Loss of Cdc42 Interacting Protein 4, a Member of the Membrane-Associated BAR Family, Decreases Platelet Microparticle Formation – a Possible Role for Dynamin

Abstract

Abstract 1153Our lab isolated Cdc42 interacting protein 4 (CIP4) in a yeast two-hybrid screen using the Src kinase Lyn as bait. CIP4 is a Bin/Amphiphysin/Rvs (BAR) protein with a C-terminal SH3 domain that interacts with Wiskott Aldrich Syndrome protein (WASp) or dynamin. BAR proteins contain a domain that interacts with membrane lipids generating membrane curvature or tubulation. WASp and dynamin, on the other hand, function in actin polymerization and membrane vesicle scission respectively. WASp is defective in boys with the eponymous syndrome and is characterized by thrombocytopenia, which are small in size. Dynamin-3 has been shown to function during megakaryocyte development (Reems et al Exp Hematol. 36:12, 2008). We have observed that CIP4−/− mice displayed thrombocytopenia comparable to that observed in WAS-/ mice. Also, cultured megakaryocytes from CIP4 and WASp knockout mice showed decreased proplatelet extensions.As a result of those observations, we hypothesized that defects in membrane dynamics due to loss of CIP4 might decrease platelet production. To better understand the role of CIP4 in platelet biogenesis, CHRF-288-11 cell line, a megakaryoblastic cell line was used for studying CIP4’s interaction with WASp and dynamin. CIP4’s interaction with WASp and dynamin-3 was investigated by co-immunoprecipitation and microscopy in CHRF cells. The cells were induced to form proplatelets by 100 ng/ml phorbol myristate acetate (PMA) or fibronectin-coated plates. Activated cells showed CIP4/dynamin-3 co-precipitation in a time-dependent manner. Also, immunofluorescent images showed diffuse distribution of CIP4 and dynamin-3 prior to activation with PMA or fibronectin. However after activation, co-localization of both proteins were observed at the membrane. These findings suggest possible interaction of CIP4-dynamin in platelet biogenesis. Platelet microparticles may serve as a surrogate marker for membrane deformation, rigidity, and cleavability. PMP are formed during cellular activation or apoptosis from various cell types such as platelets. The process of PMP formation is incompletely understood. We hypothesized that circulating levels of platelet microparticles (PMP) would be affected by the loss of CIP4 and its affects on membrane remodeling. Using flow cytometric analysis we observed decreased circulating levels of CD41+/ AnnexinV+ PMP in 3–6 month old C57BL/6 male CIP4 −/− and CIP4−/−,WAS-/ mice when compared to controls. However, WAS-/ mice showed similar levels to control mice.We conclude that CIP4, a membrane deforming protein, promotes PMP formation. However, this is independent of its interaction with WASp. Because of dynamin's role in promoting membrane scission and its association with CIP4, we are currently investigating the role of dynamin in PMP formation. Disclosures:No relevant conflicts of interest to declare.