Control of cell polarity and migration by non-centrosomal microtubules

项目摘要/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构 用于发育过程和维持体内平衡，以及在身体对外部侮辱和 疾病状态包括心脏病和肿瘤转移。在细胞迁移过程中，肌动蛋白细丝组装并成为 连接到焦点粘连(FA)复合体，而MT经历由MT-局部控制的动态不稳定性- 相关蛋白(MAP)。这两个过程使细胞能够建立前沿和后缘，并 具有方向性持久性的迁移。尽管我们对地图的功能含义的理解取得了许多进展 关于MT和MT-FA的相互作用，MT组织在空间和时间上的确切情况仍不清楚 与FA协调，促进细胞定向运动。最近的一项发现表明，非中心体MTS是 驱动极化细胞迁移的充分和必要的因素都已经建立了一种范式转变，这表明非 中心体MT以一种不同于由中心体成核的MT的方式发挥作用。这一发现 强调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动力学及其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知识的理解 过程，包括信号分子如何同时调节蛋白质家族以完成复杂的任务，如 引导持续的细胞迁移。小的GTP酶，rac1，是一种关键的信号蛋白，在空间上受控制 促进FA的形成、MT的生长和肌动蛋白细丝的组装，从而导致前沿推进。Rac1信令是 在分子马达蛋白的补充下，肌球蛋白-II，它组织肌动蛋白应激纤维，促进FA成熟， 并产生将电池的后缘向前拉的力。因此，rac1和myosin-II在空间和时间上是 控制以驱动细胞的定向移动。一种靶向MT效应蛋白MCAK被rac1局部抑制到 促进前沿MT生长和细胞极性，MCAK对MT动力学的影响对Myosin-II敏感 伸缩性。尽管如此，rac1和myosin-II如何对FA、MTS和肌动蛋白的组织做出贡献是 不是很清楚。初步证据表明，FA相关的MTS主要是非中心体的 起源和rac1活性增强了两个不同的MAP家族CAMSAPs和Septins之间的关联，这两个家族 增加非中心体MT向FA的生长。在这里，我们将检验这一假设，即rac1和myosin-II促进 CAMSAP和Septins与非中心体MTS的关系，后者抑制MCAK介导的MT拆解和 推动MT-FA互动。我们的方法将包括一个本科生研究团队使用荧光 活内皮细胞显微镜确定：(1)rac1和myosin-II如何调节CAMSAP和Septin的相互作用 对于非中心体MTS，(2)非中心体MTS的MCAK分解如何控制MT动力学和FA大小， 以及(3)Septins如何促进MT向FA的生长。这些研究将为细胞领域提供关键的进展 Rac1和Myosin-II与控制非中心体MT的细胞骨架效应蛋白的功能连接迁移 在健康和疾病中生长为FAs和调节细胞迁移。 好了！ 好了！

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. ! !