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耦合胞吐作用和内吞作用所涉及的机制。在这些细胞中，致密核心囊泡 (DCV) 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，而内吞作用则回收 DCV 膜以供再利用。最近的研究表明，这些过程的耦合定量地控制分泌，通过调节信号强度提供器官之间的特定信号传导。在肾上腺髓质中，耦合的胞吐作用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。阐明耦合的工作原理对于定义生理事件至关重要，这些生理事件是涉及高血压和全身应激的内分泌病理学的潜在目标。拟议的研究重点是分泌载体膜蛋白（SCAMP）在肾上腺髓质来源的嗜铬细胞瘤（PC12）细胞和小鼠肾上腺嗜铬细胞的外切/内切偶联中的作用。 SCAMP2 是一种异构体，与三种在胞吐作用中起作用的蛋白质（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，并且还参与 DCV 胞吐作用中融合孔的打开和扩张。其他 SCAMP 结合复合蛋白 (SCAMP1) 和动力蛋白 (SCAMP 1 & 5) - 相互作用被认为支持 DCV 胞吐作用和内吞作用。这些发现导致了这样的假设：SCAMP 组织并耦合外吞/内吞作用的打开和关闭步骤。四个目标将评估这一假设的各个方面。 1) 将绘制 SCAMP 与参与胞吐作用的 Arf6、PLD1、PIP5K 和复合蛋白的相互作用，并主要使用安培分析法测试对胞吐作用的影响。 2) 肽和全长 SCAMP 螯合外切/内吞作用所需的磷酸肌醇 PIP2 的能力，将使用 SCAMP 缺陷或表达脂质螯合缺陷的 SCAMP 突变体的细胞的生物物理测定和荧光显微镜进行评估。 3) 内吞作用中的 SCAMP 功能，特别是涉及动力相互作用，将主要通过荧光显微镜检查，以分析动力-SCAMP 相互作用受到干扰时的动力募集和 DCV 膜恢复。 4) 将使用电流分析法、示踪剂摄取和电子显微镜分析缺乏 SCAMP1 的嗜铬细胞中的胞吐作用和外吞耦合，以分析被认为与嗜铬细胞生理学相关的缺陷。

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.