Membrane-Cytoskeletal Remodeling in Platelet Biogenesis

 描述(由申请人提供)：项目摘要：血小板生产和功能缺陷在心血管、出血和炎症性疾病中起重要作用。对血小板生物发生的更全面的了解将对人类健康产生更多的见解和进展。我导师的实验室在酵母双杂交筛选中发现了一种新的细胞骨架成分--Cdc42相互作用蛋白4(CIP4)，该筛选以Src激酶Lyn为诱饵。CIP4是一种杆状蛋白，协调细胞膜和细胞骨架的重塑。CIP4通过其SH3结构域与Wiskott-Aldrich综合征蛋白(WASP)或Dynamin相互作用。Wiskott-Aldrich综合征的特征是血小板减少。我的实验室培育出了表现出血小板减少症的CIP4基因敲除(KO)小鼠。我确定CIP4-KO小鼠巨核细胞中血小板减少的机制包括减少血小板形成和减少分界膜系统，这在WASP-KO小鼠巨核细胞中没有观察到。此外，与WASP-KO小鼠不同，CIP4 KO小鼠表现出向血浆中释放的血小板微粒子减少。我推测CIP4依赖的血小板减少症与动力素有关。令人惊讶的是，动力素基因敲除的细胞显示出更多的微粒释放。由于Dynamin的主要作用是促进胞内囊泡的形成，因此这种由Dynamin介导的膜断裂的变化将是新的。我提出的研究的目的是确定CIP4-Dynamin途径在正常的血小板生物发生中调节膜重塑的机制以及这可能如何影响血小板的生物发生。我推测，动力蛋白的丢失影响了巨核细胞的膜内转运，导致更多的膜可用于分界膜系统内陷和微粒子释放。为了解决这一假设，我提出了以下两个具体目标：1)建立动力蛋白的丢失促进分界膜系统的形成；2)从动力蛋白活性降低的巨核细胞/血小板产生的微粒中确定血栓形成的可能性。这一建议将细胞生物学、生物化学、生物物理学和先进的成像技术与动物模型相结合，建立了一条在血小板生物发生和血栓形成中膜重构的新途径。这个K08奖项为我提供了密集的实验室培训、指导和委员会监督，使我能够成功地发展成为一名独立的内科科学家。研究和培训计划将在西北大学在Seth Corey和Susan Quaggin博士的指导下进行，研究生院的课程是细胞生物学，一个由血小板生物发生(Joseph Italiano)、血栓形成(小平)和造血(Liz Eclund)方面的知名研究人员组成的委员会提供建议。

 DESCRIPTION (provided by applicant): Project Summary Defects in platelet production and function play substantial roles in cardiovascular, bleeding, and inflammatory diseases. More complete understanding of platelet biogenesis will yield insights and advances in human health. My mentor's lab discovered a new cytoskeletal component, Cdc42-interacting protein 4 (CIP4), in a yeast two-hybrid screen with the Src kinase Lyn as bait. CIP4 is a BAR protein that coordinates membrane and cytoskeletal remodeling. Through its SH3 domain, CIP4 interacts with Wiskott-Aldrich Syndrome Protein (WASP) or dynamin. Wiskott-Aldrich Syndrome is characterized by thrombocytopenia. My lab generated CIP4-knockout (KO) mice that displayed thrombocytopenia. I determined that the mechanism for thrombocytopenia in CIP4-KO mouse megakaryocytes involves decreased proplatelet formation and reduced demarcation membrane system, which are not observed in WASP-KO mouse megakaryocytes. In addition, unlike WASP-KO mice, CIP4 KO mice show decreased platelet-microparticle release into the plasma. I hypothesize that CIP4-dependent thrombocytopenia involves dynamin. Surprisingly, cells with dynamin knockdown showed increased microparticle release. Since dynamin's chief role is to promote endocytic vesicle formation, this variation of membrane scission mediated by dynamin would be novel. The goal of my proposed research is to determine the mechanism by which CIP4-dynamin pathway regulates membrane remodeling in normal platelet biogenesis and how this may impact platelet biogenesis. I hypothesize that loss of dynamin, affects membrane intracellular trafficking in megakaryocytes, resulting in more membrane being available for demarcation membrane system invagination and for microparticle release. To address this hypothesis, I propose the following two specific aims: 1) establish that loss of dynamin promotes formation of the demarcation membrane system and 2) define the thrombogenic potential from microparticles produced by megakaryocytes/platelets with reduced dynamin activity. This proposal combines cell biology, biochemistry, biophysics, and advanced imaging with animal modeling to establish a new pathway of membrane remodeling in platelet biogenesis and thrombogenesis. This K08 award provides me with intensive laboratory training, mentorship, and committee oversight so that I can successfully develop into an independent physician- scientist. Research and training plan will be carried at Northwestern University under the mentorships of Drs. Seth Corey and Susan Quaggin with graduate school coursework in cell biology and advice from a committee of well-established investigators in platelet biogenesis (Joseph Italiano), thrombogenesis (Xiaoping Du) and hematopoiesis (Liz Eklund).