Role of Phospholipase D1 in regulated exocytosis

性状（由申请方提供）：分泌膜囊泡的调节性胞吐作用通过囊泡在有助于胞吐作用的特定位点募集至质膜，然后分阶段融合至质膜中进行。受调节的胞吐作用的普遍缺陷引起人类的单系统或多系统疾病，并且受调节的胞吐作用的促进（例如在糖尿病中）或抑制（例如在过敏反应中）是许多治疗方式的基础。我们的一般假设是，脂质环境的操纵是这一过程中的关键因素。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（PLD 1）在这一过程中发挥作用晚于囊泡融合到质膜通过生产磷脂酸（PA），磷脂酶D行动的脂质产品。许多调查途径现在都是时候探讨，以确定其运作机制。我们建议实现以下具体目标，以解决这些问题：1。PLD 1激活和PA产生与其促进调节胞吐作用的时间和空间关系是什么？我们将研究是否PLD 1激活需要急性分泌时，PLDV激活剂参与PLD 1促进过程中，PA在融合过程中产生。2.增加PA水平如何促进受调节的胞吐作用？我们将使用电子显微镜检查阻碍在缺乏PLD 1的细胞中完成融合的囊泡的形态，以确定融合过程在哪一步被阻断。我们还将研究PLD 1及其产物PA可能发挥作用的潜在机制，包括通过刺激产生PI 4，5 P2的酶家族PI 4P 5 KI来调节PI 4，5 P2的产生;募集CAPS的胞外融合位点，CAPS是融合所需的PI 4，5 P2结合蛋白;并通过促进熔合孔的形成或膨胀而对熔合过程本身产生潜在影响。从拟议的实验的结果将大大进一步我们的了解，通过PLD 1促进分泌调节胞吐过程中的机制。

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.