Actin filament formation is required for the platelet shape change that accompanies activation and clot formation. The applicant, Dr. Elaine Bearer, has used platelets to identify and clone a novel membrane-associated actin-binding protein, 2E4, that has the biochemical characteristics expected of an actin polymerizer. Two other proteins, Arp2 and VASP also induce actin polymerization. Platelets spread on glass elaborate three distinct actin structures: filopodia, lamellipodia, and stress fibers: and others have shown that three G-proteins, cdc42, rac and rho, specifically activate filopodia, lamellipodia, and stress fibers, respectively, in platelets and other cells. Cdc42 directly interacts with the Arp2/3 complex in other cells and Dr. Bearer has shown in preliminary experiments, that rac associates with 2E4 and rho with VASP. Further, Arp2/3 is located on filopodia, 2E4 on lamellipodia and VASP on stress fibers in immunofluorescence experiments. The applicant therefore hypothesizes that each of the three G-proteins activates its associates actin polymerizer leading to one of the three different actin structures observed in platelets.
The specific aims are to test this hypothesis in three ways.
Aim 1 focuses on the characterization of 2E4. Dr. Bearer will: (1) analyze the effect of 2E4 on actin polymerization, (2) look for and effects of VASP and Arp2/3 on 2E4 activity, (3) measure binding interactions between 2E4 and F-actin by sedimentations, (4) measure the effect of 2E4-actin binding on actin structure using fluorescence and electron microscopy, and (5) screen for 2E4-interacting proteins using the yeast 2-hybrid method and affinity chromatography.
Aim 2 is directed at the role of the three G-proteins (rac, rho and cdc42) in actin polymerizers (2E4, Arp2/3, VASP) in platelets extracts. The contributions of each of these proteins and gelsolin will be measured using immunodepletion or dominant negative G-proteins to perturb the system.
The third Aim relates these results to """"""""intact"""""""" platelets. The role of G-protein activation/inhibition on the formation of specific actin structures and on localization of 2E4, Arp2/3 and VASP in spread platelets and living fibroblasts will be assessed. Constitutively active mutant G-proteins will be introduced using a published platelet permeabilization technique and actin polymerization will be monitored by fluorescence microscopy. In other studies the dynamics of green fluorescent protein (GFP)-labeled 2E4 and Arp2 will be followed by real time video microscopy in cultured fibroblasts.
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