Temporal and Spatial Signaling in Chemotaxis

描述(申请人提供)：趋化性，细胞沿细胞外梯度定向移动，是一个有趣和重要的过程，指导细胞在发育中的胚胎。在成人中，趋化性是正确和免疫反应以及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，不适当的细胞迁移也是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)在内的病理条件的基础。在趋化细胞中，尽管受体和连接的G蛋白仍然均匀地分布在细胞周围，但信号是高度极化的。PI3Ks和PTEN分别与细胞的前膜和后膜结合，由此产生的PI(3，4，5)P3的积累促进了细胞前部伪足的产生。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，我们将筛选结构性结合的版本，并使用这些试剂以生化方法鉴定膜结合位点。为了找到结合的调节因素，我们将筛选基因组中导致或能够逆转每种酶的构成膜定位的突变。第二，定位的PI(3，4，5)P3是如何促进伪足延伸的？我们最近的证据表明，PI(3，4，5)P3的升高通过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，并将通过与识别PKB靶标的磷酸特异性抗体免疫沉淀的蛋白质的质谱学来确定PKB底物。将通过基因中断和过度表达研究来评估每个假定的靶点在介导化学诱导剂诱导的反应中的作用。我们还将使用经过验证的基于FRET的记者对正在进行趋化作用的活细胞中的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方向性响应有贡献？为了识别更多的极性蛋白质，我们将表达一个与GFP融合的阵列化的cDNA文库，并肉眼检查那些位于前面或后面的高度极化的细胞。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研究来评估。最后，我们将测试这些蛋白质在适当极化的哺乳动物细胞中是否具有保守功能。与公共健康相关：我们的研究重点放在引导细胞迁移的基本机制上，例如那些关闭伤口或聚集在感染部位的细胞。这种细胞的“归巢”反应对许多正常功能至关重要，在疾病中常常会发生改变。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条件的新策略，如癌症转移、动脉粥样硬化、哮喘和关节炎。

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.