SSAT Involvement alpha9 Integrin-Mediated Cell Migration

SSAT 参与 α9 整合素介导的细胞迁移

• 批准号: 6884521
• 负责人: GREGORY W deHART
• 金额: $ 4.83万
• 依托单位: UNIVERSITY OF CALIFORNIA, SAN FRANCISCO
• 依托单位国家: 美国
• 财政年份: 2005
• 资助国家: 美国
• 起止时间: 2005-09-15 至 2006-09-14
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6884521
• 关键词: acyltransferase; binding proteins; biological signal transduction; cell migration; confocal scanning microscopy; fluorescent dye /probe; immunofluorescence technique; immunoprecipitation; integrins; intracellular transport; polyamines; postdoctoral investigator; protein localization; protein protein interaction; protein quantitation /detection; protein structure function; protein transport; spermidine; spermine; video microscopy; yeast two hybrid system

DESCRIPTION (provided by applicant): The alpha9beta1 integrin is expressed in many different tissues, including smooth muscle, epidermis, and neutrophils. Data from alpha9 integrin subunit-knockout mice have implicated this integrin in lymphangiogenesis, thus indicating its potential role in vascular development. In addition, in vitro experiments have shown that the alpha9 integrin subunit can mediate enhanced cell migration in both neutrophils and non-leukocytic cells. In an effort to investigate the molecular mechanisms behind alpha9 integrin-mediated cell migration, spermidine/spermine-N'-acetyltransferase (SSAT), a regulator of cellular polyamine levels, was identified as one alpha9 integrin cytoplasmic domain-binding protein that is specifically crucial for this process. This proposal will investigate how SSAT mediates a9-dependent cell migration. First, to determine if SSAT affects alpha9-specific migration by modulating levels of polyamines inside the cell, transwell migration assays will be performed with cells expressing various mutant SSAT constructs or pharmacologically depleted of polyamines. Next, immunofluorescence and live confocal microscopy of ectopically-expressed fluorescent protein conjugates will be used to determine localization of the alpha9 integrin and SSAT in migrating cells. Finally, the details of the cell migration signal downstream of SSAT-alpha9 integrin interactions will be investigated, using yeast two-hybrid screening to identify other SSAT-binding proteins that are crucial for a9 integrin-mediated migration. The proposed experiments should provide insight into a novel pathway for regulating integrin function.

描述（由申请人提供）： α9β1 整合素在许多不同的组织中表达，包括平滑肌、表皮和中性粒细胞。来自 α9 整合素亚基敲除小鼠的数据表明该整合素与淋巴管生成有关，从而表明其在血管发育中的潜在作用。此外，体外实验表明，α9整合素亚基可以介导中性粒细胞和非白细胞中细胞迁移的增强。为了研究 α9 整合素介导的细胞迁移背后的分子机制，亚精胺/精胺-N'-乙酰转移酶 (SSAT)（细胞多胺水平的调节剂）被确定为一种对该过程特别重要的 α9 整合素胞质结构域结合蛋白。该提案将研究 SSAT 如何介导 a9 依赖性细胞迁移。首先，为了确定 SSAT 是否通过调节细胞内多胺的水平来影响 α9 特异性迁移，将对表达各种突变 SSAT 构建体或药理学上耗尽多胺的细胞进行 Transwell 迁移测定。接下来，异位表达的荧光蛋白缀合物的免疫荧光和活体共聚焦显微镜将用于确定 α9 整合素和 SSAT 在迁移细胞中的定位。最后，将研究 SSAT-α9 整合素相互作用下游细胞迁移信号的细节，使用酵母双杂交筛选来鉴定对 a9 整合素介导的迁移至关重要的其他 SSAT 结合蛋白。所提出的实验应该提供对调节整合素功能的新途径的见解。