Identification of a Novel Formin‑GAP Complex and Its Role in Macrophage Migration and Phagocytosis by Frank Marshall Mason, Jr. Department of Cell Biology Duke University Date:_______________________ Approved: ___________________________ Scott Soderling, Supervisor ___________________________ Vann Bennett, Chair ___________________________ John Klingensmith ___________________________ Terry Lechler ___________________________ Ann Marie Pendergast Dissertation submitted in partial fulfillment of the requirements for the degree of Doctor of Philosophy in the Department of Cell Biology in the Graduate School of Duke University 2011 i v ABSTRACT Identification of a Novel Formin‑GAP Complex and Its Role in Macrophage Migration and Phagocytosis by Frank Marshall Mason, Jr. Department of Cell Biology Duke University Date:_______________________ Approved: ___________________________ Scott Soderling, Supervisor ___________________________ Vann Bennett, Chair ___________________________ John Klingensmith ___________________________ Terry Lechler ___________________________ Ann Marie Pendergast An abstract of a dissertation submitted in partial fulfillment of the requirements for the degree of Doctor of Philosophy in the Department of Cell Biology in the Graduate School of Duke University 2011 Copyright by Frank Marshall Mason, Jr. 2011 Abstract Essential and diverse biological processes such as cell division, morphogenesis and migration are regulated by a family of molecular switches called Rho GTPases. These proteins cycle between active, GTP‑bound and inactive, GDP‑bound states, and this cycle is regulated by families of proteins called Rho GEFs and GAPs. GAPs are proteins that stimulate the intrinsic GTPase activity of Rho‑family proteins, potentiating the active to inactive transition. GAPs target specific spatiotemporal pools of GTPases by responding to cellular cues and utilizing protein‑protein interactions. By dissecting these interactions and pathways, we can infer and then decipher the biological functions of these GAPs. This work focuses on the characterization of a novel Rho‑family GAP called srGAP2. In this study, we identify that srGAP2 is a Rac‑specific GAP that binds a Formin‑family member, Formin‑like 1 (FMNL1). FMNL1 is activated by Rac and polymerizes, bundles and severs actin filaments. srGAP2 specifically inhibits actin severing by active FMNL1, and the assembly of an srGAP2‑FMNL1 complex is regulated by Rac. Work on FMNL1 shows that it plays important roles in regulating phagocytosis and adhesion in macrophages. To learn more about srGAP2 and its role in regulating FMNL1, we studied macrophages isolated from an srGAP2 KO mouse we have recently generated. This work demonstrates that loss of srGAP2 decreases the iv ability for macrophages to invade through extracellular matrix but increases phagocytosis. These results suggest that these two processes might be coordinated in vivo by srGAP2 and that srGAP2 might be a critical regulator of the innate immune system. v Dedication To my wife and best friend, who makes me smile every day and loves me for the person I am. To my parents, who have always supported me. I could never fully express my gratitude. vi Contents Abstract .......................................................................................................................................... iv List of Figures ............................................................................................................................... xi List of Abreviations .................................................................................................................... xiii Acknowledgements .................................................................................................................... xv 1 Introduction ............................................................................................................................. 1 1.1 Rho‑Family GTPases and Their Regulation .............................................................. 1 1.1.1 Rho‑family GTPases ................................................................................................. 1 1.1.2 Guanine Nucleotide Exchange Factors ................................................................. 6 1.1.3 Rho GTPase Activating Proteins ............................................................................ 9 1.2 Actin and Actin Remodeling Proteins ...................................................................... 15 1.2.1 Actin ......................................................................................................................... 15 1.2.2 Arp2/3 and Arp2/3‑Activating Proteins .............................................................. 18 1.2.3 Formins .................................................................................................................... 21 1.2.4 WH2‑Containing Proteins ..................................................................................... 24 1.2.5 Actin Severing Proteins ......................................................................................... 26 1.3 srGAP (WRP) Family of Rho GAPs .......................................................................... 28 1.3.1 F‑BAR and IF‑BAR domains ................................................................................. 30 1.3.2 SH3 domains ........................................................................................................... 32 1.3.3 srGAP1 ..................................................................................................................... 34 1.3.4 WRP .......................................................................................................................... 34 vii 1.3.5 srGAP2 ..................................................................................................................... 35 2 Materials and Methods ........................................................................................................ 37 2.1 Cell Culture .................................................................................................................. 37 2.2 Plasmids and Constructs ............................................................................................ 37 2.3 Antibodies for Staining and Immunoblots .............................................................. 38 2.4 Immunoprecipitations and Pulldowns .................................................................... 38 2.5 Immunostaining .......................................................................................................... 39 2.6 Yeast Two‑Hybrid ....................................................................................................... 40 2.7 Protein Production and Purification ......................................................................... 40 2.8 Production and Purification of Profilin .................................................................... 41 2.9 Actin Severing Assays ................................................................................................ 42 2.10 Preparation of NEM‑Treated Myosin ..................................................................... 44 2.11 Actin Filament Binding and Bundling Assays ...................................................... 45 2.12 Pyrene Actin Assays ................................................................................................. 46 2.13 GAP Assays and PAK Pulldowns ........................................................................... 48 2.14 Induction and Isolation of Peritoneal Macrophages ............................................ 49 2.15 2D Matrix Degradation by Macrophages .............................................................. 50 2.16 Preparation of Radiolabeled Protein for Peptide Array Overlay ....................... 53 2.17 Isolation of Bone Marrow Cells (Macrophages) ................................................... 53 2.18 3D‑Invasion of Bone Marrow Macrophages ......................................................... 55 2.19 Phagocytosis ............................................................................................................... 57 3 Bi‑modal regulation of a formin by srGAP2 ..................................................................... 59 viii 3.1 Introduction .................................................................................................................. 59 3.2 Results ........................................................................................................................... 61 3.2.1 srGAP2 binds Formin‑like 1, Formin‑like 3, and Afadin ................................. 61 3.2.2 srGAP2 regulates signaling between Rac and FMNL1 .................................... 66 3.2.3 Dynamic formation of the srGAP2/FMNL1 complex ....................................... 69 3.2.4 Both srGAP2 and FMNL1 co‑localize with F‑actin during Fc‑γ receptor phagocytosis ........................................................................................................................ 72 3.2.5 Direct regulation of FMNL1 actin severing, but not bundling or severing by the srGAP2 SH3 domain .................................................................................................... 73 3.3 Discussion ..................................................................................................................... 81 4 The Role of srGAP2 in Macrophages ................................................................................