Spatial regulation of cytoskeleton during chemotaxis

趋化过程中细胞骨架的空间调节

• 批准号: 7059425
• 负责人: CHANG Y CHUNG
• 金额: $ 26.54万
• 依托单位: VANDERBILT UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2003
• 资助国家: 美国
• 起止时间: 2003-05-01 至 2008-04-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7059425
• 关键词: SDS polyacrylamide gel electrophoresis; Wiskott Aldrich syndrome; actins; binding proteins; biological signal transduction; cell motility; chemoattractants; chemotaxis; confocal scanning microscopy; cyclic AMP; cytoskeleton; enzyme linked immunosorbent assay; fluorescence resonance energy transfer; gene mutation; immunoprecipitation; macrophage; neutrophil; phosphotransferases; polymerase chain reaction; polymerization; protein protein interaction; protein structure function; site directed mutagenesis

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.

描述（由申请人提供）：趋化性，化学梯度的细胞运动是真核细胞中各种各样的生物学过程的核心，包括在伤口愈合过程中巨噬细胞和中性粒细胞的迁移，胸腺细胞的归位，神经crest细胞的迁移以及dictyostelium的聚集。趋化运动的第一步是在细胞的前缘介导F-肌动聚合的介导剂介导的增加，这为假脚架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传导机制将是了解定向细胞运动的关键步骤。 Wiskott-Aldrich综合征蛋白（WASP）和相关蛋白已成为将多个信号通路与F-肌动蛋白聚合联系起来的关键下游成分。 WASP基因的突变引起Wiskott-Aldrich综合征（WAS），一种人类X连锁的免疫缺陷。尽管随机运动的速度相对正常，但发现患者的嗜中性粒细胞和巨噬细胞的趋化性是有缺陷的。尽管许多重要的研究集中在使用体外生化研究来检查黄蜂激活的机理，但对于黄蜂在定向运动的调节中的作用知之甚少。用酵母的两杂化筛选cdc42作为诱饵，鉴定出编码与人黄蜂同源的蛋白质（DDWASP）的dictyostelium基因。在本应用中提出的初步结果中，DDWASP在趋化过程中的肌动蛋白细胞骨架调节中似乎起着重要作用。我们假设DDWASP的空间和时间激活的动态调节对于dictyotostelium趋化过程中肌动蛋白细胞骨架的空间调节至关重要。我们建议测试以下假设：细胞主要通过动态调节DDWASP亚细胞定位，从而获得F-肌动蛋白组装的极化激活。为了剖析调节DDWASP定位和激活的信号通路，我们将表征负责调节空间定位和DDWASP激活的域，并确定其与其他信号传导组件的相互作用。这项研究的结果将有助于我们了解信号通路中特定成分的激活如何收敛到激活DDWASP依赖性的F-肌动蛋白聚合以及细胞如何连续重塑肌动蛋白细胞骨架以沿化学量吸收剂源的方向形成前缘。这项研究还将阐明人类黄蜂的突变如何导致细胞骨架缺陷，白细胞功能障碍和造血恶性肿瘤。