Novel Roles for Phosphoinositide Signaling in alpha-Granule Biogenesis

磷酸化磷脂酰肌醇（磷酸肌醇）是一种膜结合型磷脂酰肌醇。 磷脂影响巨核细胞生成所需的多种不同过程， 激活血小板。我们最近在《发育细胞》上发表了磷酸肌醇 在神经元细胞中通过募集效应蛋白如GOLPH 3启动细胞内运输 它们参与高尔基体生物发生过程中质膜的泡状融合和出芽。 由于巨核细胞α-颗粒来源于高尔基体网络和多囊泡细胞， 因此，我假设磷酸肌醇信号传导对于细胞内运输是必要的 是α颗粒生物发生所必需的。磷脂酰肌醇转移蛋白（PITP）是 一个小的蛋白质家族的成员，结合和转移磷酸肌醇单体从一个 细胞隔室彼此相连，从而使磷酸肌醇合成成为可能。我们取得了 出乎意料的观察结果是，在细胞内发现的两种主要的PITP同种型， 在巨核细胞中，PITPα和PITPβ发挥着以前未被认识到的但重要的作用， 将货物从多囊泡体运输到α颗粒。PITP介导的丢失 磷酸肌醇合成产生的形态缺陷类似于在人类中观察到的， 灰色血小板综合征。该提案的总体假设是磷酸肌醇信号传导 由PITP介导的是必要的膜动力学和蛋白质运输所需的 巨核细胞α颗粒的生物发生和维持。在项目目标1中，我们将 严格分析单个PITP亚型的离散生化特性， 巨核细胞我们的初步数据表明，两个PITP亚型控制磷酸肌醇 通过生物化学上不同的机制发出信号。在目标2中，我们将确定如何 磷酸肌醇信号传导有助于α颗粒生物发生和功能。结合 在项目2中，我们将检验以下假设： 巨核细胞和血小板的微结构域调节效应蛋白，如NBEAL 2（ 突变的蛋白质负责灰色血小板综合征）。这种信号级联调节 NBEAL 2介导膜动力学和蛋白质运输的能力。我们还将分析在 通过离体流变学和超显微镜研究详细描述了α颗粒的功能作用， 将在项目2中进行，在项目3中进行体内血栓形成研究，以及体内炎症研究 研究项目4。

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. In conjunction with Project 2, 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 the 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 that will be performed with Project 2, in vivo thrombosis studies with Project 3, and in vivo inflammation studies with Project 4.