound Rho in the pseudopodia relative to the cell body demonstrates that there is differential activation of Rho in M S V - M D C K - I N V cells. As well, Rho activation in the pseudopodia is preferential to Rac activation as shown by a series of FRET experiments using single molecule activated RhoGTPase sensors. However, the inhibition of R O C K can lead to local Rac activation in these distinct protrusive domains indicating that there is a regulatory balance between members of the RhoGTPase family. 80 Many different membrane protrusions have been characterized, most playing some role in cell motility. Among these are lamellipodia and filopodia, which are regulated differentially by Rac and cdc42 GTPases (Nobes and Hall, 1995a; Ridley et al., 1992). Rac has been shown to induce actin polymerization that drives leading edge protrusion as well as facilitating the development of small focal complexes that serve as temporary anchors to the extracellular matrix during cell migration (Beningo et al., 2001; Nobes and Hall, 1995b). The induction of tightly packed actin bundles to produce thin filopodial protrusions is regulated by cdc42 and these structures are thought to function primarily in directional sensing and in response to chemotactic agents (Kozma et al., 1995; Nobes and Hall, 1999). The functional role of Rho has traditionally been in developing stress fibres, promoting focal adhesions and increasing contractile force at the rear of the cell through the activation of myosin (Ridley, 2001; Ridley and Hall, 1992). However, recent studies have shown that Rho also plays a role in promoting pseudopodial protrusion in MSV-MDCK-PNV cells (Jia et al., 2006). The lack of stress fibres and focal adhesions in M S V - M D C K - I N V cells maybe a consequence of the rapid temporal regulation of Rho activation. The pseudopodia, where the highest level of Rho activation occurs, are highly dynamic and perhaps Rho proteins are activated only long enough to stimulate protrusion, but not to allow actin microfilament stabilization or focal complex maturation. It has been shown that lysophosphatidic acid (LPA) induces pseudopodial extension in M S V - M D C K - I N V and that this phenotype is associated with increased cell motility (Jia et a l , 2006). This type of motility is characterized by transient rounding and blebbing of the cell membrane followed by cell spreading and the development of 81 multiple actin rich pseudopodia. A similar mode of motility has been described by Sahai and Marshall (2003) for A374 melanoma cells that display a rounded, blebbed morphology, increased Rho activation compared to elongated cells and sensitivity to R O C K inhibition during migration. This could also be likened to the mesenchymal and amoeboid modes of motility associated with invasive cancer cells (Sahai, 2005). Mesenchymal motility is distinguished by an elongated cell morphology (Friedl and Wolf, 2003), Rac activation at the leading edge (Ridley et al., 2003), and protease dependent degradation of the extracellular matrix (Nabeshima et al., 2002). Amoeboid cell motility exhibits weak and diffuse focal adhesions (Friedl, 2004), Rho/ROCK dependency (Sahai and Marshall, 2003) and protease independent migration (Wolf et al., 2003). Therefore it is plausible that Rho proteins are involved in discrete signaling pathways that, depending on cell type and extracellular environment, can either promote or discourage tumor cell invasion. Evidence for a negative role for Rho/ROCK signaling in invasion is the fact that inhibition of R O C K promotes motility and invasion in astrocytomas (Salhia et al., 2005). As well, in a separate study the up-regulation of AP-1 transcription factor in rat fibroblasts indirectly suppresses Rho/ROCK signaling leading to increased membrane protrusion and mesenchyme-like invasion (Spence et al., 2006). However, many groups have also shown that activation of the Rho/ROCK pathway leads to enhanced invasivity, including the demonstration that Rho activation is necessary for enhanced N F - K B (nuclear factor-kappa B ) transcriptional activity leading to invasion in human prostate cancer cells (Hodge et al., 2003). Additionally, the RhoGAP protein, DLC1 (deleted in liver cancer), inactivates Rho and leads to a decrease in invasion in 82 hepatocellular carcinoma cells (Wong et al., 2005). Thus, it is evident that there are multiple consequences of Rho/ROCK signaling. In the M S V - M D C K - I N V cell line activation of Rho/ROCK signaling leads to the formation of transient, blebbed pseudopodial protrusions that facilitate tumor cell motility. The consequences of RhoGTPase signaling in cells are large and diverse. The functional specificity of these consequences can be facilitated by the localization of signaling proteins to specific cellular domains. This compartmentalization of signaling makes it possible to differentially regulate numerous cell systems. Previous studies have shown concentrated pools of activated Rac in membrane ruffles that form a decreasing gradient of activation from the leading edge of motile fibroblasts (Kraynov et al., 2000). Other groups have shown RhoA activation directly at the edge of membrane protrusions with low levels found in the cell body of HEK293T cells (Pertz et al., 2006). Using a FRET based probe for activated Rho and Rac proteins we have shown an increase in Rho activation in the pseudopodial domains of M S V - M D C K - I N V cells in comparison to the cell body. Rac activation in untreated cells displays no significant differences between the pseudopod and cell body regions. This supports evidence that local Rho signaling is involved in regulating pseudopodial protrusions (Jia et al., 2005). Extension of this result shows that upon R O C K inhibition there is a decrease in Rho activation in the pseudopodia. This effect is also seen when cells were cotransfected with a dominant active Rac construct. However, it should also be noted that upon dominant active Rac expression cell morphology changed drastically displaying a round spread phenotype with characteristic lamellipodia. Rho activation was measured in regions of membrane protrusion, in this case lamellipodia rather than characteristic M S V - M D C K - I N V cell 83 pseudopodia. Activation of Rac can be stimulated by R O C K inhibition (Salhia et al., 2005), which correlates well with our data demonstrating that treatment of cells with Y27632 leads to increased Rac activation in the pseudopodia. Together, this is indicative crosstalk between Rho and Rac signaling as initially observed by Sander et al. (1999) when Rac activation led to inhibition of Rho in migrating cells. This could occur via activation of Pakl , an effector of Rac, which inhibits guanine nucleotide exchange factor (GEF), Netl , that is specific for RhoA (Alberts et al., 2005). Altogether, our evidence points to the pseudopodia of M S V - M D C K - I N V cells as a Rho regulated signaling domain that is maintained by balancing the activation Rho and Rac directed signaling pathways. Given the delicate signaling balance that occurs in the pseudopodia it is reasonable to assume that this involves the contribution of many other proteins. This is demonstrated by identification of a protein complement in the pseudopodial domain that is unique from that in the cell body. Previous studies have used similar techniques to ours for isolating the pseudopodial fraction of cells, however we are the first to use 1 um pore filters (as opposed to 3-8 pm) (Beckner et al., 2005; Cho and Klemke, 2002; Jia et al., 2005). The small size of the pore, which allows only pseudopodia to pass through and not cell body components such as nuclei, limits the potential for contamination of this fraction with cell body associated proteins. With this said, we were able to categorize many of the proteins that were designated as present in the pseudopodia. As could be expected the largest and most abundant group were cytoskeletal and associated proteins, P-actin being a main contributor. Although P-actin and its binding partners are important in developing and 84 maintaining pseudopodia, microtubules have also been shown to extend into these protrusions and we have identified tubulin as a major constituent of the pseudopodia. In these cells it is difficult to identify a dominant pseudopod, as is the case with many other membrane protrusions (eg. lamellipodia), due to their transient nature. The microcomposition of individual pseudopods may vary temporarily, however, the major constituents, such as actin and tubulin are likely present at comparable levels in all pseudopodia. The presence of many proteins identified in the pseudopodial screen were validated both by immunofluorescence, included in this thesis, and by western blot (Jia et al., 2005). Several adhesion proteins were detected including vinculin, talin, integrin p i , and paxillin as well as the downstream regulatory protein F A K and its activated form phospho-FAK. While focal adhesions do not appear to be present in the pseudopodia, as seen in the immunofluorescence images of M S V - M D C K - I N V cells labeled for adhesion markers, this does not mean that transient focal complex formation is not occurring. Fluorescence recovery after photobleach experiments (FRAP) investigate the turnover of actin in the pseudopodial domain. The half time of recovery for actin in both untreated and Y28632 treated cells is between 14s and 15s, which is comparable to actin turnover observed by other researchers (Guha et al., 2005). As well, the percentage of actin recovery in both control and treated cells is greater than 75% (mobile fraction). This indicates firstly, that there is a significant amount of actin turnover in the pseudopodia and secondly, that this turnover can occur independently from R O C K signaling pathways. As already shown, R O C K inhibition leads to an increase in Rac activation in the pseudopodia. Rac activation in many cells leads to increased actin dynamics and lamellipodial protrusion (Nobes and Hall, 1995a; Rottner et al., 1999). Therefore, while 85 Rac may induce a conformational change in the pseudopodia, it is reasonable to assume that signaling downstream of Rac could still maintain actin turnover. Other proteins that were picked up in the proteomic screen and validated by immunofluorescence include intracellular chaperones, Hsp90 and HSC70, ubiquitin-proteasome associated ATPase, p97, as well as an endoplasmic reticulum protein, BiP and the proteins found in the nuclear envelope, lamin A / C and nucleoporin complex proteins. Molecular chaperones and ubiquitin-proteasome associated proteins are important for essential cellular functions such as protein folding, translocation and turnover (DeLaBarre and Brunger, 2005; Whitesell and Lindquist, 2005). Therefore the presence of such proteins in the pseudopodia is evidence for a dynamic signaling domain that recruits its own cellular machinery. The presence of BiP in the pseudopodia has been confirmed however its functional role in unclear. BiP is also known as glucose-related protein-78 (GRP-78) and has been shown to confer protection against stress conditions in solid tumors (Miyake et al., 2000; Reddy et al., 2003). As well, BiP/GRP78 was apparently shown to be preferentially expressed on the surface of metastatic tumors cells (Arap et al., 2004). Therefore, it likely has a role in mediating the stress response in tumor cells. The function of the nuclear proteins that were identified in the screen has not been elucidated, however, immunofluorescence labeling confirmed their attendance in the pseudopodia, i f not their enrichment. Pseudopodial isolation and analysis has been performed in similar studies and shows the upregulation of many cytoskeletal proteins, glycolytic enzymes and molecular chaperones (Beckner et al., 2005). Elongation factor l a (EFla) was also identified in the pseudopodial fraction and in addition to being necessary for protein elongation during translation, it likely has a 86 number of other cellular functions. E F l a is the second most abundant protein in the cell, next to actin, and comprises 1-2% of cellular protein (Dharmawardhane et al., 1991; Slobin, 1980). It associates with both actin filaments (Liu et al., 1996b) and microtubules (Durso and Cyr, 1994) and has been shown to be an important factor in regulating cell polarity (Fulton, 1993; Kislauskis et al., 1994). In addition to playing a role in P-actin mRNA localization (Liu et al., 2002), which will be discussed later on, E F l a may help to mediate the cytoskeleton in non-translational events. For example, in response to chemotactic agents, dramatic changes occur in E F l a localization increasing its association with actin filaments (Dharmawardhane et al., 1991). As well, it has been linked with the induction of microtubule depolymerization events (Shiina et al., 1994). Therefore, with evidence pointing to a broad role in cytoskeletal regulation, it is not surprising that E F l a has been associated with tumors having high metastatic potential (Pencil et al., 1993; Taniguchi et al., 1991) and that a truncated and mutated form of the protein has been identified as a prostatic carcinoma oncogene (Shen et al., 1995). Thus the presence of E F l a in the pseudopodia most likely facilitates a number of processes including cytoskeletal rearrangement, mRNA translocation and protein translation. Functional implication of RNA targeting to the pseudopodial domain The polarization of a cell or the asymmetric distribution of its components is facilitated by the distribution of proteins to specific intracellular sites. Many proteins contain signaling sequences that serve as cytoplasmic road maps and direct their translocation within the cell. For example, several regions are required for p21-ras 87 GTPase to localize to the plasma membrane, including a C A A X box and either a polybasic region or a palmitic acid tag (Hancock et al., 1990). Another method of attaining cellular polarization is through the localization of mRNA prior to protein translation. Localizing mRNA can be an extremely efficient system as shown in neurons that can target approximately different 400 mRNA molecules to their dendrites (Eberwine et al., 2001). There are two main reasons why targeting of mRNA may be advantageous. Firstly, targeting of an mRNA molecule not only ensures its correct localization but also mediating translation confers an additional level of regulation. This is important during polarization so that separate cellular compartments can engage in dissimilar processes. For example, in the drosophila embryo, the translation of oskar or nanos mRNA in an inappropriate location can lead to the development of a second abdomen in place of a head and thorax (Ephrussi et al., 1991; Gavis and Lehmann, 1992). Secondly, the targeting of mRNA relinquishes the responsibility of translation regulation to the region of the cell that it is localized. This allows for a rapid response to stimuli affecting that region and the independent regulation of distinct cellular compartments. For example, different groups of mRNAs are targeted to growth cones and dendrites in mammalian neurons (Steward and Scoville, 1976). This is necessary for the response to axon guidance cues and the ability to influence synaptic plasticity, respectively. Although the exact mechanism governing differential mRNA localization is unclear, it is possible that unique zipcodes in the 3'UTRs of these molecules could dictate targeting (Ferrandon et al., 1994). Studies show that P-actin mRNA localizes to the leading edge of fibroblasts and that this event is dependent on Rho signaling (Latham et al., 2001). As well as there is 88 evidence that E F l a is involved in transport and anchoring of mRNA in cell protrusions (Liu et al:, 2002). The fact that the pseudopodial domains of our cell model are Rho/ROCK dependent and enriched in E F l a protein provides a good indication that mRNA is present in the pseudopodia. The first indication of this was the immunofluorescence labeling of cytosolic mRNA by propidium iodide. Given that treatment of the cells with RNAse completely erased all cytoplasmic labeling, the fluorescence that was observed extending away from the cell and into the actin rich regions of the pseudopodia is therefore mRNA. The interesting result is that following treatment with R O C K inhibitor, Y27632, mRNA is no longer localized to the pseudopodial protrusions. As previously noted, Y27632 treatment does affect the morphology of the pseudopodia, however, the cells still maintain distinct actin rich protrusions and it is in these structures that mRNA localization has decreased. Therefore we have shown that mRNA translocation to M S V - M D C K - I N V cell pseudopodia is dependent on R O C K signaling. This is in agreement with a study by Latham et al. (2001) showing that the targeting of P-actin in fibroblasts is mediated by a Rho/ROCK pathway. The effect of Rho/ROCK signaling is supported by the cellular response to mRNA localization during FRAP experiments. While R O C K inhibition does significantly decrease the amount of mRNA observed in the pseudopodia, there is a residual labeling appearing in this domain in some cells. Whether it is mRNA that has yet to be relocalized or degraded or whether the localization of certain mRNA species is independent of Rho/ROCK signaling is unknown. However, it was shown that the inhibition of R O C K slowed the return of this residual mRNA to the pseudopodia. This is in comparison to P-actin protein where turnover was not affected by treatment with 89 Y27632. Rho/ROCK signaling may therefore play a larger role in mRNA trafficking than in pseudopodial actin dynamics. This prediction is complemented by the fact that while R O C K inhibition alters cell morphology it has little effect on the presence of actin densities in the protrusions. Therefore, the cell may be relying on separate protein localization mechanisms to distribute protein within the cell. This could explain why in motility assays following R O C K treatment M S V - M D C K - I N V cells display a decreased motility trend (Jia et al., 2005), while other cell lines, that may be differentially regulated, show increased motility with R O C K inhibition (Li et al., 2006; Salhia et al., 2005). The question arose as to the specificity of the mRNAs that were evident in the pseudopodia. Therefore a microarray analysis was performed on the pseudopodial fraction and a large number of mRNAs were identified as enriched in this region. A selection of mRNAs with a high fold increase in comparison to the cell body was selected for experimentation by in situ hybridization. The presence of P-actin, Shp2, M-ras, Arp 2/3 p41, RhoA and syntrophin a l in the pseudopodia was confirmed using this method, however, functional experiments have not yet been undertaken. While the significance of specific mRNAs in the pseudopodia needs clarification it is important to note that their combined presence is different from the mRNA complement comprising the cell body fraction. The presence of P-actin mRNA in the pseudopodia is not surprising as this domain is enriched in P-actin protein and other studies have shown its localization to leading edge protrusions. There are several reasons why P-actin mRNA localizes to pseudopodia. These help to explain the role of mRNA localization in the development of pseudopodial protrusions. Firstly, the p-actin mRNA isoform demonstrates a leading 90 edge localization pattern as opposed to a-actin and y-actin mRNA species that localize primarily around the nucleus (Hill and Gunning, 1993; Taneja and Singer, 1990). The fact that P-actin mRNA is localized in the same region where P-actin protein is polymerized and used to form filaments that promote membrane protrusion is a good indication that it is contributing to local protein synthesis. This would be beneficial in M S V - M D C K - I N V cell pseudopodia as the transient nature of this domain requires constant actin filament polymerization and depolymerization events. Secondly, going on the assumption that an average cell has 2500 P-actin mRNA molecules and that each of these can produce 1.5 P-actin proteins/second, a typical cell could produce 3900 P-actin proteins per second or 2.4xl0 5 P-actin proteins/minute (Kislauskis et al., 1997). The predicted requirement for G-actin at the leading edge of a moving cell is 3.6xl0 6 actin proteins/minute (Chan et al., 1998). Therefore, using these calculations for P-actin supply and demand, the mRNA present in the cell could produce 7% of the protein required for polymerization events. If these mRNAs were dispersed through out the cell, spontaneous translation would have a limited effect on actin requirements. However, if P-actin mRNA is concentrated in a functional region it could contribute more significantly to meeting actin protein requirements. This rationale corresponds well with the pseudopodia in our model in that the actin rich domains are separated from the cell body by a narrow membrane extension and the localization of mRNA is this domain would be able to contribute considerably to local protein requirements. Thirdly, the ability to locally regulate mRNA translation would allow efficient and directed cellular responses. For example, EGF stimulation of metastatic MTLn3 at the membrane induces rapid local nucleation events that lead to enhanced lamellipod protrusion (Chan et al., 91 1998). Therefore, cellular responses are made more efficient by localized regions reacting to external cues first and then directing the remainder of the cell. This is what appears to be taking place in M S V - M D C K - I N V cell pseudopodia. The function of pseudopodial localization of mRNAs whose presence was validated by in situ hybridization experiments is unknown. However, knowledge about the roles played by their protein products may lend itself to speculation of mRNA functionality. The role of P-actin mRNA has been discussed and the possible roles of p41 (a subunit of the Arp2/3 complex) and RhoA mRNA are apparent. That is briefly, the Arp2/3 complex is a necessary constituent in the actin filament nucleation process and therefore the presence of all its subunits would be necessary for efficient actin polymerization. As already discussed Rho proteins likely play a key role in pseudopodial regulation and therefore it would seem intuitive to have RhoA mRNA present for local translation events. Corroborating evidence shows increased RhoA mRNA expression in testicular germ cell tumors that increases with respect to tumor progression (Kamai et al., 2001). The possible roles for m-ras, Shp2 and syntrophin are not quite as obvious, however, the literature regarding these proteins points to some interesting options. Traditionally, the ras family of proteins has been involved in cell growth, differentiation, transformation and apoptosis (Bollag and McCormick, 1991; Lowy and Willumsen, 1993). However, m-ras was identified by cloning cDNAs from skeletal muscle cells and was shown to induce actin rich microspikes upon injection into fibroblasts (m is for muscle and microspikes) (Matsumoto et al., 1997). Interestingly, two hours after microinjection cells displayed a loss of actin stress fibres and developed multiple actin rich foci. Other studies have shown that overexpression of m-ras is 92 sufficient to induce E M T in epithelial cells (Ward et a l , 2004) and that this transformation is dependent on autocrine HGF signaling (Zhang et al., 2004). These results correspond with the M S V - M D C K - I N V cell phenotype that lacks actin stress fibres, displays multiple actin rich domains and is regulated by an autocrine HGFR activation loop (Vadnais et al., 2002). Therefore m-ras could play a role in regulating actin cytoskeletal dynamics and is potentially involved in signaling upstream of Rho as shown for other ras proteins (Wozniak et al., 2005). Shp-2 is a well known non-receptor tyrosine phosphatase that is unique among cytosolic phosphatases in that, along with Shp-1, contains tandem SH2 (src homology 2) domains in its N-terminal (Poole and Jones, 2005). It is the binding of phosphopeptides to these SH2 domains that is thought to activate phosphatase activity in Shpl/2 proteins. Shp-2 activation has been linked to the regulation of RhoA in several studies, however, it is evident that this regulation appears be both positive (Inagaki et al., 2000) and negative (Kodama et al., 2000) with the distinguishing factor being unknown. One known regulatory mechanism involves the upstream effector of RhoA, p l 9 0 R h o G A P . p l 9 0 R h o G A P is a GTPase activating protein (GAP) that catalyzes GTP hydrolysis on activated RhoA. Therefore, dephosphorylation of p l 9 0 R h o G A P b y Shp-2, inactivates the GAP and promotes RhoA activation (Sordella et al., 2003). Conversely, Shp-2 also dephosphorylates the non-receptor tyrosine kinase, src, and activates it. Activated src phosphorylates p i Q Q R h o G A P which stimulates GTPase activity and suppresses RhoA (Arthur et al., 2000). Additionally, Shp-2 may be responsible for dephosphorylating and inactivating certain RhoGEFs (guanine nucleotide exchange factor), which are responsible for exchanging GDP for GTP on Rho proteins (Kodama et al., 2000; Schoenwaelder et al., 2000). It is 93 interesting to note that activation of src is also necessary for ZBP1 (zipcode binding protein 1) mediated translation of p-actin mRNA (Huttelmaier et al., 2005). Therefore, assuming it is locally translated, the presence of Shp-2 mRNA in the pseudopodia may have a dramatic role in regulating the activation of RhoA. Syntrophin a l is a peripheral membrane protein that is primarily expressed in striated muscle cells (Froehner, 1984). It has been shown to associate with dystrophin and as part of a complex is proposed to stabilize the sarcolema by linking the actin skeleton to the extracellular matrix (Ervasti and Campbell, 1993b). Syntrophin a l is able to bind directly to F-actin (Iwata et al., 1998) and colocalize with F-actin fibres in Chinese hamster ovarian cells (Iwata et al., 2004). Interestingly, upon stimulation with L P A syntrophin a l translocates to the plasma membrane (Iwata et al., 2004). Therefore, while the exact role is not clear, it is evident that syntrophin is associated with the actin cytoskeleton and is at some point regulated by Rho activation. This could support a functional role for the presence of its mRNA in the pseudopodia. The localization of mRNA in the pseudopodia M S V - M D C K - I N V cells has been established. However, the mechanism by which mRNA molecules are transported within these cells was not clear. Active transport along components of the cytoskeleton has been ascertained in a number of model systems. Mechanisms involving transport along microtubules have been described for myelin basic protein (MBP) mRNA (Carson et al., 1997; Worboys, 1994). The delivery of M B P mRNA to myelin membranes of oligodendrocytes was shown to be dependent on the presence of intact microtubules and the plus end microtubule motor protein, kinesin. The use of both microtubule and microfilament disrupting agents (eg. Nocodazole or cytochalasin) demonstrated that the 94 mRNA localization was selectively dependent on microtubules rather than actin microfilaments. As well, the requirement for kinesin indicates that mRNA is actively transported to its intracellular destination. In contrast, Latham et al. (2001) show that transport of P-actin mRNA to the leading edge of fibroblasts is dependent on the presence of intact actin microfilaments and functional myosin II B motor protein. They postulate that mRNA -containing complexes can bind to myosin and be transported along microfilaments to protrusions. This hypothesis is supported by the observed mRNA localization to the leading edge following activation of the Rho/ROCK signaling pathway. The explanation for this being that R O C K signaling results either in the direct phosphorylation and activation of myosin light chain (MLC) or the phosphorylation and inactivation of myosin light chain phosphatase (MLCP). The effect of both of these events is the activation of myosin and the induction of actomyosin contraction in the cell. Therefore a model of local Rho and R O C K activation that stimulates myosin activity and subsequent mRNA transport is attractive. Our investigation into the method of mRNA transport in M S V - M D C K - I N V cells demonstrates microtubule independence, as evidenced by the maintained pseudopodial localization in the presence of nocodazole. Given this result we predict that the mechanism of transport involves actin microfilaments, however, this will have to be confirmed by observing the effect of microfilament disrupting agents and/or inhibition of myosin motor proteins. Interestingly, there is a slight increase in pseudopodial mRNA localization seen following long term treatment with nocodazole. It is known that microtubule depolymerization induces the activation of Rho/ROCK mediated signaling (Enomoto, 1996; Liu et al., 1998). Hence, it would be tempting to speculate that 95 following microtubule disruption, the Rho/ROCK pathway is further stimulated, leading to increased myosin activation and increased mRNA transport to the pseudopodia. This prediction could be further supported by investigating the effects of nocodazole treatment on Rho activation in the pseudopodia using the Rho-GTP sensing FRET probes. In summary, this investigation has further characterized the M S V - M D C K - I N V cell line. The Rho/ROCK mediated pseudopodia contain distinct protein and mRNA complements that facilitate regulation by localized signaling cascades. 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