Spatial regulation of cytoskeleton during chemotaxis

描述(申请人提供)：趋化性，细胞沿化学梯度向上移动，是真核细胞中各种生物学过程的中心，包括创伤愈合过程中巨噬细胞和中性粒细胞的迁移，胸腺细胞的归巢，神经脊细胞的迁移，以及网状网状茎的聚集。趋化运动的第一步是趋化剂介导的细胞前沿F-肌动蛋白聚合的增加，这为伪足延伸和细胞运动提供了动力。剖析控制F-肌动蛋白组织的信号机制将是理解定向细胞运动的关键一步。Wiskott-Aldrich综合征蛋白(WASP)及其相关蛋白已成为连接多条信号通路与F-肌动蛋白聚合的关键下游成分。WASP基因的突变会导致Wiskott-Aldrich综合征(Was)，这是一种人类X连锁免疫缺陷。IS患者的中性粒细胞和巨噬细胞的趋化性被发现是有缺陷的，尽管随机运动的速度相对正常。虽然许多重要的研究都集中在利用体外生化研究来研究WASP的激活机制，但对WASP在定向运动调节中的作用知之甚少。以镉42为诱饵，通过酵母双杂交筛选，鉴定了一个与人WASP同源的网柄菌基因(DdWASP)。在我们在这一应用中展示的初步结果中，DdWASP似乎在趋化过程中对肌动蛋白细胞骨架的调节中发挥了重要作用。我们假设DdWASP在空间和时间上的激活调控对于网柄苔藓趋化过程中肌动蛋白细胞骨架的空间调控是必不可少的。我们提出的假设是，细胞获得F-肌动蛋白组装的极化激活主要是通过动态调节DdWASP亚细胞定位来实现的。为了剖析调控DdWASP定位和激活的信号通路，我们将表征负责调控DdWASP空间定位和激活的结构域(S)，并确定它们与其他信号成分的相互作用。这项研究的结果将有助于我们理解信号通路中特定成分的激活如何收敛到激活依赖于DdWASP的F-肌动蛋白聚合，以及细胞如何不断重塑肌动蛋白细胞骨架以形成趋化剂源方向的前沿。这项研究还将阐明WASP在人类中的突变如何导致细胞骨架缺陷、白细胞功能障碍和血液系统恶性肿瘤。

DESCRIPTION (provided by applicant): Chemotaxis, cell movement up a chemical gradient, is central to a wide variety of biological processes in eukaryotic cells, including migration of macrophage and neutrophils during wound healing, homing of thymocytes, migration of neural crest cells, and aggregation of Dictyostelium. The first step of chemotactic movement is a chemoattractant mediated increase in F-actin polymerization at the leading edge of the cell, which provides the motive force for pseudopod extension and cell movement. Dissecting signaling mechanisms controlling F-actin organization would be a key step toward understanding directed cell movement. The Wiskott-Aldrich Syndrome protein (WASP) and related proteins have emerged as key downstream components linking multiple signaling pathways to F-actin polymerization. Mutations in the WASP gene cause Wiskott-Aldrich Syndrome (WAS), a human X-linked immunodeficiency. Chemotaxis of neutrophils and macrophages from WAS patients was found to be defective despite relatively normal speed of random motility. While many important studies have focused on using in vitro biochemical studies to examine the mechanism of WASP activation, little is known about the role of WASP in the regulation of directional motility. A Dictyostelium gene encoding a protein (DdWASP) homologous to human WASP was identified by a yeast two-hybrid screen with Cdc42 as bait. In our preliminary results presented in this application, DdWASP appears to play an important role in the regulation of actin cytoskeleton during chemotaxis. We hypothesize that dynamic regulation of the spatial and temporal activation of DdWASP is essential for the spatial regulation of actin cytoskeleton during Dictyostelium chemotaxis. We propose to test the hypothesis that cells acquire polarized activation of F-actin assembly mainly by dynamic regulation of DdWASP subcellular localization. To dissect signaling pathways regulating DdWASP localization and activation, we will characterize the domain(s) responsible for the regulation of spatial localization and activation of DdWASP and identify their interactions with other signaling components. Results from this study will help us understand how activation of specific components in signaling pathways converges to activate DdWASP-dependent F-actin polymerization and how cells continuously remodel the actin cytoskeleton to form the leading edge in the direction of a chemoattractant source. This study will also elucidate how mutations in WASP in humans lead to the cytoskeletal defects, leukocytes dysfunction, and hematopoietic malignancies.