Temporal and Spatial Signaling in Chemotaxis

趋化性中的时间和空间信号传导

基本信息

批准号：
7904703
负责人：
Peter N Devreotes
金额：
$ 22.13万
依托单位：
JOHNS HOPKINS UNIVERSITY
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：
2009-09-01 至 2011-03-31
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/7904703
关键词：
Actins Adult Arthritis Asthma Atherosclerosis Back Binding Binding Sites Biochemical Biological Biological Assay Cell Line Cell fusion Cell-Free System Cells Chemotactic Factors Chemotaxis Chimeric Proteins Chronic Complex Cytokinesis Cytoskeleton Defect Disease Dissociation Embryo Enzymes Event GTP-Binding Proteins Genes Genome Genomics Goals Grant Homing Homologous Gene Image Immune response Immunoprecipitation Infection Inflammatory Lead Libraries Life Link Maintenance Mammalian Cell Mass Spectrum Analysis Mediating Membrane Molecular Mutation Neoplasm Metastasis PTEN gene Phospho-Specific Antibodies Phosphorylation Phosphotransferases Physiological Physiology Process Production Proteins Pseudopodia Public Health Reaction Reagent Receptor Cell Reporter Research Role Series Signal Transduction Site Spatial Distribution Testing Tissues base cell motility cell type crosslink extracellular gain of function gene discovery gene function loss of function overexpression phosphoinositide-3,4,5-triphosphate polarized cell polymerization receptor reconstitution repaired response wound

项目摘要

DESCRIPTION (provided by applicant): Chemotaxis, the directed movement of cells along extracellular gradients, is an intriguing and vital process that guides cells in developing embryos. In the adult, chemotaxis is required for correct and immune responses and in tissue maintenance and repair. In addition to these roles in normal physiology, inappropriate cell migration is the basis for pathological conditions including cancer metastasis and chronic inflammatory diseases such as atherosclerosis, asthma and arthritis. In chemotaxing cells, although receptors and linked G-proteins remain uniformly distributed around the cell perimeter, signaling is highly polarized. PI3Ks and PTEN bind to the membrane at the front and back of the cell, respectively, and the resulting accumulation of PI(3,4,5)P3 promotes pseudopodia production at the front. These observations raise new questions. First, how does receptor/G-protein signaling localize these key enzymes? By randomly mutagenizing PI3K and PTEN, we will screen for versions that bind constitutively and use these reagents to identify the membrane binding sites biochemically. To find regulators of binding, we will screen for mutations in the genome that cause, or can reverse, constitutive membrane localization of each enzyme. Second, how does localized PI(3,4,5)P3 promote pseudopodia extension? We have recent evidence to suggest that elevated PI(3,4,5)P3 regulates the cytoskeleton by signaling through PKB. We will use cell-free systems to define the mechanisms by which receptor/G-protein signaling controls PKB activity and will identify PKB substrates by mass- spectrometry of proteins that immunoprecipitate with phosphospecific antibodies that recognize PKB targets. The role of each putative target in mediating chemoattractant-induced responses will be assessed by gene disruption and overexpression studies. We will also image PKB activation and kinase activity in living cells undergoing chemotaxis using validated FRET-based reporters. Third, what additional strongly polarized components contribute to the directional response? To identify more polarity proteins, we will express an arrayed library of cDNAs fused to GFP and visually inspect highly polarized cells for those that localize to the front or back. The function of these genes in chemotactic signaling will be assessed by both loss of function and gain of function studies. Finally, we will test whether these proteins have conserved functions in appropriate polarized mammalian cells. Relevance to public health: Our research focuses on the basic mechanisms that guide migrating cells, such as those closing a wound or gathering at the site of an infection. This cellular "homing" response is critical for many normal functions and is often altered in diseases. Our studies will provide basic information and likely lead to new strategies for intervening in pathological conditions involving cell migration such as cancer metastasis, atherosclerosis, asthma, and arthritis.

描述（由申请人提供）：趋化性，即细胞沿着细胞外梯度的定向运动，是引导胚胎发育中细胞的一个有趣且重要的过程。在成人中，正确的免疫反应以及组织维护和修复需要趋化性。除了在正常生理学中的这些作用之外，不适当的细胞迁移也是病理状况的基础，包括癌症转移和慢性炎症性疾病，如动脉粥样硬化、哮喘和关节炎。在趋化细胞中，尽管受体和连接的 G 蛋白仍然均匀分布在细胞周边，但信号传导是高度极化的。 PI3Ks 和 PTEN 分别与细胞前部和后部的膜结合，由此产生的 PI(3,4,5)P3 积累促进前部伪足的产生。这些观察提出了新的问题。首先，受体/G 蛋白信号如何定位这些关键酶？通过随机诱变 PI3K 和 PTEN，我们将筛选组成型结合的版本，并使用这些试剂通过生化方法识别膜结合位点。为了找到结合的调节因子，我们将筛选基因组中导致或可以逆转每种酶的组成性膜定位的突变。其次，局部化的PI(3,4,5)P3如何促进伪足延伸？我们最近的证据表明，升高的 PI(3,4,5)P3 通过 PKB 信号传导来调节细胞骨架。我们将使用无细胞系统来定义受体/G 蛋白信号传导控制 PKB 活性的机制，并将通过与识别 PKB 靶标的磷酸特异性抗体进行免疫沉淀的蛋白质质谱分析来识别 PKB 底物。将通过基因破坏和过度表达研究来评估每个假定靶标在介导化学引诱剂诱导的反应中的作用。我们还将使用经过验证的基于 FRET 的报告基因对经历趋化性的活细胞中的 PKB 激活和激酶活性进行成像。第三，哪些额外的强极化分量有助于定向响应？为了识别更多极性蛋白质，我们将表达与 GFP 融合的 cDNA 阵列文库，并目视检查高度极化的细胞，看看是否存在位于正面或背面的细胞。这些基因在趋化信号传导中的功能将通过功能丧失和功能获得研究来评估。最后，我们将测试这些蛋白质在适当的极化哺乳动物细胞中是否具有保守功能。与公共卫生的相关性：我们的研究重点是引导细胞迁移的基本机制，例如闭合伤口或聚集在感染部位的细胞。这种细胞“归巢”反应对于许多正常功能至关重要，并且在疾病中经常发生改变。我们的研究将提供基本信息，并可能导致干预涉及细胞迁移的病理状况（例如癌症转移、动脉粥样硬化、哮喘和关节炎）的新策略。