Role of Phospholipase D1 in regulated exocytosis

描述（由申请人提供）：分泌膜囊泡的调节胞吐是通过在有利于胞吐的特定部位将囊泡招募到质膜上进行的，然后分阶段融合到质膜上。调节胞外分泌的普遍性缺陷导致人类单一或多系统疾病，促进（如糖尿病）或抑制（如过敏反应）调节胞外分泌是多种治疗方式的基础。我们的一般假设是，在这个过程中，对液体环境的操纵是一个关键因素。已发表的报告和我们的初步证据表明，信号转导酶磷脂酶D1 （PLD1）在囊泡融合到质膜的过程中通过产生磷脂酸（PA）发挥作用，磷脂酸是PLD作用的脂质产物。现在，探索许多调查途径，以确定其发挥作用的机制是及时的。针对这些问题，我们提出以下具体目标：PLD1的激活和PA的产生与它们促进受调节的胞外分泌有什么时空关系？我们将研究在分泌时是否需要急性激活PLD1，哪些PLDVs激活因子参与PLD1促进的过程，以及在融合事件中何处产生PA。2. 如何通过增加PA水平促进调节胞吐？我们将使用电子显微镜检查在缺乏PLD1的细胞中阻碍完成融合的囊泡形态，以确定融合过程在哪一步被阻止。我们还将研究PLD1及其产物PA可能发挥作用的潜在机制，包括通过刺激PI4P5KI（产生PI4、5P2的酶家族）来调节PI4、5P2的产生；融合所需的pi4,5p2结合蛋白CAPS的胞外融合位点募集；以及通过促进熔合孔的形成或膨胀对熔合过程本身的潜在影响。这些实验的结果将大大加深我们对PLD1在受调节的胞吐过程中促进分泌的机制的理解。

DESCRIPTION (provided by applicant): Regulated exocytosis of secretory membrane vesicles proceeds via recruitment of the vesicles to the plasma membrane at specific sites conducive for exocytosis, followed by staged fusion into the plasma membrane. Generalized defects in regulated exocytosis cause single or multi-system disease in humans, and the promotion (e.g in diabetes) or inhibition (e.g. in anaphylaxis) of regulated exocytosis underlies numerous therapeutic modalities. Our general hypothesis is that manipulation of the iipid environment is a key element in this process. Published reports and our preliminary evidence suggest that the signal-transducing enzyme Phospholipase D1 (PLD1) plays a role late in this process during fusion of the vesicles into the plasma membrane via production of phosphatidic acid (PA), the Iipid product of PLD action. Many avenues of investigation are now timely to explore to determine the mechanism through which it functions. We propose to carry out the following specific aims to address these questions: 1. What are the temporal and spatial relationships of PLD1 activation and PA generation to their facilitation of regulated exocytosis? We will examine whether PLD1 activation is required acutely at the time of secretion, which of PLDVs activators participate in the PLD1-facilitated process, and where PA is produced during the fusion event. 2. How is regulated exocytosis facilitated by increasing levels of PA? We will use electron microscopy to examine the morphology of vesicles hindered from completing fusion in cells lacking PLD1 to determine which step the fusion process is blocked at. We will also examine potential mechanisms though which PLD1 and its product PA may be functioning, including regulation of the production of PI4,5P2 by stimulation of PI4P5KI, the enzyme family that generates PI4,5P2; recruitment to exocytic fusion sites of CAPS, a PI4,5P2-binding protein that is required for fusion; and potential affects on the fusion process itself though promotion of fusion pore formation or expansion. The results from the proposed experiments will substantially further our understanding of the mechanisms through which PLD1 promotes secretion during regulated exocytosis.