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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

基本信息

批准号：
8000857
负责人：
John David Castle
金额：
$ 2.16万
依托单位：
UNIVERSITY OF VIRGINIA
依托单位国家：
美国
项目类别：
财政年份：
2009
资助国家：
美国
起止时间：
2009-12-22 至 2010-03-31
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/8000857
关键词：
Address Adrenal Glands Adrenal Medulla Biogenic Amines Biological Assay Catecholamines Cell membrane Cell physiology Cell surface Cells Chromaffin Cells Clathrin-Coated Vesicles Coupled Coupling Cytoplasmic Granules Defect Dense Core Vesicle Docking Dynamin Dynamin I Electron Microscopy Endocrine Endocytosis Event Exocytosis Fluorescence Microscopy Goals Hormones Hypertension Knockout Mice Length Link Lipids Maps Mediating Membrane Membrane Proteins Modeling Monomeric GTP-Binding Proteins Mouse Cell Line Mus Neuroendocrine Cell Neurotransmitters Organ Organism PC12 Cells Pathology Peptides Pheochromocytoma Phosphatidylinositol 4,5-Diphosphate Phosphatidylinositols Phosphotransferases Physiological Physiology Process Protein Binding Protein Isoforms Proteins Recovery Recycling Role Secretory Vesicles Signal Transduction Site Stimulus Stress Surface System Testing Tracer Work cholinergic effusion inositol 4-phosphate insight intercellular communication link protein macromolecule man mutant peptide hormone phosphatidylinositol 4-phosphate phospholipase D1 polypeptide protein function response secretory carrier membrane protein 1 uptake

项目摘要

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(由申请者提供)：长期目标是了解神经内分泌细胞的胞吐和内吞耦合作用的机制。在这些细胞中，致密核小泡(DCV)的胞吐作用被用来分泌调节人类大部分内部功能的激素和生物胺，而内吞作用则回收DCV膜以供重新利用。最近的研究表明，这些过程的耦合在数量上控制着分泌，通过调节信号强度来提供器官之间的特定信号。在肾上腺髓质中，胞吐和胞内作用共同控制儿茶酚胺、肾上腺素和去甲肾上腺素的分泌。阐明偶联是如何起作用的，对于定义生理事件至关重要，这些事件是涉及高血压和全身应激的内分泌病理的潜在靶点。建议的研究集中在分泌载体膜蛋白(SCAMPs)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外向/内向偶联中的作用。其中一个异构体SCAMP2与三种在胞吞作用中起作用的蛋白质相互作用--小G蛋白Arf6、磷脂酶D1(PLD1)和磷脂酰肌醇4-磷酸5-激酶(PIP5K)--也参与DCV胞吞作用中融合孔的打开和扩张。其他SCAMP结合复合素(SCAMP1)和动力素(SCAMPs1和5)-被认为支持DCV胞吐和内吞的相互作用。这些发现导致了一种假设，即SCAMP组织并耦合外/内吞作用的开放和关闭步骤。四个目标将评估这一假说的各个方面。1)SCAMP与Arf6、PLD1、PIP5K和参与胞吐作用的复合蛋白的相互作用将主要用安培方法来定位和测试其对胞吐的影响。2)多肽和全长SCAMP对磷脂酰肌醇PIP2的截留能力将通过生物物理测试和表达SCAMP缺失或表达突变体的细胞的荧光显微镜来评估。3)SCAMP在细胞内吞作用中的作用，特别是涉及Dynamin相互作用的功能，将主要通过荧光显微镜来分析Dynamin-SCAMP相互作用受到干扰时的Dynamin募集和DCV膜恢复。4)利用电流法、示踪剂摄取和电子显微镜分析缺失SCAMP1的嗜铬细胞的胞吐作用和胞外-内吞偶联，以分析与嗜铬细胞生理有关的缺陷。