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）： 这项研究的目的是开发可用于研究整联蛋白的相互作用的测定方法，整联蛋白是一种与某些形式的肿瘤转移有关的细胞受体蛋白，具有细胞外基质蛋白，也用于研究细胞膜中整合素簇的研究。该测定将首先使用表达S2细胞的整合素进行开发，然后将表达突变蛋白的组织进行原位研究。从果蝇上进行的遗传筛选获得了要研究的突变蛋白，并将用于阐明整联蛋白作用的分子机制。由于脊椎动物和无脊椎动物整合素之间的相当大的结构同源性，果蝇被用作研究脊椎动物整合素的模型生物。原位荧光标记技术将用于标记整联蛋白和配体，以便细胞不需要固定在底物上，并启用时间依赖性和距离依赖性的结合数据。一般测定格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，并且总内反射荧光将用作检测方案，以限制从块状细胞材料中限制自动荧光。