In situ Assay for Integrin Clustering and Ligand Binding

整合素聚类和配体结合的原位测定

• 批准号: 7066075
• 负责人: EMILY A SMITH
• 金额: $ 1.05万
• 依托单位: UNIVERSITY OF ARIZONA
• 依托单位国家: 美国
• 财政年份: 2005
• 资助国家: 美国
• 起止时间: 2005-06-01 至 2006-07-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7066075
• 关键词: Drosophilidae; arthropod genetics; extracellular matrix proteins; fluorescence resonance energy transfer; fluorescent dye /probe; gene mutation; genetic screening; integrins; postdoctoral investigator; protein localization; protein protein interaction; protein structure function; receptor binding

DESCRIPTION (provided by applicant): The goal of this research is to develop assays that can be used to study the interactions of integrins, a class of cell receptor proteins implicated in some forms of tumor metastasis, with extracellular matrix proteins, and also for the study of integrin clustering in the cell membrane. The assay will first be developed using integrin expressing S2 cells, and then tissue expressing mutant integrins will be studied in situ. The mutant integrins to be studied were obtained from genetic screens performed on Drosophila, and will be used to elucidate the molecular mechanism of integrin action. Drosophila is used as a model organism for studying vertebrate integrins due to the considerable structural homology between vertebrate and invertebrate integrins. In situ fluorescent labeling techniques will be utilized to tag the integrin and ligand so that the cells do not need to be fixed to a substrate, and to enable both time-dependent and distance-dependent binding data to be obtained. The general assay format will use fluorescence resonance energy transfer to quantitatively study the interactions between donor and acceptor tagged integrins and/or ligands, and total internal reflection fluorescence will be used as the detection scheme to limit autofluorescence from the bulk cellular material.

描述（由申请人提供）： 本研究的目标是开发可用于研究整合素（一类与某些形式的肿瘤转移有关的细胞受体蛋白）与细胞外基质蛋白相互作用的测定方法，以及用于研究细胞膜中整合素簇集的测定方法。该试验将首先使用表达整合素的S2细胞开发，然后将原位研究表达突变整合素的组织。待研究的突变整合素是从对果蝇进行的遗传筛选中获得的，并将用于阐明整合素作用的分子机制。果蝇是研究脊椎动物整联蛋白的模式生物，这是由于脊椎动物整联蛋白与无脊椎动物整联蛋白具有相当大的结构同源性。将利用原位荧光标记技术来标记整联蛋白和配体，使得细胞不需要固定到基底上，并且能够获得时间依赖性和距离依赖性结合数据。一般测定形式将使用荧光共振能量转移来定量研究供体和受体标记的整联蛋白和/或配体之间的相互作用，并且将使用全内反射荧光作为检测方案来限制来自大量细胞材料的自发荧光。