Membrane-cytoskeleton interactions in megakaryocytes and platelets - PROJECT ABSTRACT Abnormal platelet production and function, due to genetic factors, cancer therapy, or unknown causes, pose significant clinical risks, including bleeding and thrombotic events. Platelets are produced primarily in the bone marrow by megakaryocytes through intricate processes involving polyploidization and extensive membrane and cytoskeletal rearrangements. These include the formation of the demarcation membrane system (DMS), the surface-connected membrane reservoir necessary for proplatelet extension and release within bone marrow sinusoids. Megakaryocytes also form podosomes, which serve as mechanosensing structures to identify the most conducive sites for initiating trans-endothelial proplatelet extension. The precise molecular mechanisms responsible for these unique membrane and cytoskeletal rearrangements remain poorly understood. The membrane-shaping F-BAR protein PACSIN2 stands central in the interplay between membranes and the cytoskeleton. PACSIN2 contains an N-terminal F-BAR domain tubulating membranes and a C-terminal SH3 domain interacting with the endocytic GTPase dynamin 2 (DNM2) and actin-nucleation-promoting factor WASp. Through its F-BAR domain, PACSIN2 also interacts with the cytoskeletal and scaffolding protein filamin A (FlnA), a critical regulator of platelet production and function. Single nucleotide polymorphisms in PACSIN2 have been associated with key platelet parameters in humans. Our recent data have discovered pivotal insights into the role of PACSIN2 in megakaryocyte and platelet biology. PACSIN2 is an internal component of the initiating DMS in megakaryocytes, where its membrane tubulation activity is regulated by FlnA (PMCID: PMC4492198). Pacsin2–/– mice display a mild thrombocytopenia with slightly enlarged platelets and marked platelet-intrinsic thrombus formation defects (PMCID: PMC10841284). Pacsin2–/– megakaryocytes have a mildly defective DMS, reduced ploidy, and impaired podosome and proplatelet formation. Pacsin2–/– platelets display elevated integrin β1 activity. Deletion of integrin β1 within megakaryocytes effectively normalizes the thrombocytopenia and thrombus formation defects. We hypothesize that PACSIN2 regulates membrane-cytoskeletal interactions and integrin β1 activity to govern the formation and organization of the DMS and podosomes during megakaryocyte maturation. We propose three aims to investigate the molecular mechanisms underlying how PACSIN2 modulates platelet production and function. In Aim 1, we will characterize megakaryocyte maturation and platelet production in the presence or absence of PACSIN2 and integrin β1. In Aim 2, we will identify the PACSIN2 effector proteins modulating actin assembly/remodeling at sites of podosome formation in megakaryocytes by proteomics and overexpression methodologies. In Aim 3, we will define the mechanisms by which PACSIN2 modulates integrin β1 activity to regulate thrombus formation and platelet hemostatic function. We anticipate that our studies will yield basic information on how PACSIN2 contributes to megakaryocyte and platelet biology.
