The Centrosome as a master controller of platelet production.

血小板是特化的无核细胞，在止血、血管生成、免疫和血管生成中发挥重要作用。 炎症血小板减少症（血小板计数<150 × 109/L）是一个主要的临床问题， 包括免疫性（特发性）血小板减少性紫癜，骨髓增生异常综合征， 化疗、手术和遗传疾病。对血小板的需求-以及对更好理解的需求 他们的机械形成-是在历史上最高的。该计划将采用多管齐下的方法， 研究巨核细胞（MK），以发现治疗策略和分子靶点， 前血小板形成和增加血小板计数。MK是前体细胞，其通过以下方式产生血小板： 将其细胞质重塑为珠状前血小板过程，其功能为血小板组装线 生产虽然我们知道细胞骨架力学为血小板的产生提供动力，但关于血小板生成的许多问题仍然存在。 血小板的生物发生仍然没有答案。我们知道，基于微管的力对前血小板至关重要， 然而，令人惊讶的是，人们对触发血小板聚集的机制缺乏了解。 生产我们假设中心体的调节，通过超纺锤体的形成和KIFC 1马达 参与，是至关重要的启动血小板生产。我们将使用一种新颖的高内涵显微镜 筛选以确定驱动血小板生成的小分子和信号通路。使用前血小板 图像分析，我们将测试数千种药物分子候选人的能力，刺激或抑制 血小板生成;将建立靶向途径分析、二次筛选和剂量反应曲线 以识别复合“命中”。虽然我们知道前血小板突起从骨髓中延伸出来， 内皮屏障，并存款血小板进入血液，我们不知道如何。因此，我们将采用生物- 工程和一个独特的微流体骨髓芯片上测试的想法，肌动蛋白驱动的巨核细胞， 足体提供了穿透内皮的机制。这个芯片也将被用来研究 在生理条件下，细胞器被运输到组装的血小板中， 这个超级纺锤体组件作为一个主要的运输枢纽来分配这些细胞器。我们将 通过研究血管巯基异构酶如何在新的血小板颗粒生物学中发挥作用， 它们被包装、运输并从血小板中排出。我们预计， 研究将1）促进对启动和调节血小板的机制的理解 形成，和2）确定新的治疗靶点和方法，以加速血小板生成， 血小板减少症患者。鉴于MK油田的相对不成熟，R35结构是必要的 并将提供重要的时间和重点，以扩大现有的知识基础。该提案将协调 一个多元化的合作者群体，为该领域提供新的数据和理论，并支持初级科学家， 来自具有广泛翻译经验的实验室的持续指导和久经考验的领导力。

Platelets are specialized anucleate cells that play an essential role in hemostasis, angiogenesis, immunity, and inflammation. Thrombocytopenia (platelet counts <150x109/L) is a major clinical problem encountered across a number of conditions including immune (idiopathic) thrombocytopenic purpura, myelodysplastic syndromes, chemotherapy, surgery, and genetic disorders. The demand for platelets—and for an improved understanding of their mechanistic formation—is at an all-time high. This program will use a multi-prong approach to investigate megakaryocytes (MKs) to discover therapeutic strategies and molecular targets that drive proplatelet formation and increase platelet counts. MKs are precursor cells that generate platelets by remodeling their cytoplasm into beaded proplatelet processes, which function as the assembly lines for platelet production. While we know that cytoskeletal mechanics power platelet production, many questions about platelet biogenesis remain unanswered. We know that microtubule-based forces are critical for proplatelet elongation; however, there is a surprising lack of understanding of the mechanisms that trigger platelet production. We hypothesize that centrosome regulation, via super spindle formation and KIFC1 motor involvement, is critical for the initiation of platelet production. We will use a novel high-content microscopy screen to identify the small molecules and signaling pathways that drive platelet production. Using proplatelet image analysis, we will test thousands of drug molecule candidates for their ability to stimulate or inhibit platelet production; target pathway analysis, secondary screens, and dose-response curves will be established to identify compound “hits.” While we know that proplatelet protrusions extend from bone marrow, breach the endothelial barrier, and deposit platelets into the blood, we do not know how. Therefore, we will employ bio- engineering and a unique microfluidic bone marrow on-a-chip to test the idea that actin-driven megakaryocyte podosomes provide a mechanism to penetrate the endothelium. This chip will also be used to study how organelles are transported into assembling platelets under physiological conditions, and to test the hypothesis that super spindle assembly functions as a major transport hub for distributing these organelles. We will determine if vascular thiol isomerases play a role in new platelet granule biology through investigating how they are packaged, transported, and exocytosed from platelets. We expect that findings from this investigation will 1) advance the understanding of the mechanisms that initiate and regulate platelet formation, and 2) identify novel therapeutic targets and approaches to accelerate platelet production in patients with thrombocytopenia. The R35 structure is necessary given the relative immaturity of the MK field and will provide vital time and focus to expanding the current base of knowledge. This proposal will coordinate a diverse group of collaborators, provide the field with novel data and theory, and support junior scientists with consistent mentorship and proven leadership from a laboratory with broad ranging translational experience.