Exocytosis and Coupled Endocytosis in Neuroendocrine Cells

神经内分泌细胞的胞吐作用和耦合内吞作用

• 批准号: 7148305
• 负责人: John David Castle
• 金额: $ 30.91万
• 依托单位: UNIVERSITY OF VIRGINIA
• 依托单位国家: 美国
• 财政年份: 2006
• 资助国家: 美国
• 起止时间: 2006-07-15 至 2011-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7148305
• 关键词: PC12 cells; chromaffin cells; dynamin; endocytosis; exocytosis; fluorescence microscopy; genetically modified animals; intracellular transport; laboratory mouse; membrane transport proteins; neuroendocrine system; phosphatidylinositols; phospholipase D; protein protein interaction; secretion; tissue /cell culture

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.

描述（由申请人提供）：长期目标是了解神经内分泌细胞中偶联胞吐和胞吞作用的机制。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，而内吞作用则使DCV膜重新利用。最近的见解表明，这些过程的耦合控制分泌定量，通过调节信号强度提供器官之间的特定信号。在肾上腺髓质中，胞吐和胞吞作用控制着儿茶酚胺、肾上腺素和去甲肾上腺素的分泌。阐明耦合如何工作是必不可少的定义生理事件，是潜在的目标，内分泌病理学涉及高血压和全身性应激。目前的研究主要集中在分泌性载体膜蛋白（SCAMPs）在肾上腺髓质源性嗜铬细胞瘤（PC 12）细胞和小鼠肾上腺嗜铬细胞中的胞吞/胞吐偶联中的作用。一种亚型SCAMP 2与在胞吐中发挥作用的三种蛋白质--小G蛋白Arf 6、磷脂酶D1（PLD 1）和磷脂酰肌醇4-磷酸5-激酶（PIP 5 K）--相互作用，并且还参与DCV胞吐中融合孔的打开和扩张。其他SCAMP结合复合蛋白（SCAMP 1）和发动蛋白（SCAMP 1和5）-被认为支持DCV胞吐和胞吞作用的相互作用。这些发现导致了这样的假设，即SCAMPs组织和耦合外吞/内吞的开放和关闭步骤。四个目标将评估这一假设的各个方面。1)将绘制SCAMP与参与胞吐的Arf 6、PLD 1、PIP 5 K和复合蛋白的相互作用，并主要使用电流分析法测试对胞吐的影响。2)肽和全长SCAMP螯合外吞/内吞所需的磷酸肌醇PIP 2的能力将使用缺乏SCAMP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来评估。3)SCAMP在内吞作用中的功能，特别是涉及发动蛋白相互作用的功能，将主要通过荧光显微镜检查，以分析发动蛋白募集和DCV膜恢复，其中发动蛋白-SCAMP相互作用受到干扰。4)将使用电流分析法、示踪剂摄取和电子显微镜在缺乏SCAMP 1的嗜铬细胞中分析胞吐和胞吐-胞吞偶联，以分析被认为与嗜铬细胞生理学相关的缺陷。