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，即细胞沿着细胞外梯度的定向运动，是一个吸引人的和重要的过程，在胚胎发育中引导细胞。在成人中，趋化性是正确的免疫反应和组织维护和修复所必需的。除了在正常生理学中的这些作用之外，不适当的细胞迁移是包括癌症转移和慢性炎性疾病如动脉粥样硬化、哮喘和关节炎的病理状况的基础。在趋化细胞中，尽管受体和连接的G蛋白保持均匀分布在细胞周边，但信号是高度极化的。PI 3 Ks和PTEN分别在细胞的前部和后部与膜结合，并且所产生的PI（3，4，5）P3的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PI 3 K和PTEN，我们将筛选组成型结合的版本，并使用这些试剂以生物化学方式鉴定膜结合位点。为了找到结合的调节剂，我们将筛选基因组中引起或可以逆转每种酶的组成性膜定位的突变。第二，PI（3，4，5）P3如何促进伪足的延伸？我们最近的证据表明，PI（3，4，5）P3的升高通过PKB信号转导调节细胞骨架。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靶点的磷酸特异性抗体免疫沉淀的蛋白质的质谱来鉴定PKB底物。将通过基因破坏和过表达研究来评估每个假定靶点在介导化学引诱剂诱导的反应中的作用。我们还将图像PKB激活和激酶活性的活细胞进行趋化性使用验证FRET为基础的记者。第三，还有哪些强极化分量对定向响应有贡献？为了鉴定更多的极性蛋白，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中定位于前部或后部的蛋白。这些基因在趋化性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是否在适当的极化哺乳动物细胞中具有保守的功能。与公共卫生的相关性：我们的研究重点是引导迁移细胞的基本机制，例如那些关闭伤口或聚集在感染部位的细胞。这种细胞“归巢”反应对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，干预病理条件涉及细胞迁移，如癌症转移，动脉粥样硬化，哮喘和关节炎。