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The mechanisms regulating actin dynamics and polarized membrane transport during cell migration

细胞迁移过程中调节肌动蛋白动力学和极化膜运输的机制

基本信息

批准号：
10693336
负责人：
Kristin Artinger
金额：
$ 31.61万
依托单位：
UNIVERSITY OF COLORADO DENVER
依托单位国家：
美国
项目类别：
财政年份：
2018
资助国家：
美国
起止时间：
2018-02-01 至 2026-08-31
项目状态：
未结题

来源：
https://reporter.nih.gov/project-details/10693336
关键词：
3-Dimensional AGFG1 gene Actins Binding Binding Proteins Biochemical Biological Assay C-terminal CISH gene Cellular biology Complex Cullin Proteins Cytoskeleton Data Defect Development Extracellular Matrix Family Focal Adhesions Foundations Glutathione Goals Guanosine Triphosphate Phosphohydrolases Immunoprecipitation In Vitro Intracellular Membranes Invaded Knowledge Location Malignant Neoplasms Maps Matrix Metalloproteinases Mediating Membrane Membrane Protein Traffic Microscopy Migration Assay Modeling Molecular Mutagenesis Neoplasm Metastasis Neural Crest Cell Organ PP5 protein-serine-threonine phosphatase Pathway interactions Process Property Protein Subunits Proteins Pseudopodia Publishing Regulation Research Role Shapes Signal Transduction Site Testing Three-Dimensional Imaging Time Tissues Traction Transmembrane Transport Vertebrates Work Zebrafish cancer cell cancer therapy cell motility design imaging approach in vitro Assay in vivo in vivo Model malignant breast neoplasm member migration monomer multidisciplinary novel protein phosphatase 6 rab GTP-Binding Proteins ras Oncogene scaffold trafficking ubiquitin-protein ligase zebrafish development

项目摘要

Project Summary One of the most fundamental challenges in cell biology is understanding how cells migrate in three- dimensional extracellular matrices (ECM) at specific times and to specific locations. Cell migration helps shape all tissues and organs during development and the disruption of the normal mechanisms that normally control migration dramatically enhance the lethality of cancers. Our recent studies identified members of Rab40 sub- family as important regulators of cell migration. Rab proteins are the largest family of small monomeric GTPases belonging to the Ras oncogene superfamily. Among them, Rab40 sub-family is unique because it contains a suppressor of cytokine signaling (SOCS) box located at the C-terminal end of the protein. Importantly, we and others have shown that Rab40 sub-family of proteins bind to Cullin5 via the SOCS box to form a ubiquitin E3 ligase complex that regulate various aspects of cell migration in vitro and in vivo. Consequently, Rab40 proteins emerged as important coordinators between membrane trafficking, cytoskeleton dynamics and cell signaling, and understanding the molecular machinery governing functions of Rab40 sub- family during cell migration is a major focus of this proposal. In all vertebrates Rab40 family consists of two members, Rab40b and Rab40c. Our recent work has shown that Rab40b and Rab40c are important for targeted secretion of matrix metalloproteinases, as well as regulation of actin dynamics during breast cancer migration, invasion and metastasis. We also have shown that Rab40b/Cullin5 complex mediate ubiquitylation of several regulators of cell migration, including Rap2, while Rab40c/Cullin5 mediates protein phosphatase 6 (PP6) complex ubiquitylation and inactivation. Based on our published and preliminary data, we hypothesize that Rab40b and Rab40c mediate coordination between signaling, membrane trafficking, and actin dynamics during cell migration. The following aims are designed to test this hypothesis by combining of the unique expertise from Dr. Rytis Prekeris (Rab GTPases and actin dynamics), Dr. Kristin Artinger (zebrafish and neural crest cell migration), and Dr. Traci Lyons (breast cancer). First, we will define the roles of Rab40b/Cullin5 and Rap2 complexes in regulating membrane and actin dynamics during cell migration by mapping Rab40b-dependent Rap2 ubiquitylation sites and dissecting how Rap2 regulates actin and focal adhesion site dynamics at the leading edge and/or invadopodia. Second, we will elucidate the roles of Rab40c/Cullin5-PP6 complex in regulating signaling during cell migration. To that end, we will use protein binding assays in combination with protein mutagenesis and various microscopy approaches to determine biochemical properties of PP6 binding to Rab40c and the consequences of this binding on PP6 complex stability and activity. Third, we will determine the functions of Rab40b/Cullin5-Rap2 and Rab40c/Cullin5-PP6 pathways during neural crest cell migration in vivo using zebrafish since zebrafish model allows direct and real- time in vivo analysis of cell migration.

项目概要细胞生物学中最基本的挑战之一是了解细胞如何以三种方式迁移：在特定时间和特定位置的维度细胞外基质（ECM）。细胞迁移有助于塑造发育过程中的所有组织和器官以及通常控制的正常机制的破坏移民极大地提高了癌症的致死率。我们最近的研究确定了 Rab40 亚组的成员家族作为细胞迁移的重要调节者。 Rab 蛋白是最大的小单体家族 GTPases 属于 Ras 癌基因超家族。其中，Rab40亚家族比较独特，因为它包含位于蛋白质 C 末端的细胞因子信号传导抑制器 (SOCS) 盒。重要的是，我们和其他人已经证明 Rab40 蛋白亚家族通过 SOCS 盒与 Cullin5 结合，形成泛素 E3 连接酶复合物，调节体外和体内细胞迁移的各个方面。因此，Rab40 蛋白成为膜运输、细胞骨架之间的重要协调者。动力学和细胞信号传导，并了解控制 Rab40 子功能的分子机制细胞迁移过程中的家庭是该提案的主要焦点。在所有脊椎动物中，Rab40 家族由两个组成成员 Rab40b 和 Rab40c。我们最近的工作表明 Rab40b 和 Rab40c 对于基质金属蛋白酶的靶向分泌以及乳腺癌期间肌动蛋白动力学的调节迁移、侵袭和转移。我们还表明 Rab40b/Cullin5 复合物介导泛素化包括 Rap2 在内的多种细胞迁移调节因子，而 Rab40c/Cullin5 介导蛋白磷酸酶 6 (PP6) 复杂的泛素化和失活。根据我们发布的初步数据，我们假设 Rab40b 和 Rab40c 介导信号传导、膜运输和肌动蛋白之间的协调细胞迁移过程中的动力学。以下目标旨在通过结合以下内容来检验这一假设： Rytis Prekeris 博士（Rab GTPases 和肌动蛋白动力学）、Kristin Artinger 博士（斑马鱼和肌动蛋白动力学）的独特专业知识神经嵴细胞迁移）和 Traci Lyons 博士（乳腺癌）。首先，我们将定义角色 Rab40b/Cullin5 和 Rap2 复合物在细胞迁移过程中调节膜和肌动蛋白动力学绘制 Rab40b 依赖性 Rap2 泛素化位点并剖析 Rap2 如何调节肌动蛋白和焦点前缘和/或侵袭伪足的粘附位点动态。其次，我们将阐明以下角色： Rab40c/Cullin5-PP6 复合物在细胞迁移过程中调节信号传导。为此，我们将使用蛋白质结合测定结合蛋白质诱变和各种显微镜方法来确定 PP6 与 Rab40c 结合的生化特性以及这种结合对 PP6 复合物的影响稳定性和活性。第三，我们将确定Rab40b/Cullin5-Rap2和Rab40c/Cullin5-PP6的功能使用斑马鱼体内神经嵴细胞迁移的途径，因为斑马鱼模型允许直接和真实的细胞迁移的体内时间分析。