微丝细胞骨架在神经前体细胞迁移中的动态变化及功能研究

The actin cytoskeleton is essential for various biological events including cell migration. In response to extracellular signals, a migrating cell reorganizes its actin cytoskeleton, which is asymmetrically enriched at the leading edge and powers cell migration. Although numerous studies from the cultured system have provided the conceptual framework in our understanding of cell migration, the complex extracellular environment cannot be comprehensively recapitulated in cell cultures. We have developed live imaging and conditional genome-editing tools for the C. elegans Q neuroblast, making it an appealing experimental system to study actin dynamics and cell migration in intact animals. We have uncovered the function of actin-binding protein Coronin and Anillin in Q cell migration, and have identified a conserved MIG-13-mediated signaling pathway that assembles the actin cytoskeleton at the leading edge, and have isolated the Hippo-RhoG signaling axis in the maintenance of actin polarity in migrating Q cells. The proposed study will dissect the mechanism underlying MIG-13 activation, will systematic examine the roles of actin-binding proteins in the C. elegans genome in the regulation of actin dynamics in the migrating Q cells. We will also combined biochemical analysis and genetic screens to isolate novel factors that are responsible for actin organization and cell migration. Our results will advance our understanding of actin dynamics, cell migration and neural development and will also provide insights to cell migration-associated diseases.

微丝细胞骨架是广泛存在于真核细胞中的重要亚细胞结构，参与包括细胞迁移在内多种生物学过程。虽然体外实验系统为研究微丝动态和细胞迁移做出了重要贡献，但细胞所处的复杂在体微环境难以被全面模拟。申请人发展了活体显微成像和条件性基因组编辑技术，以线虫Q神经前体细胞为在体系统研究微丝动态和细胞迁移的调控机制，揭示了Coronin和 Anillin等微丝结合蛋白在细胞迁移中的功能，鉴定了保守跨膜蛋白MIG-13介导的信号通路决定迁移细胞中微丝的极性排布，Hippo-RhoG信号通路继而维持微丝极性的稳定性。以此为基础，申请人将深入解析MIG-13信号通路影响微丝动态变化的作用机制，以线虫基因组中微丝结合蛋白为切入点研究微丝骨架在细胞迁移中的功能，结合生化分析和遗传筛选发现参与微丝动态调控的未知因子。预期结果将为哺乳类微丝骨架、细胞迁移和神经发育研究提供新线索，为相关疾病的预防治疗提供理论参考。

微丝细胞骨架是广泛存在于真核细胞中的重要亚细胞结构，参与包括细胞迁移在内多种生物学过程。在细胞定向迁移过程中，细胞前导端级联信号通路激活微丝细胞骨架迅速组装，推动细胞膜向前扩张。与此相对应，胞体后端信号途径必须抑制微丝骨架聚合。我们在Q神经前体细胞迁移的研究显示，跨膜蛋白MIG-13/Lrp12通过Arp2/3成核促进因子WAVE和WASP指导微丝骨架组装引导细胞向前迁移。为探究MIG-13活性的调控机制，本课题综合多种研究手段，发现线虫酪氨酸激酶SRC-1，可以直接磷酸化MIG-13受体，在迁移细胞前导端激发其促进微丝组装的活性。继而，我们还发现了一种受体类酪氨酸磷酸酶PTP-3，体内实验表明，PTP-3蛋白在神经前体迁移过程中参与维持F-actin的极性。体外实验中，PTP-3可以去磷酸化由SRC-1产生的MIG-13磷酸化修饰。重要的是，GFP敲入动物中SRC-1和MIG-13均呈现沿迁移神经前体细胞质膜的均匀分布，而内源性的PTP-3特异性聚集定位于迁移细胞的后部。此外，我们发现膜骨架蛋白Spectrin在纤毛结构组装和迁移细胞后部通过提供更多的细胞膜物理刚性限制微丝细胞骨架扩增，而Coronin蛋白家族微丝细胞骨架结合蛋白POD-1在迁移细胞中通过调控微丝细胞骨架解聚维持其动态的功能机制。以上，本项目揭示多个参与指导微丝细胞骨架动态级联信号的关键因子，及其在不同层面调控极性细胞迁移的分子功能机制。以上结果分别发表在PNAS （两篇）、PLOS Biology、Journal of Cell Science等国际学术杂志上。