Exocytosis and Coupled Endocytosis in Neuroendocrine Cells

描述（由申请人提供）：长期目标是了解神经内分泌细胞中胞外和内吞作用耦合的机制。在这些细胞中，密集核囊泡（DCVs）的胞吐作用用于分泌调节人体大部分内部功能的激素和生物胺，内吞作用使DCV膜循环再利用。最近的研究表明，这些过程的耦合定量控制分泌，通过调节信号强度在器官之间提供特定的信号。在肾上腺髓质中，胞外和内吞作用控制儿茶酚胺、肾上腺素和去甲肾上腺素的分泌。阐明耦合如何起作用对于确定包括高血压和全身应激在内的内分泌病理的潜在靶点生理事件至关重要。目前的研究主要集中在分泌载体膜蛋白（SCAMPs）在肾上腺髓质源性嗜铬细胞瘤（PC12）细胞和小鼠肾上腺嗜铬细胞的外/内吞偶联中的作用。其中一种异构体SCAMP2与三种在胞外作用中起作用的蛋白——小G蛋白Arf6、磷脂酶D1 （PLD1）和磷脂酰肌醇4-磷酸5激酶（PIP5K）相互作用，并参与DCV胞外作用中融合孔的打开和扩张。其他SCAMPs结合络合蛋白（SCAMP1）和动力蛋白（SCAMPs 1和5）-相互作用被认为支持DCV胞外作用和内吞作用。这些发现导致了SCAMPs组织和耦合外/内吞作用的开启和关闭步骤的假设。四个目标将评估这一假设的各个方面。1) SCAMP与参与胞吐的Arf6、PLD1、PIP5K和络合蛋白的相互作用将被绘制，并主要使用电流计检测对胞吐的影响。2)肽和全长SCAMPs隔离外/内吞作用所需的磷酸肌肽PIP2的能力将通过生物物理实验和荧光显微镜对缺乏SCAMPs或表达脂质隔离缺陷的SCAMP突变体的细胞进行评估。3) SCAMP在胞吞作用中的功能，特别是涉及动力蛋白相互作用，将主要通过荧光显微镜来分析动力蛋白募集和DCV膜恢复，其中动力蛋白-SCAMP相互作用受到干扰。4)在缺乏SCAMP1的染色质细胞中，将使用电流法、示踪剂摄取和电子显微镜分析胞外作用和外内吞偶联，以分析被认为与染色质细胞生理有关的缺陷。

DESCRIPTION (provided by applicant): The long-range objective is to understand the mechanisms involved in coupled exocytosis and endocytosis in neuroendocrine cells. In these cells, exocytosis of dense-core vesicles (DCVs) is used to secrete hormones and biogenic amines that regulate most of man's internal functions, and endocytosis recycles DCV membranes for reutilization. Recent insight indicates that coupling of these processes controls secretion quantitatively, providing for specific signaling between organs through regulating signaling strength. In adrenal medulla, coupled exocytosis and endocytosis controls secretion of catecholamines, adrenalin and noradrenalin. Clarifying how coupling works is essential for defining physiological events that are potential targets in endocrine pathologies involving hypertension and systemic stress. Proposed studies focus on the roles of Secretory Carrier Membrane Proteins (SCAMPs) in exo-/endocytic coupling in adrenal medulla- derived pheochromocytoma (PC12) cells and mouse adrenal chromaffin cells. One isoform, SCAMP2, interacts with three proteins that function in exocytosis - small G protein Arf6, phospolipase D1 (PLD1), and phosphatidyl inositol 4-phosphate 5-kinase (PIP5K) - and also participates in opening and dilating fusion pores in DCV exocytosis. Other SCAMPs bind complexin (SCAMP1) and dynamin (SCAMPs 1 & 5) - interactions which are thought to support DCV exocytosis and endocytosis. These findings have led to the hypothesis that SCAMPs organize and couple opening and closing steps of exo-/endocytosis. Four aims will evaluate aspects of this hypothesis. 1) SCAMP interactions with Arf6, PLD1, PIP5K, and complexin involved in exocytosis will be mapped and tested for effects on exocytosis mainly using amperometry. 2) Ability of peptides and full-length SCAMPs to sequester phosphoinositide PIP2, required for exo-/endocytosis, will be evaluated using biophysical assays and fluorescence microscopy of cells deficient in SCAMPs or expressing SCAMP mutants defective in lipid sequestration. 3) SCAMP function in endocytosis, particularly involving dynamin interaction, will be examined mainly by fluorescence microscopy to analyze dynamin recruitment and DCV membrane recovery where dynamin-SCAMP interactions are perturbed. 4) Exocytosis and exo- endocytic coupling will be analyzed in chromaffin cells lacking SCAMP1 using amperometry, tracer uptake, and electron microscopy to analyze defects thought to be related to chromaffin cell physiology.