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：
7642392
负责人：
John David Castle
金额：
$ 29.41万
依托单位：
UNIVERSITY OF VIRGINIA
依托单位国家：
美国
项目类别：
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/7642392
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（由申请人提供）：远程目标是了解神经内分泌细胞中偶联胞吐作用和内吞作用的机制。在这些细胞中，致密核囊泡的胞吐作用（DCV）用于分泌调节大多数人内部功能的激素和生物胺，以及内吞作用回收DCV膜以进行再生。最近的见解表明，这些过程的耦合数量地控制分泌，从而通过调节信号强度来提供器官之间的特定信号传导。在肾上腺髓质中，偶联的胞吐作用和内吞作用控制着儿茶酚胺，肾上腺素和去甲肾上腺素的分泌。阐明耦合的工作原理对于定义是涉及高血压和全身压力的内分泌病理中潜在靶标的生理事件至关重要的。拟议的研究重点是分泌载体膜蛋白（SCAMP）在肾上腺髓质衍生的嗜铬细胞瘤（PC12）细胞和小鼠肾上腺染色体细胞中的外胞/内吞耦合中的作用。一种同工型SCAMP2与三种在胞吐作用中起作用的蛋白质 - 小G蛋白ARF6，凤凰酶D1（PLD1）和磷脂酰肌醇4-磷酸5-激酶（PIP5K） - 以及参与DCV杂胞菌病的开放式融合孔的开放和扩张孔。其他SCAMP结合复合蛋白（SCAMP1）和Dynamin（SCAMPS 1和5） - 被认为支持DCV胞吐作用和内吞作用的相互作用。这些发现导致了一个假设，即Scamps组织了外吞和伴侣的伴侣开放和闭合步骤。四个目标将评估该假设的各个方面。 1）将与ARF6，PLD1，PIP5K和复合素进行SCAMP相互作用，并测试主要使用安培法对胞吐作用的影响。 2）将使用生物物理测定和荧光显微镜评估SCAMP或表达SCAMP突变体在脂质序列中有缺陷的细胞的肽和内吞作用所必需的肽和全长SCAMP的能力。 3）将主要通过荧光显微镜检查内吞作用，尤其是涉及动力蛋白相互作用的SCAMP功能，以分析动态蛋白的募集和DCV膜恢复，在这些促进蛋白相互作用受到干扰。 4）将在缺乏Amperometry，Tracer摄取和电子显微镜的缺乏SCAMP1的铬蛋白细胞中分析胞吐作用和外吞偶联，以分析被认为与铬蛋白细胞生理学有关的缺陷。