Temporal and Spatial Signaling in Chemotaxis

趋化性中的时间和空间信号传导

• 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
• 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

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）：趋化性是细胞沿细胞外梯度的定向运动，是一个有趣而重要的过程，可引导细胞发展胚胎。在成年人中，正确和免疫反应以及组织维护和修复需要趋化性。除了正常生理学中的这些作用外，不适当的细胞迁移是病理状况的基础，包括癌症转移和慢性炎症性疾病，例如动脉粥样硬化，哮喘和关节炎。在趋化细胞中，尽管受体和连接的G蛋白保持在细胞周围周围均匀分布，但信号传导高度极化。 PI3K和PTEN分别与细胞正面和背面的膜结合，PI的产生积累（3,4,5）P3促进了正面的假地产生。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，我们将筛选出组成性结合并使用这些试剂以生化识别膜结合位点的版本。为了找到结合的调节剂，我们将筛选在基因组中导致或可能逆转每个酶的膜定位的突变。其次，局部PI（3,4,5）P3如何促进假地延伸？我们最近有证据表明，升高的PI（3,4,5）P3通过通过PKB发出信号来调节细胞骨架。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，并通过质谱鉴定PKB底物，这些蛋白质的蛋白质质谱法可以用识别PKB靶标的磷酸型抗体进行免疫沉淀。每个假定靶标在介导化学吸引剂引起的反应中的作用将通过基因破坏和过表达研究来评估。我们还将使用经过验证的基于FRET的记者进行趋化细胞中的PKB激活和激酶活性。第三，哪些其他强化的组件有助于方向响应？为了识别更多的极性蛋白，我们将表达一个融合GFP的cDNA库，并在视觉上检查高度极化的细胞，以定位于前后或背部。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研究的增益来评估。最后，我们将测试这些蛋白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：我们的研究重点是指导迁移细胞的基本机制，例如关闭伤口或在感染部位聚集的细胞。这种细胞“归纳”反应对于许多正常功能至关重要，并且经常在疾病中改变。我们的研究将提供基本信息，并可能导致新的策略，以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。