Asef2 Regulates Cell Migration through Myosin II Contractility

描述（由申请人提供）：细胞迁移是许多生物学和病理学过程的核心，包括胚胎发生、炎症反应、组织修复和再生、癌症、关节炎、动脉粥样硬化、骨质疏松症和先天性发育性脑缺陷。然而，尽管它的重要性，迁移目前还没有很好地了解分子水平。迁移可被视为多步骤循环，其包括突起的延伸（通常由外源性试剂诱导）、突起前缘附近附着物（粘附物）的形成和解体、细胞体向前移位以及细胞后部回缩。这些过程的协调调节依赖于肌动蛋白细胞骨架的重塑。Asef 2是最近鉴定的蛋白质，其已显示激活Rac，Rac是肌动蛋白细胞骨架的关键调节剂;然而，Asef 2在调节细胞迁移中的功能目前还不清楚。该提案的目标是研究Asef 2通过其调节潜在过程的能力在控制细胞迁移中的作用。目的1将通过改变蛋白表达和产生影响其活性的突变体来探测Asef 2和Rac在迁移中的功能。为此，将在二维（2D）I型胶原上和三维（3D）I型胶原凝胶中分析迁移，所述I型胶原是促进细胞附着的细胞外基质蛋白。由于组织和器官是三维的，因此3D矩阵将用于概括细胞的体内环境。目的2将通过检验Asef 2通过Rac和肌动蛋白马达蛋白肌球蛋白II控制迁移的假设来探索Asef 2调节迁移的机制。为了支持这一假设，初步结果显示Asef 2增加收缩性，并且已知肌球蛋白II对肌动球蛋白收缩性很重要。在这个目标中，表达构建体，突变体，和抑制剂将被用来确定是否Asef 2通过肌球蛋白II调节迁移。在目标3中，将使用全内反射荧光显微镜（一种先进的显微镜技术）探索Asef 2在调节粘附组装和拆卸（粘附周转）中的作用。此外，类似于目标2中概述的方法将用于确定Asef 2是否通过Rac和肌球蛋白II调节粘附动力学。拟议的研究将显着提高我们对调节细胞迁移的分子因子的理解。这可能导致各种病理性疾病的新治疗方法，这些疾病可归因于不受控制的细胞迁移。

DESCRIPTION (provided by applicant): Cell migration is central to numerous biological and pathological processes, including embryogenesis, the inflammatory response, tissue repair and regeneration, cancer, arthritis, atherosclerosis, osteoporosis, and congenital developmental brain defects. Yet, despite its importance, migration is currently not well understood on a molecular level. Migration may be viewed as a multi-step cycle, which includes extension of a protrusion, usually induced by an exogenous agent, formation and disassembly of attachments (adhesions) near the leading edge of the protrusion, translocation of the cell body forward, and retraction of the cell rear. The coordinated regulation of these processes is dependent on remodeling of the actin cytoskeleton. Asef2 is a recently identified protein that has been shown to activate Rac, which is a key regulator of the actin cytoskeleton; however, the function of Asef2 in modulating cell migration is currently not well understood. The goal of this proposal is to investigate the role of Asef2 in controlling cell migration through its ability to regulate underlying processes. Aim 1 will probe the function of Asef2 and Rac in migration by altering protein expression and by generating mutants that affect their activity. In this aim, migration wil be analyzed on two dimensional (2D) type I collagen, which is an extracellular matrix protein that promotes cell attachment, and in three dimensional (3D) type I collagen gels. Since tissues and organs are three dimensional, 3D matrices will be used to recapitulate the in vivo environment of cells. Aim 2 will explore the mechanism by which Asef2 regulates migration by testing the hypothesis that Asef2 controls migration through Rac and the actin motor protein myosin II. In support of this hypothesis, preliminary results show that Asef2 increases contractility, and myosin II is known to be important for actomyosin contractility. In this aim, expression constructs, mutants, and inhibitors will be employed to determine if Asef2 regulates migration through myosin II. In Aim 3, the role of Asef2 in modulating adhesion assembly and disassembly (adhesion turnover) will be explored using total internal reflection fluorescence microscopy, an advanced microscopy technique. Moreover, an approach similar to that outlined in Aim 2 will be used to determine if Asef2 regulates adhesion dynamics through Rac and myosin II. The proposed studies will significantly enhance our understanding of the molecular factors that regulate cell migration. This could lead to new therapeutic approaches for the various pathological disorders, which can be attributed to uncontrolled cell migration.