Cytoskeletal Mechanisms of Platelet Formation

描述（由申请人提供）：血小板在以下方面发挥重要作用： 止血。这个提议的目的是研究细胞骨架 使用小鼠巨核细胞（MK）培养物的血小板形成机制 系统血小板的形成遵循一系列确定的形态发生形状变化 由MK细胞骨架内的微管（MT）和肌动蛋白丝驱动。它 从MK的大伪足延伸开始， MT的皮质束伸长成最终形成 在其球根状末端有突出的MT线圈。MT的线性阵列， 前血小板轴作为血小板移位的轨道 形成血小板。反复弯曲和分支， 前血小板在血小板形成中起关键作用。这种依赖肌动蛋白的 放大前血小板末端的过程对于产生 珠状外观的前血小板。 该MK培养系统将用于研究MT和肌动蛋白的作用 纤维在血小板形成机制中的作用。目标我将定义如何MT 迫使前血小板突起延长并决定边缘MT线圈 在血小板形态发生过程中形成。MT细胞骨架的动态变化 将被观察到，因为它在活的MK中起作用，以确定 MT组装和/或在前血小板伸长和MT螺旋形成中滑动。在 目的2，结构和生物化学方法将用于表征和 明确细胞器和颗粒沿着MT转运到 正在形成血小板目的3将探讨肌动蛋白依赖性 前血小板弯曲/分支。最后一个目的是检测血小板 在分子水平上的形态发生，并定义当膜骨架的 盘状血小板聚集并被vWf锁定 受体-细丝蛋白-肌动蛋白连接。了解细胞骨架力学 血小板形成可以提供关于血小板是如何产生的见解， 产生治疗血小板减少症的策略。

DESCRIPTION (provided by applicant): Blood platelets play an essential role in hemostasis. The purpose of this proposal is to investigate the cytoskeletal mechanics of platelet formation using a mouse megakaryocyte (MK) culture system. Platelet formation follows a defined set of morphogenetic shape changes driven by microtubules (MTs) and actin filaments within the MK cytoskeleton. It begins with the extension of large pseudopodia from the MK that appear to use cortical bundles of MTs to elongate into proplatelets which ultimately form prominent coils of MTs at their bulbous ends. Linear arrays of MTs that line the proplatelet shaft serve as tracks for the translocation of platelet components into developing platelets. Repeated bending and branching of proplatelets plays a crucial role in platelet formation. This actin-dependent process, which amplifies proplatelet ends, is essential for generating the beaded appearance of proplatelets. This MK culture system will be used to investigate the role of MTs and actin filaments in the mechanics of platelet formation. Aim I will define how MT forces elongate proplatelet processes and determine how the marginal MT coil forms during platelet morphogenesis. Dynamic changes in the MT cytoskeleton will be observed as it functions in living MKs to establish the contribution of MT assembly and/or sliding in proplatelet elongation and MT coil formation. In Aim 2, structural and biochemical methods will be used to characterize and define the mechanism of organelle and granule transport along MTs into developing platelets. Aim 3 will examine the mechanism of actin-dependent proplatelet bending/branching. In the last aim, we will examine platelet morphogenesis at the molecular level and define when the membrane skeleton of the discoid platelet assembles and is locked into place by the vWf receptor-filamin-actin linkage. Understanding the cytoskeletal mechanics of platelet formation may provide insights into how platelets are produced and yield strategies for treatment of thrombocytopenia.