Deficient thrombopoiesis, due to genetic causes, secondary to cancer therapy, or from unknown etiology, poses significant risks of mortality, mostly due to bleeding. This study will address the gap of knowledge on the role of clathrin- and caveolae-dependent receptor-mediated endocytosis (RME) in megakaryocyte (MK) and platelet biology, focusing on the role of the large housekeeping GTPase dynamin 2 (DNM2) and the F-BAR protein PACSIN2 in platelet formation and function. We anticipate that our studies will yield basic information on how RME contributes to MK and platelet biology and lead to the development of approaches to reestablish thrombopoiesis in the setting of thrombocytopenia. Blood platelets are produced in the bone marrow by MKs, in a process that requires extensive intracellular membrane rearrangements, including the formation of the demarcation membrane system (DMS). The precise mechanisms underlying these unique membrane rearrangements are poorly understood. We have generated fundamental insights into the cellular and molecular mechanisms involved in the formation of the MK DMS: 1) Dnm2fl/fl Pf4-Cre mice specifically lacking DNM2 in MKs develop severe macrothrombocytopenia, splenomegaly, myelofibrosis and bleeding due to impaired RME and altered DMS formation in bone marrow MKs; 2) the F-BAR protein PACSIN2 is an internal component of a well-defined plasma membrane invagination that characterizes the initiating DMS; and 3) PACSIN2 deletion significantly improves the severe thrombocytopenia of Dnm2fl/fl Pf4-Cre mice, demonstrating that DNM2 and PACSIN2 work in concert to regulate DMS formation and platelet production. The overall hypothesis of the proposed research is that DNM2- and PACSIN2-dependent RME regulates the formation of the MK DMS, thereby regulating platelet production and function. In this research proposal, we will focus on the role of DNM2 and PACSIN2 in RME required for MK DMS formation and platelet function. We will characterize clathrin- and caveolae-dependent RME in mouse bone marrow MKs and compare these in the absence of DNM2, PACSIN2, or both proteins using biochemical and two- and three-dimensional microscopy approaches (Aim 1). We will extend these studies to examine the role of DNM2 membrane binding and fission activity and PACSIN2 ability to recruit actin regulatory proteins at sites of RME by protein transfection into MKs and mutational approaches (Aim 2). Lastly, we will determine how clathrin- and caveolae-dependent RME contributes to platelet hemostatic function in the absence of DNM2, PACSIN2, or both proteins (Aim 3).
The objectives of the proposed research are to enhance understanding of the fundamental process of receptor-mediated endocytosis in megakaryocyte and platelet biology. The information acquired with this project may lead to improved treatments of platelet disorders and bone marrow diseases, such as thrombocytopenia and myelofibrosis.
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