The mechanisms regulating actin dynamics and polarized membrane transport during 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阶段迁移- 在特定时间和特定位置对细胞外基质(ECM)进行立体定向。细胞迁移有助于塑造 发育过程中的所有组织和器官以及正常控制的正常机制的破坏 移民极大地提高了癌症的致命性。我们最近的研究确定了Rab40亚家族的成员 家族是细胞迁移的重要调节因子。Rab蛋白是最大的小单体家族 GTP酶属于RAS癌基因超家族。其中，Rab40子家族独树一帜，因为它 含有位于蛋白质C末端的细胞因子信号转导抑制因子(SOCS)盒。 重要的是，我们和其他人已经证明了Rab40亚家族蛋白通过SoCs盒与cullin5结合，从而 形成泛素E3连接酶复合体，调节细胞在体外和体内迁移的各个方面。 因此，Rab40蛋白成为膜转运、细胞骨架之间的重要协调者 动力学和细胞信号转导，以及了解Rab40亚基调控功能的分子机制 细胞迁移过程中的家庭是这一提议的主要焦点。在所有脊椎动物中，Rab40家族由两个 成员，Rab40b和Rab40c。我们最近的工作表明，Rab40b和Rab40c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Rab40b/cullin5复合体介导泛素化 包括Rap2在内的几种细胞迁移调节因子，而Rab40c/Cullin5介导蛋白磷酸酶6 (PP6)复合体泛素化和失活。根据我们公布的初步数据，我们假设 Rab40b和Rab40c介导信号、膜转运和肌动蛋白之间的协调 细胞迁移过程中的动态变化。以下目标旨在通过组合以下各项来检验这一假设 Rytis Prekeris博士(Rab GTP酶和肌动蛋白动力学)、Kristin Artinger博士(斑马鱼和 神经脊细胞迁移)和Traci Lyons博士(乳腺癌)。首先，我们将定义 Rab40b/cullin5和Rap2复合体在细胞迁移过程中调节细胞膜和肌动蛋白动态的作用 定位Rab40b依赖的Rap2泛素化位点并剖析Rap2如何调节肌动蛋白和斑点 前缘和/或内侧足底的粘连部位动态。第二，我们将阐明 Rab40c/Cullin5-PP6复合体在调节细胞迁移信号中的作用。为此，我们将使用蛋白质 结合蛋白质诱变和不同显微镜方法的结合分析 PP6与Rab40c结合的生化特性及其对PP6复合体的影响 稳定性和活跃性。第三，我们将确定Rab40b/Cullin5-Rap2和Rab40c/Cullin5-PP6的功能 由于斑马鱼模型允许直接和真实的细胞迁移，因此利用斑马鱼在体内迁移神经脊细胞的途径 体内细胞迁移时间分析。

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.