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：
10536451
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/10536451
关键词：
3-Dimensional AGFG1 gene Actins Binding Binding Proteins Biochemical Biological Assay C-terminal CISH gene Cells Cellular biology Complex Cytoskeleton Data Defect Development Extracellular Matrix Family Focal Adhesions Foundations Glutathione Goals Guanosine Triphosphate Phosphohydrolases Immunoprecipitation In Vitro Intracellular Membranes Knowledge Location Malignant Neoplasms Maps Matrix Metalloproteinases Mediating Membrane Membrane Protein Traffic Microscopy Migration Assay Modeling Molecular Mutagenesis Neoplasm Metastasis Neural Crest Cell Organ Pathway interactions Process Property Protein Family Protein Subunits Proteins Pseudopodia Publishing Regulation Research Role Shapes Signal Transduction Site Testing Three-Dimensional Imaging Time Tissues Transmembrane Transport Vertebrates Work Zebrafish base cancer cell cancer therapy cell motility design imaging approach in vitro Assay in vivo in vivo Model malignant breast neoplasm member migration 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.

项目摘要细胞生物学中最根本的挑战之一是了解细胞如何在三个中迁移在特定时间和特定位置，尺寸细胞外矩阵（ECM）。细胞迁移有助于形状在开发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迁移极大地增强了癌症的杀伤力。我们最近的研究确定了Rab40子的成员家族作为细胞迁移的重要调节剂。 Rab蛋白是最大的小型单体家族属于Ras Oncogene超家族的GTPases。其中，Rab40子家庭是独一无二的包含位于蛋白质C末端的细胞因子信号传导（SOCS）盒的抑制剂。重要的是，我们和其他人已经表明，蛋白质的Rab40子家庭通过SOCS盒与Cullin5结合到Cullin5与形成一种泛素E3连接酶复合物，可在体外和体内调节细胞迁移的各个方面。因此，Rab40蛋白在膜运输，细胞骨架之间作为重要协调剂出现动力学和细胞信号传导，以及理解Rab40 sub--的分子机制细胞迁移期间的家庭是该提案的主要重点。在所有脊椎动物中，rab40家族由两个会员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，入侵和转移。我们还表明，rab40b/cullin5复合物介导了泛素化包括RAP2在内的几个细胞迁移的调节剂，而Rab40c/Cullin5介导蛋白磷酸酶6 （PP6）复杂的泛素化和灭活。基于我们发布的初步数据，我们假设 RAB40B和RAB40C介导信号，膜运输和肌动蛋白之间的协调细胞迁移期间的动力学。以下目的旨在通过结合结合来检验该假设 Rytis Prekeris博士的独特专业知识（RAB GTPases和Actin Dynamics），Kristin Artinger博士（Zebrafish和神经rest细胞迁移）和Traci Lyons博士（乳腺癌）。首先，我们将定义 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方面通过映射依赖RAB40B的RAP2泛素化位点并解剖RAP2如何调节肌动蛋白和焦点前缘和/或Invadopodia的粘附位点动力学。其次，我们将阐明 RAB40C/CULLIN5-PP6复合物在调节细胞迁移过程中信号传导方面。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该结合对PP6复合物的后果稳定性和活动。第三，我们将确定RAB40B/CULLIN5-RAP2和RAB40C/CULLIN5-PP6的功能神经rest细胞在体内使用斑马鱼在体内迁移的途径，因为斑马鱼模型允许直接和实地细胞迁移的体内分析时间。