Temporal and Spatial Signaling in Chemotaxis

描述（由申请人提供）：趋化性，细胞沿着细胞外梯度定向运动，是一个有趣的和重要的过程，指导胚胎发育中的细胞。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。除了在正常生理中的这些作用外，不适当的细胞迁移是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）等病理条件的基础。在趋化细胞中，虽然受体和连接的g蛋白仍然均匀分布在细胞周围，但信号是高度极化的。pi3k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的积累促进了前部伪足的产生。这些观察结果提出了新的问题。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，我们将筛选具有组成性结合的版本，并使用这些试剂进行生物化学鉴定膜结合位点。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膜定位的突变。第二，定位的PI(3,4,5)P3是如何促进假足延伸的？我们最近有证据表明，升高的PI(3,4,5)P3通过PKB信号调节细胞骨架。我们将使用无细胞系统来确定受体/ g蛋白信号控制PKB活性的机制，并将通过识别PKB靶标的磷酸化特异性抗体免疫沉淀蛋白的质谱法鉴定PKB底物。每个假定的靶点在化学引诱剂诱导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的报告基因，对发生趋化的活细胞中的PKB激活和激酶活性进行成像。第三，哪些额外的强极化分量对定向响应有贡献？为了鉴定更多极性蛋白，我们将表达与GFP融合的cdna序列文库，并目视检查那些位于前部或后部的高度极化细胞。这些基因在趋化信号中的功能将通过功能丧失和功能获得的研究来评估。最后，我们将测试这些蛋白是否在适当的极化哺乳动物细胞中具有保守功能。与公共卫生相关：我们的研究重点是引导迁移细胞的基本机制，例如那些愈合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，在疾病中经常发生改变。我们的研究将提供基本信息，并可能为干预涉及细胞迁移的病理条件（如癌症转移、动脉粥样硬化、哮喘和关节炎）提供新的策略。

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.