Control of cell polarity and migration by non-centrosomal microtubules

项目概要/摘要。 细胞迁移需要肌动蛋白和微管（MT）细胞骨架的协调和调节重塑 对于发育过程和自我平衡的维持，以及在身体对外部侮辱的反应期间， 以及包括心脏病和肿瘤转移的疾病状态。在细胞迁移过程中，肌动蛋白丝组装， 与粘着斑（FA）复合物连接，而MT经历由MT局部控制的动态不稳定性。 相关蛋白（MAPs）。这两个过程使细胞能够建立前沿和后沿， 以定向持久性迁移。尽管我们在理解功能性影响方面取得了许多进展， MT和MT-FA相互作用的MAP，MT组织在空间和时间上的精确程度仍然未知 与FA协调以促进定向细胞运动。最近的一项发现表明，非中心体MT是 已经建立了一个范式转变，这表明非极化细胞迁移是足够的，也是必需的。 中心体MT被引发以不同于由中心体成核的MT的方式发挥功能。这一发现 强调了需要确定细胞骨架蛋白如何识别和调节非中心体与 中心体MT动态以及对极性和迁移的影响。这种知识上的差距影响了我们对 基本过程，包括信号分子如何同时调节蛋白质家族， 复杂的任务，如引导持续的细胞迁移。小的GT3，Rac 1，是一种关键的信号蛋白， 空间控制，以促进FA形成，MT生长和肌动蛋白丝组装，导致前缘 提前Rac 1信号传导由分子马达蛋白肌球蛋白II补充，肌球蛋白II组织肌动蛋白应力纤维， 促进FA成熟，并产生将细胞的后缘向前拉的力。因此，Rac 1和肌球蛋白-II 在空间和时间上受到控制，以驱动细胞定向运动。一种靶向MT效应蛋白MCAK， 局部抑制Rac 1，以促进前沿MT生长和细胞极性，和MCAKs对MT动力学的影响是 对肌球蛋白II收缩性敏感。尽管有这些知识，Rac 1和肌球蛋白-II是如何促进组织的， FA、MT和肌动蛋白还不太清楚。初步证据表明，FA相关的MT是 主要是非中心体起源，Rac 1活性增强了两个不同家族的关联， MAP、CAMSAP和septins，它们增加非中心体MT向FA的生长。在这里，我们将测试假设 Rac 1和肌球蛋白-II促进CAMSAP和septins与非中心体MT的结合， MCAK介导的MT分解并驱动MT-FA相互作用。我们的方法将包括一个本科生团队， 研究人员使用活内皮细胞的荧光显微镜来确定：（1）Rac 1和肌球蛋白II如何调节 CAMSAP和septin与非中心体MT的相互作用，（2）MCAK如何分解非中心体MT 控制MT动力学和FA大小，以及（3）septins如何促进MT生长为FA。这些调查将提供 通过将Rac 1和肌球蛋白-II与细胞骨架效应物功能性连接，在细胞迁移领域取得了重要进展 控制非中心体MT生长为FA的蛋白质以及健康和疾病中细胞迁移的调节。

Project Summary/Abstract. The coordinated and regulated remodeling of the actin and microtubule (MT) cytoskeleton is required for cell migration for developmental processes and homeostatic maintenance, as well as during the body’s response to external insults and disease states including heart disease and tumor metastasis. During cell migration, actin filaments assemble and become linked to focal adhesion (FA) complexes, while MTs undergo dynamic instability that is locally controlled by MT- associated proteins (MAPs). These two processes enable cells to establish a leading-edge and a trailing-edge, and to migrate with directional persistence. Despite many advances in our understanding of the functional implications of MAPs on MTs and MT-FA interactions, it remains unknown how exactly MT organization is spatially and temporally coordinated with FAs to promote directional cell movement. A recent discovery showing that non-centrosomal MTs are both sufficient and required to drive polarized cell migration has established a paradigm shift, suggesting that non- centrosomal MTs are primed to function in a way that is distinct from MTs nucleated by the centrosome. The finding underscores the need to determine how cytoskeletal proteins identify and regulate non-centrosomal versus centrosomal MT dynamics and effects on polarity and migration. This gap in knowledge impacts our understanding of fundamental processes, including how signaling molecules simultaneously regulate families of proteins to achieve complex tasks, such as guiding persistent cell migration. The small GTPase, Rac1, is a key signaling protein that is spatially controlled to promote FA formation, MT growth, and actin filament assembly, resulting in leading edge advance. Rac1 signaling is complemented by the molecular motor protein, myosin-II, which organizes actin stress fibers, promotes FA maturation, and generates forces that pull the trailing-edge of the cell forward. Thus, Rac1 and myosin-II are spatially and temporally controlled to drive directional cell movement. One targeted MT effector protein, MCAK, is locally inhibited by Rac1 to promote leading-edge MT growth and cell polarity, and MCAKs effects on MT dynamics are sensitive to myosin-II contractility. Despite this knowledge, how Rac1 and myosin-II contribute to the organization of FAs, MTs, and actin is not well understood. Preliminary evidence demonstrates that FA-associated MTs are predominantly of non-centrosomal origin and that Rac1 activity enhances the association of two different families of MAPs, CAMSAPs and septins, which increase non-centrosomal MT growth into FAs. Here, we will test the hypothesis that Rac1 and myosin-II promote the association of CAMSAP and septins with non-centrosomal MTs, which inhibits MCAK-mediated MT disassembly and drives MT-FA interactions. Our approach will incorporate a team of undergraduate researchers using fluorescence microscopy of live endothelial cells to determine: (1) how Rac1 and myosin-II regulate CAMSAP and septin interactions with non-centrosomal MTs, (2) how MCAK disassembly of non-centrosomal MTs controls MT dynamics and FA size, and (3) how septins promote MT growth into FAs. These investigations will provide critical advances to the field of cell migration by functionally linking Rac1 and myosin-II with cytoskeletal effector proteins that control non-centrosomal MT growth into FAs and the regulation of cell migration in health and disease.