Signalling Processes in Dictyostelium

描述（由申请人提供）：趋化性或定向细胞向小分子配体的运动在许多细胞和生理反应中起关键作用，包括癌细胞的转移、免疫系统反应中中性粒细胞和巨噬细胞的运动、发育期间胚胎细胞的迁移和发育期间网骨藻的聚集。在这些不同的细胞类型和过程中，响应细胞能够将浅的细胞外化学引诱物梯度放大为非常陡峭的细胞内梯度，从而将定向信号转化为定向细胞运动。这一要求的实现发生通过一个集成电路的信号通路，是高度保守的，通过进化之间的网骨藻和man.Recent的研究结果建立Ras和相关的GTTRAP1是重要的定向传感，伪足的形成，细胞极化，和细胞附着之间。Ras和Rap1都优先在趋化的网骨藻细胞的前沿被激活，并且废除它们的功能会损害这一过程。 该建议的重点是进一步检查Ras和Rap1的作用，使用网骨藻细胞，这是服从生化，遗传和细胞生物学方法。我们建议确定的机制，Ras和Rap1的激活调节和空间限制的趋化细胞的前沿。这将通过RasGEF复合物的分析和检查Ras和Rap1的特异性GTdR激活蛋白（GAP）的破坏导致的趋化性缺陷来实现。通过检查激活的Ras和Rap1菌株，其中正常的Ras和Rap1活性被改变的空间和时间调节，我们将阐明细胞定向在一个趋化梯度的机制。我们建议确定Ras和Rap1如何通过识别下游效应子来帮助介导趋化性。这些功能将通过分析其无效突变的帮助下，真正的类型的荧光报告和生化分析，揭示不同的组件的定向传感，伪足形成，和细胞极化。本申请中提出的工作应该为控制这种高度进化保守的细胞生物学过程的机制提供新的和重要的见解，从而提供所需的背景来阐明各种人类疾病的细胞基础，包括影响先天免疫和癌细胞转移的那些。

DESCRIPTION (provided by applicant): Chemotaxis, or directed cell movement toward a small molecule ligand, plays a key role in many cellular and physiological responses, including metastasis of cancer cells, movement of neutrophils and macrophages in immune system reactions, migration of embryonic cells during development, and aggregation of Dictyostelium during development. In each of these varied cell types and processes, the responding cells are able to amplify a shallow extracellular chemoattractant gradient into a very steep intracellular gradient and thus translate the directional signal into directional cell movement. Fulfillment of this requirement occurs through an integrated circuit of signaling pathways that are highly conserved through evolution between Dictyostelium and man. Recent findings establish that the Ras and the related GTPase Rap1 are important for directional sensing, pseudopod formation, cell polarization, and cell attachment. Both Ras and Rap1 are preferentially activated at the leading edge of chemotaxing Dictyostelium cells and abrogation of their function impairs this process. This proposal focuses on the further examination of the roles of Ras and Rap1 of using Dictyostelium cells, which are amenable to biochemical, genetic, and cell biological approaches. We propose to identify the mechanisms by which the activations of Ras and Rap1 are regulated and spatially restricted to the leading edge of chemotaxing cells. This will be achieved through the analysis of a RasGEF complex and by examining defects in chemotaxis resulting from disruptions of specific GTPase activating proteins (GAPs) for Ras and for Rap1. Through the examination of the spatial and temporal regulation of activated Ras and Rap1 strains in which normal Ras and Rap1 activity is altered, we will elucidate the mechanisms by which cells orient themselves in a chemoattractant gradient. We propose to determine how Ras and Rap1 help mediate chemotaxis through the identification of downstream effectors. The function of these will be examined through the analysis of their null mutations with the aid of real-type fluorescent reporters and biochemical assays that reveal different components of directional sensing, pseudopod formation, and cell polarization. The work proposed in this application should provide new and important insights into mechanisms that control this highly evolutionarily conserved cell biological process, and thus provide the needed background to elucidate the cellular basis underlying a variety of human diseases, including those affecting innate immunity and metastasis of cancer cells.