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

• 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
• 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

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 细胞生物学中最基本的挑战之一是了解细胞如何在三个- 三维细胞外基质（ECM）在特定的时间和特定的位置。细胞迁移有助于形状 所有组织和器官在发育过程中和正常机制的破坏，通常控制 迁移大大提高了癌症的致命性。我们最近的研究确定了Rab 40亚基的成员， 家族作为细胞迁移的重要调节因子。Rab蛋白是最大的小单体蛋白家族， 属于Ras癌基因超家族的GTP酶。其中，Rab 40亚家族是独一无二的，因为它 含有位于蛋白质C末端的细胞因子信号传导抑制因子（SOCS）盒。 重要的是，我们和其他人已经证明Rab 40蛋白亚家族通过SOCS盒与Cullin 5结合， 形成泛素E3连接酶复合物，在体外和体内调节细胞迁移的各个方面。 因此，Rab 40蛋白作为膜运输、细胞骨架和细胞外基质之间的重要协调者而出现。 动力学和细胞信号传导，并了解Rab 40亚单位的分子机制管理功能， 在细胞迁移过程中的家庭是这个建议的主要焦点。在所有脊椎动物中Rab 40家族由两个 Rab 40 b和Rab 40 c。我们最近的工作表明Rab 40 b和Rab 40 c对于 乳腺癌中基质金属蛋白酶的靶向分泌以及肌动蛋白动力学的调节 迁移、侵袭和转移。我们还发现Rab 40 b/Cullin 5复合物介导泛素化， Rab 40 c/Cullin 5介导蛋白磷酸酶6， (PP6)复杂的泛素化和失活。根据我们公布的和初步的数据，我们假设 Rab 40 b和Rab 40 c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。以下目的旨在通过结合以下因素来检验这一假设： Rytis Prekeris博士（Rab GTP酶和肌动蛋白动力学）、Kristin Artinger博士（斑马鱼和 神经嵴细胞迁移）和Traci里昂博士（乳腺癌）。首先，我们将定义 Rab 40 b/Cullin 5和Rap 2复合物在细胞迁移过程中调节膜和肌动蛋白动力学的作用 绘制Rab 40 b依赖的Rap 2泛素化位点，并分析Rap 2如何调节肌动蛋白和局灶性 在前缘和/或侵入伪足处的粘附位点动力学。其次，我们将阐明 Rab 40 c/Cullin 5-PP 6复合物在细胞迁移信号转导中的作用为此，我们将使用蛋白质 结合试验结合蛋白质诱变和各种显微镜方法来确定 PP 6与Rab 40 c结合的生物化学性质以及这种结合对PP 6复合物的影响 稳定性和活性。第三，我们将确定Rab 40 b/Cullin 5-Rap 2和Rab 40 c/Cullin 5-PP 6的功能 由于斑马鱼模型允许直接和真实的- 细胞迁移的体内时间分析。