Multifunctional nucleolus in Plant cell growth and development.
Masters Defense
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
10. The Migration Cycle: Where is FAK Involved? Leading Edge Protrusion polarization Trailing EdgeRetraction FA Disassembly/assembly FAK/Rho? FAK/ERK? Fak/Rac?
18. What molecular mechanisms explain the polarization defect of FAK-depleted SMCs? Activity of the Rho subfamily GTPases
19.
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. 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.
23.
24.
25.
26.
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. 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. 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
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. Cortactin colocalizes intensely with Dia2 A272D WT FL A272D GFP cortactin merge phalloidin Does A272D associate more strongly with cortactin than WT Dia2?
38.
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
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