Novel Roles for Phosphoinositide Signaling in alpha-Granule Biogenesis

项目摘要 磷酸化磷脂酰肌醇(磷脂酰肌醇)是一种膜结合蛋白 影响巨核细胞生成所需的多种不同过程的磷脂 激活血小板。我们最近在《发育细胞》上发表了磷脂酰肌醇 在神经细胞中通过招募效应蛋白如GOLPH3来启动细胞内转运 参与高尔基体生物发生过程中质膜的囊泡融合和萌发。 由于巨核细胞α颗粒来源于跨高尔基网络和多泡 身体，我假设磷脂酰肌醇信号是细胞内运输所必需的 α颗粒的生物发生所必需的。磷脂酰肌醇转移蛋白(PITPs)是 结合和转移磷脂酰肌醇单体的小蛋白家族的成员 从而使肌醇磷脂的合成成为可能。我们已经做出了 意想不到的观察到两种主要的PITP亚型在 巨核细胞、PITPα和PITPβ在 货物从多泡体到α颗粒的运输。PITP介导的丢失 肌醇磷脂的合成产生的形态缺陷类似于人类 格雷血小板综合征。这一提议的总体假设是，肌醇磷脂信号 由PITPs介导是膜动力学和蛋白质运输所必需的 巨核细胞α颗粒的生物发生和维持。在项目的目标1中，我们将 严格分析不同PITP亚型的离散生化特性 巨核细胞。我们的初步数据显示，这两种PITP亚型控制着肌醇磷脂 通过生物化学不同的机制发出信号。在目标2中，我们将确定如何 肌醇磷脂信号在α颗粒的生物发生和功能中起重要作用。我们将测试 巨核细胞离散微区内肌醇磷脂合成假说 血小板调节效应蛋白，如NBEAL2(导致灰色的突变蛋白 血小板综合征)。这一信号级联调控NBEAL2的S介导膜的能力 动力学和蛋白质贩运。我们还将详细分析α-Granules的功能角色 通过体外流变学和超微结构研究、体内血栓形成研究和体内 炎症研究。

Project Summary Phosphorylated phosphatidylinositols (phosphoinositides) are a type of membrane bound phospholipid that impact multiple diverse processes required for megakaryopoiesis and the activation of platelets. We have recently published in Developmental Cell that phosphoinositides in neuronal cells initiate intracellular trafficking by recruiting effector proteins such as GOLPH3 that are involved in vesicular fusion and budding of plasma membranes during Golgi biogenesis. Since megakaryocyte α-granules are derived from the trans-Golgi network and Multi-Vesicular Bodies, I hypothesize that phosphoinositide signaling is necessary for the intracellular trafficking required for the biogenesis of α-granules. PhosphatidylInositol Transfer Proteins (PITPs) are members of a small protein family that bind and transfer phosphoinositide monomers from one cellular compartment to another and thereby enable phosphoinositide synthesis. We have made the unexpected observation that the two predominant PITP isoforms found within megakaryocytes, PITPα and PITPβ play previously unrecognized but essential roles in the trafficking of cargo from the Multi-Vesicular Body to α-granules. Loss of PITP-mediated phosphoinositide synthesis produces morphologic defects similar to what is seen in humans with Gray Platelet Syndrome. The overall hypothesis of this Proposal is that phosphoinositide signaling mediated by PITPs is necessary for the membrane dynamics and protein trafficking required for the biogenesis and maintenance of megakaryocyte α-granules. In Aim 1 of the Project, we will rigorously analyze the discrete biochemical properties of individual PITP isoforms in megakaryocytes. Our preliminary data shows that the two PITP isoforms control phosphoinositide signaling through biochemically distinct mechanisms. In Aim 2, we will determine how phosphoinositide signaling contributes to alpha granule biogenesis and function. We will test the hypothesis that phosphoinositide synthesis within discrete microdomains of megakaryocytes and platelets regulates effector proteins such as NBEAL2 (the mutated protein responsible for Gray Platelet Syndrome). This signaling cascade modulates NBEAL2’s ability to mediate membrane dynamics and protein trafficking. We will also analyze in detail the functional roles of α-granules with ex vivo rheologic and ultramicroscopy studies, in vivo thrombosis studies, and in vivo inflammation studies.