The Role of Focal Adhesion Kinase in Vascular Smooth Muscle Cell Migration Lee Mangiante Masters Thesis Defense Cellular a...
Outline <ul><li>Background:   Vascular SMCs and FAK </li></ul><ul><li>Results:   FAK mediates SMC migration to PDGF   </li...
Background Vascular SMCs and FAK
Vascular Smooth Muscle Cells (SMCs) <ul><li>Comprise the medial layer of all arteries </li></ul><ul><li>Regulate blood pre...
SMCs in Atherosclerosis <ul><li>Injury to the vessel wall triggers the inflammatory response </li></ul><ul><li>Inflammator...
PDGF-BB homodimer <ul><li>Isoforms:  A, B, C, and D </li></ul><ul><li>Receptors:  PDGFRA, PDGFRB </li></ul><ul><li>BB is a...
Focal Adhesion Kinase (FAK) <ul><li>Nonreceptor tyrosine kinase found at focal adhesions </li></ul><ul><li>Transduces adhe...
A Unique Role for FAK in SMC Biology <ul><li>Germline deletion of FAK is lethal at E 8.5 – 10; embryos exhibit “leaky vasc...
FAK in Migration: Extant Questions <ul><li>Known: </li></ul><ul><li>FAK depletion    Impaired wound closure, transwell mi...
The Migration Cycle: Where is FAK Involved?  Leading Edge Protrusion polarization Trailing EdgeRetraction FA Disassembly/a...
Overall Research Goal: <ul><li>Determine the role of FAK in aortic SMC migration toward platelet-derived growth factor-BB ...
Results FAK mediates SMC migration to PDGF
Deletion of  fak  in VSMCs: the  fak  flox/flox   mouse LacZ   Cre = FAK = phalloidin (F-actin) ATP-Binding Dom. loxP   Ex...
FAK is required for 3D migration to PDGF <ul><li>FAK- SMCs are spread, focal adhesions appear normal </li></ul><ul><li>FAK...
What are the cytoskeletal characteristics of FAK-depleted SMCs treated with PDGF? <ul><li>Immunofluorescent staining: </li...
PDGF-induced dorsal ruffling is FAK-independent Lac Z  (FAK+)  Cre  (FAK-) 2.5 min 20x = cortactin = phalloidin Peripheral...
PDGF-induced cell polarization is FAK-dependent LacZ (FAK+) Cre (FAK-)
What molecular mechanisms explain the polarization defect of FAK-depleted SMCs? Activity of the Rho subfamily GTPases
Ridley, AJ.  J Cell Sci.  2001 Aug;114(Pt 15):2713-22.  Rac = PUSH Rho = PULL Rho GTPase Signaling Pathways <ul><li>Rho, R...
Rac-PI3K Signaling is unperturbed by FAK depletion Pulldown: GTP-Rac1 IB: pAKT Live cell: GFP-WAVE2 AKT ,  WAVE 1/ 2 PI3K ...
Myosin activation, but not global RhoA activity, is attenuated by FAK depletion ROCK GTP- RhoA pMLC contractility MLC phos...
Dia2 localizes to focal adhesions  dependently  of FAK <ul><li>In LacZ-infected SMCs, Dia2 commonly targets to  peripheral...
<ul><li>Dia2 is enriched at peripheral/dorsal ruffles following PDGF treatment,  regardless of FAK content </li></ul><ul><...
<ul><li>What is the biological significance of Dia2 at membrane ruffles vs. focal adhesions? </li></ul><ul><ul><li>What is...
FAK  Dia2  Stable Microtubules?  No <ul><li>Palazzo, et al.  Science   (2004): FRNK overexpression abolishes stable MT’s...
<ul><li>Chan et al. (1996)  Identified cortactin as a  formin binding protein  by screening a mouse limb expression librar...
Cortactin: a structurally distinct Arp2/3 activator A  = acidic region; facilitates Arp2/3 binding P  =  proline-rich doma...
Structure and Regulation of Dia2 GBD = GTPase Binding Domain DID = Diaphanous Inhibitory Domain FH1, FH2 = Formin Homology...
Dia2 and cortactin interact independently of F-actin I. GST pulldown (SMC lysates) II. Co-IP (COS-7 lysates) FH1  = prolin...
Moving Forward Dia2 and Cortactin
Main Questions <ul><li>What regulates Dia2-cortactin binding? </li></ul><ul><ul><li>Extracellular cues? </li></ul></ul><ul...
Why would Dia2 and cortactin associate? <ul><li>Formins  and  Arp2/3  are traditionally seen as two separate actin nucleat...
Shifting the actin paradigm: <ul><li>New evidence suggests that DRFs may interact with the WANP complex  </li></ul><ul><li...
Dia2/WANP interactions <ul><li>Beli et al.  Nat Cell Biol.  (2008):   Dia2 N-term/C-term bind to the Scar homology domain/...
Is Dia2 passive or active? <ul><li>Passive:  WAVE2 sequesters Dia2 to prevent filipodia formation  </li></ul><ul><li>Activ...
“ Active” (open) mutants of Dia2 GBD GBD ID DID DID FH1 FH1 FH2 FH2 DAD DAD A  D Δ GBD A272D All kept in the  “open”  con...
Cortactin colocalizes intensely with Dia2 A272D WT FL A272D GFP cortactin merge phalloidin Does A272D associate more stron...
Could Src regulate Dia2-cortactin binding? <ul><li>Three putative Src phosphorylation sites within the  FH2  domain </li><...
Dia2 in PDGF-Stimulated Migration  “ Protrusive Dia” polarization “ Retractile Dia” FA Disassembly/assembly FAK  coordinat...
How might Dia2 promote contractility? <ul><li>Direct mechanisms: </li></ul><ul><ul><li>Localized  actin polymerization  ev...
Future Experiments <ul><li>Further map the interaction sites on Dia2 and cortactin </li></ul><ul><li>Determine whether cor...
Appendix Knockdown of leupaxin in human aortic SMCs
Leupaxin: Structure <ul><li>Member of the paxillin family of LIM proteins </li></ul><ul><li>Contains four LIM domains (zin...
Leupaxin: Putative Functions <ul><li>First identified in leukocytes ( JBC , 1998) </li></ul><ul><li>Mostly studied in hema...
Leupaxin in SMCs  <ul><li>Enriched in arterial and visceral SMCs   </li></ul><ul><li>Binds to FAK via its LD3 motif </li><...
How does endogenous leupaxin knockdown impact SMC biology? <ul><li>Differentiation? </li></ul><ul><li>Migration? </li></ul...
Leupaxin Knockdown in Human ASMCs <ul><li>siRNA designed to 3’ UTR of human leupaxin </li></ul><ul><li>No effect on locali...
Leupaxin in Migration: 3D vs. 2D <ul><li>Transwell assay:   leupaxin knockdown SMCs cannot migrate three-dimensionally to ...
2D Motility in Sparsely Plated SMCs <ul><li>In serum media, leupaxin knockdown cells display slower random motility  </li>...
Aberrant PDGF-induced membrane ruffles Stain:  cortactin / phalloidin <ul><li>Leupaxin knockdown SMCs lack smooth, continu...
Preliminary Conclusions <ul><li>Leupaxin is required for 3D migration to serum, but not 2D wound healing </li></ul><ul><li...
Future Leupaxin Studies <ul><li>Elucidate the mechanisms by which leupaxin facilitates motility in different contexts  (2D...
Acknowledgments Committee Members William B. Coleman Adrienne Cox Financial Support Robert H. Wagner Scholarship Joseph E....
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Masters Defense

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  • Masters Defense

    1. 1. The Role of Focal Adhesion Kinase in Vascular Smooth Muscle Cell Migration Lee Mangiante Masters Thesis Defense Cellular and Molecular Pathology Joan M. Taylor, PhD
    2. 2. Outline <ul><li>Background: Vascular SMCs and FAK </li></ul><ul><li>Results: FAK mediates SMC migration to PDGF </li></ul><ul><li>Moving forward: Dia2 and cortactin </li></ul><ul><li>Appendix: Knockdown of leupaxin in human aortic SMCs </li></ul>
    3. 3. Background Vascular SMCs and FAK
    4. 4. Vascular Smooth Muscle Cells (SMCs) <ul><li>Comprise the medial layer of all arteries </li></ul><ul><li>Regulate blood pressure by modifying vessel tone </li></ul><ul><li>Proper SMC migration is critical for vasculogenesis & wound repair </li></ul><ul><li>But, can also contribute to vascular pathogenesis </li></ul>http://www.lab.anhb.uwa.edu
    5. 5. SMCs in Atherosclerosis <ul><li>Injury to the vessel wall triggers the inflammatory response </li></ul><ul><li>Inflammatory cells and damaged endothelium release SMC chemoattractants including platelet-derived growth factor (PDGF) </li></ul><ul><li>SMCs invade the intima, occluding the arterial lumen </li></ul>www.siumed.edu/ ~dking2/crr/CR026b.htm
    6. 6. PDGF-BB homodimer <ul><li>Isoforms: A, B, C, and D </li></ul><ul><li>Receptors: PDGFRA, PDGFRB </li></ul><ul><li>BB is a potent chemoattractant/mitogen for SMCs </li></ul><ul><li>In culture, stimulates formation of ring-shaped actin structures called dorsal ruffles </li></ul><ul><li>Important for SMC differentiation during development </li></ul><ul><li>Germline deletion is embryonic lethal, with failed recruitment of SMC precursors to the vasculature </li></ul><ul><li>Stimulates growth and migration of SMCs during vascular injury response </li></ul>
    7. 7. Focal Adhesion Kinase (FAK) <ul><li>Nonreceptor tyrosine kinase found at focal adhesions </li></ul><ul><li>Transduces adhesion signals from the extracellular matrix; can also cooperate in growth factor & contractile agonist signaling </li></ul><ul><li>Activates myriad pathways important in numerous biological processes </li></ul>FAT Kinase Paxillin GRAF CAS Site I Site II Y 397 SRC SH2 SI SII P  1 ASAP Pi3K SH2 Integrin Binding
    8. 8. A Unique Role for FAK in SMC Biology <ul><li>Germline deletion of FAK is lethal at E 8.5 – 10; embryos exhibit “leaky vasculature” </li></ul><ul><li>FRNK, an endogenous dominant negative for FAK, is expressed exclusively in SMCs during development and injury response </li></ul><ul><li>This suggests that FAK activity requires tight control in SMCs and may play a special role in this cell type </li></ul>
    9. 9. FAK in Migration: Extant Questions <ul><li>Known: </li></ul><ul><li>FAK depletion  Impaired wound closure, transwell migration to fibronectin, spreading, in fibroblasts, endothelials, keratinocytes… </li></ul><ul><li>FAK overexpression  increased motility/invasiveness </li></ul>Unknown: <ul><li>Structural specifics ? </li></ul><ul><li>Signaling mechanisms? </li></ul><ul><li>Smooth muscle cells? PDGF? </li></ul>
    10. 10. The Migration Cycle: Where is FAK Involved? Leading Edge Protrusion polarization Trailing EdgeRetraction FA Disassembly/assembly FAK/Rho? FAK/ERK? Fak/Rac?
    11. 11. Overall Research Goal: <ul><li>Determine the role of FAK in aortic SMC migration toward platelet-derived growth factor-BB (PDGF). </li></ul><ul><ul><li>Identify the biomechanical events controlled by FAK </li></ul></ul><ul><ul><li>Determine the spatiotemporal signaling events by which this structural regulation is accomplished </li></ul></ul>
    12. 12. Results FAK mediates SMC migration to PDGF
    13. 13. Deletion of fak in VSMCs: the fak flox/flox mouse LacZ Cre = FAK = phalloidin (F-actin) ATP-Binding Dom. loxP Exon 18 loxP Cre recombinase No FAK produced 72 Hours Post-Virus FAK ERK Lac Z Cre
    14. 14. FAK is required for 3D migration to PDGF <ul><li>FAK- SMCs are spread, focal adhesions appear normal </li></ul><ul><li>FAK depletion blocks three-dimensional migration toward PDGF, but not 10% serum </li></ul><ul><li>Migration is rescued by overexpression of wild-type FAK, but not FAK Y397F </li></ul>Stain: vinculin / phalloidin Transwell migration assay LacZ Cre
    15. 15. What are the cytoskeletal characteristics of FAK-depleted SMCs treated with PDGF? <ul><li>Immunofluorescent staining: </li></ul><ul><li>Cortactin: localizes to dorsal ruffles, lamellipodia </li></ul><ul><li>Phalloidin: binds F-actin stress fibers </li></ul>
    16. 16. PDGF-induced dorsal ruffling is FAK-independent Lac Z (FAK+) Cre (FAK-) 2.5 min 20x = cortactin = phalloidin Peripheral ruffles Dorsal ruffles
    17. 17. PDGF-induced cell polarization is FAK-dependent LacZ (FAK+) Cre (FAK-)
    18. 18. What molecular mechanisms explain the polarization defect of FAK-depleted SMCs? Activity of the Rho subfamily GTPases
    19. 19. Ridley, AJ. J Cell Sci. 2001 Aug;114(Pt 15):2713-22. Rac = PUSH Rho = PULL Rho GTPase Signaling Pathways <ul><li>Rho, Rac, and Cdc42 </li></ul><ul><li>Dynamic cycle of activation/inhibition during cell migration </li></ul><ul><li>Rac facilitates membrane protrusion </li></ul><ul><li>Rho controls cell contraction and focal adhesion dynamics </li></ul>
    20. 20. Rac-PI3K Signaling is unperturbed by FAK depletion Pulldown: GTP-Rac1 IB: pAKT Live cell: GFP-WAVE2 AKT , WAVE 1/ 2 PI3K GTP- Rac Arp2/3 Membrane protrusion Leading edge formation
    21. 21. Myosin activation, but not global RhoA activity, is attenuated by FAK depletion ROCK GTP- RhoA pMLC contractility MLC phosphatase Pulldown: GTP-RhoA IB: pMLC
    22. 22. Dia2 localizes to focal adhesions dependently of FAK <ul><li>In LacZ-infected SMCs, Dia2 commonly targets to peripheral streaks after PDGF treatment ( 85% of 26 movies) </li></ul><ul><li>This pattern is absent or less dramatic in Cre-infected SMCs ( 18% of 22 movies) </li></ul><ul><li>GFP-Dia2 colocalizes with mCherry-paxillin after PDGF treatment, suggesting that these “streaks” are indeed focal adhesions </li></ul>
    23. 23. <ul><li>Dia2 is enriched at peripheral/dorsal ruffles following PDGF treatment, regardless of FAK content </li></ul><ul><li>Dia2 also colocalizes with cortactin in serum-maintained fixed cells </li></ul>Dia2 localizes to ruffles independently of FAK Stain: cortactin / GFP-Dia2 Live cells: GFP-Dia2
    24. 24. <ul><li>What is the biological significance of Dia2 at membrane ruffles vs. focal adhesions? </li></ul><ul><ul><li>What is Dia2 doing at each location? </li></ul></ul><ul><ul><li>What signaling events drive Dia2 to each location? </li></ul></ul><ul><ul><li>Why is FAK required for one, but not the other? </li></ul></ul>
    25. 25. FAK  Dia2  Stable Microtubules? No <ul><li>Palazzo, et al. Science (2004): FRNK overexpression abolishes stable MT’s, and can be rescued by constitutively active mDia1 </li></ul>1. PDGF does not alter levels of glu-tubulin (stable MT’s) 2. FAK depletion does not abolish glu-tubulin staining
    26. 26. <ul><li>Chan et al. (1996) Identified cortactin as a formin binding protein by screening a mouse limb expression library with a formin probe </li></ul><ul><li>They proposed that the SH3 domain of cortactin bound to the proline -rich portion of the formin probe </li></ul>Can we detect such an interaction between cortactin and mDia2 in vitro ? Might this interaction regulate the “switch” between ruffle localization and FA localization?
    27. 27. Cortactin: a structurally distinct Arp2/3 activator A = acidic region; facilitates Arp2/3 binding P = proline-rich domain SH3 = Src homology; binds proline-rich motifs Repeat domain: binds F-actin (20 fold higher than Arp2/3) W = WASP homology; binds G-actin C = central region; binds/activates Arp2/3 GB = GTPase binding domain (Cdc42, Rac) B = basic region Sufficient to activate Arp2/3 CTN WASP, WAVE WASP CTN, WASP, WAVE Daly, RJ. 2004
    28. 28. Structure and Regulation of Dia2 GBD = GTPase Binding Domain DID = Diaphanous Inhibitory Domain FH1, FH2 = Formin Homology 1, 2 FH3 = Formin Homology 3 DAD = Diaphanous Autoinhibitory Domain
    29. 29. Dia2 and cortactin interact independently of F-actin I. GST pulldown (SMC lysates) II. Co-IP (COS-7 lysates) FH1 = proline rich FH2 = no prolines; binds G-actin
    30. 30. Moving Forward Dia2 and Cortactin
    31. 31. Main Questions <ul><li>What regulates Dia2-cortactin binding? </li></ul><ul><ul><li>Extracellular cues? </li></ul></ul><ul><ul><li>Upstream signaling? </li></ul></ul><ul><ul><li>Post-translational modifications? </li></ul></ul><ul><ul><li>Conformation of Dia2? </li></ul></ul><ul><li>What is the biological function of this interaction? </li></ul><ul><ul><li>Actively cooperating in actin polymerization? How and for what purpose? </li></ul></ul><ul><ul><li>Sequestering Dia2? </li></ul></ul>
    32. 32. Why would Dia2 and cortactin associate? <ul><li>Formins and Arp2/3 are traditionally seen as two separate actin nucleators </li></ul><ul><li>Arp2/3 controls protrusive machinery (lamellipodia); formins control contractile machinery (stress fibers, focal adhesions, contractile ring in yeast) </li></ul><ul><li>Arp2/3 nucleates branches from existing filaments </li></ul><ul><li>Formins generate filaments from monomeric actin </li></ul>Goode et al. Ann Rev Biochem. (2007)
    33. 33. Shifting the actin paradigm: <ul><li>New evidence suggests that DRFs may interact with the WANP complex </li></ul><ul><li>W AVE </li></ul><ul><li>A bi1 </li></ul><ul><li>N ap1 </li></ul><ul><li>P IR121 </li></ul>Arp2/3
    34. 34. Dia2/WANP interactions <ul><li>Beli et al. Nat Cell Biol. (2008): Dia2 N-term/C-term bind to the Scar homology domain/proline-rich domain of WAVE2 </li></ul><ul><li>Yang et al. PLoS Biol. (2007): N-term of Dia2 interacts with Abi1 C-term (no interaction with WAVE) </li></ul>* Neither report detected an interaction with the FH1 or FH2 domains of Dia2
    35. 35. Is Dia2 passive or active? <ul><li>Passive: WAVE2 sequesters Dia2 to prevent filipodia formation </li></ul><ul><li>Active: Dia2 provides “mother filaments” for Arp2/3; bundles branched filaments into filipodia, as shown below </li></ul>Can Dia2 interact with cortactin in its “active” (open) conformation? Yang, et al. PLoS Biol. (2007)
    36. 36. “ Active” (open) mutants of Dia2 GBD GBD ID DID DID FH1 FH1 FH2 FH2 DAD DAD A  D Δ GBD A272D All kept in the “open” conformation by disrupting the DID-DAD interaction
    37. 37. Cortactin colocalizes intensely with Dia2 A272D WT FL A272D GFP cortactin merge phalloidin Does A272D associate more strongly with cortactin than WT Dia2?
    38. 38. Could Src regulate Dia2-cortactin binding? <ul><li>Three putative Src phosphorylation sites within the FH2 domain </li></ul><ul><li>Overexpression of constitutively active Src induces tyrosine phosphorylation of Flag-Dia2 </li></ul>Does tyrosine phosphorylation of the FH2 domain by Src modify the association of cortactin and Dia2?
    39. 39. Dia2 in PDGF-Stimulated Migration “ Protrusive Dia” polarization “ Retractile Dia” FA Disassembly/assembly FAK coordinates these two activities to enable fluid forward movement of the SMC Dia 2 Dia 2 cortactin cortactin Src P ? Src P ? Dia 2 Dia 2
    40. 40. How might Dia2 promote contractility? <ul><li>Direct mechanisms: </li></ul><ul><ul><li>Localized actin polymerization events can promote SMC contractility independently of MLC </li></ul></ul><ul><ul><li>Dia1 can regulate myosin-mediated contractility by targeting microtubules to focal adhesions </li></ul></ul><ul><li>Indirect mechanisms: </li></ul><ul><ul><li>Potential crosstalk between Dia2 and ROCK </li></ul></ul><ul><ul><li>In endothelial cells, cortactin and myosin light chain kinase (MLCK) interact to form a contractile apparatus at the cell periphery. Does this occur in SMCs? </li></ul></ul>cortactin EC MLCK Merge Dudek, et al. J. Biol Chem. (2004)
    41. 41. Future Experiments <ul><li>Further map the interaction sites on Dia2 and cortactin </li></ul><ul><li>Determine whether cortactin and Dia2 associate directly or indirectly </li></ul><ul><li>Determine if cortactin binds only to Dia2, or also to Dia1 </li></ul><ul><li>Use in vitro assays to determine if Dia2-cortactin binding changes the actin polymerizing activities of either protein </li></ul><ul><li>Assess the impact of FAK depletion/inactivation on Dia2-cortactin binding </li></ul><ul><li>Elucidate the signaling events that regulate binding </li></ul>
    42. 42. Appendix Knockdown of leupaxin in human aortic SMCs
    43. 43. Leupaxin: Structure <ul><li>Member of the paxillin family of LIM proteins </li></ul><ul><li>Contains four LIM domains (zinc finger motifs; target paxillin to focal adhesions) </li></ul><ul><li>Contains three LD motifs (bind to c-Src, Lyn, and FAK) </li></ul>Turner, CE. Nat Cell Biol. 2000
    44. 44. Leupaxin: Putative Functions <ul><li>First identified in leukocytes ( JBC , 1998) </li></ul><ul><li>Mostly studied in hematopoetic cells (macrophages, B-cells, osteoclasts) </li></ul><ul><li>Also enriched in prostate cancer cells and vascular SMCs </li></ul>Promotion of SMC differentiation Vascular SMCs 123 SRF Regulation of B-cell receptor signaling B-cell lymphoma 121 Lyn Podosomal complex signaling Vascular SMCs 123 , osteoclasts 127 FAK Podosomal remodeling Osteoclasts 127 p95 PKL Podosomal complex signaling, osteoclast activation Osteoclasts 126 , protstate cancer cells 120 c-Src Regulation of antigen receptor signaling; podosomal remodeling Spleen 128 , osteoclasts 126 , prostate cancer cells 120 PTP-PEST Focal adhesion adapter protein Macrophages 116 , osteoclasts 127 , prostate cancer cells 120 PYK2 Putative Biological Function Cell Type Binding Partner
    45. 45. Leupaxin in SMCs <ul><li>Enriched in arterial and visceral SMCs </li></ul><ul><li>Binds to FAK via its LD3 motif </li></ul><ul><li>GFP-leupaxin shuttles in and out of the nucleus </li></ul><ul><li>GFP-leupaxin binds directly to serum response factor (SRF) and activates SMC gene transcription </li></ul><ul><li>FAK activity modulates leupaxin localization and function </li></ul>Sundberg-Smith, et al. Circ Res. 2008
    46. 46. How does endogenous leupaxin knockdown impact SMC biology? <ul><li>Differentiation? </li></ul><ul><li>Migration? </li></ul><ul><li>Proliferation? </li></ul><ul><li>Apoptosis? </li></ul>
    47. 47. Leupaxin Knockdown in Human ASMCs <ul><li>siRNA designed to 3’ UTR of human leupaxin </li></ul><ul><li>No effect on localization/expression of paxillin or Hic-5 </li></ul>Control knockdown leupaxin Hic-5 paxillin phalloidin
    48. 48. Leupaxin in Migration: 3D vs. 2D <ul><li>Transwell assay: leupaxin knockdown SMCs cannot migrate three-dimensionally to serum </li></ul><ul><li>Wounding assay: SMCs do not require leupaxin to close a wound on uncoated plastic </li></ul>
    49. 49. 2D Motility in Sparsely Plated SMCs <ul><li>In serum media, leupaxin knockdown cells display slower random motility </li></ul><ul><li>Cell paths are more confined in knockdown cells </li></ul><ul><li>Displacement from origin is reduced approx. 55% </li></ul>
    50. 50. Aberrant PDGF-induced membrane ruffles Stain: cortactin / phalloidin <ul><li>Leupaxin knockdown SMCs lack smooth, continuous areas of ruffling </li></ul><ul><li>Ruffles appear spiky, disconnected </li></ul>Treatment: 5’ PDGF
    51. 51. Preliminary Conclusions <ul><li>Leupaxin is required for 3D migration to serum, but not 2D wound healing </li></ul><ul><li>Leupaxin knockdown cells show reduced velocity and displacement under sparsely plated conditions </li></ul><ul><li>Leupaxin knockdown cells form spiky, ragged membrane ruffles in response to PDGF </li></ul><ul><li>Leupaxin silencing impairs cell proliferation (*not quantified ) </li></ul>
    52. 52. Future Leupaxin Studies <ul><li>Elucidate the mechanisms by which leupaxin facilitates motility in different contexts (2D? 3D? Serum? PDGF?) </li></ul><ul><li>Clarify our understanding of leupaxin in SMC proliferation and differentiation </li></ul><ul><li>Examine leupaxin expression in vivo in the developing mouse (SMC lineages) </li></ul><ul><li>Create a leupaxin knockout animal model </li></ul><ul><li>Explore the role of leupaxin in vascular disease states (mouse, human) </li></ul>
    53. 53. Acknowledgments Committee Members William B. Coleman Adrienne Cox Financial Support Robert H. Wagner Scholarship Joseph E. Pogue Fellowship Joan Taylor Laura DiMichele Jason Doherty Lisa Galante Zeenat Hakim Rebecca Sayers Liisa Smith Chris Mack Alicia Blaker Jeremiah Hinson Kashelle Lockman Matt Medlin Dean Staus Jim Bear Liang Cai Tom Marshall Microscopy Services Lab Bob Bagnell Elena Davis Vicki Madden

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