细胞迁移受体蛋白MIG-13/LRP12区域性活化的机制研究

Directional cell migration requires the establishment and maintenance of the polarity. Despite remarkable progresses in understanding polarity establishment, relatively little is known regarding the maintenance of a protrusive leading edge and a contractile rear. Our previous study reported that the C. elegans transmembrane protein LRP12 homolog, MIG-13, acted through ABL-1-WAVE and SEM-5-WASP two parallel pathways to stimulate Arp2/3-mediated actin polymerization at the leading edge, thereby establishing the polarity. However it remains elusive how to maintain the stability of polarity during cell migration. Our preliminary results showed that a tyrosine kinase SRC-1 phosphorylates MIG-13 and promotes its activity at the leading edge, whereas a receptor-like tyrosine phosphatase PTP-3 is essential for the polarity maintenance in the migrating Q cells. We found that this phosphatase is specifically enriched in the rear. Our biochemical analyses uncovered an antagonism between SRC-1 kinase and PTP-3 phosphatase on activating receptor protein MIG-13. We aim to further explore the mechanism underlying MIG-13 signaling pathway during directional cell migration. We expect that our study will identify a novel mechanism that maintains cell polarity during directional cell migration.

定向细胞迁移的进行需要在迁移方向上建立和维持极性结构的稳定，大量的研究报导了极性结构的分子组成及建立机制，然而极性结构如何被持续稳定并不清楚。申请人先前的工作揭示跨膜蛋白LRP12的线虫同源蛋白MIG-13通过ABL-1-WAVE和SEM-5-WASP两条平行通路在Q细胞前导端激活Arp2/3复合体，促进微丝骨架的极性组装。申请人拟深入研究MIG-13/LRP12激活的极性结构是如何在细胞迁移过程中被稳定的机制，初步结果表明：酪氨酸激酶SRC-1通过磷酸化MIG-13的胞内段开启下游信号通路，从而建立极性结构；受体酪氨酸磷酸酶PTP-3特异性的定位于Q细胞的两侧及尾部，体外生化实验揭示PTP-3可以去磷酸化MIG-13，对维持迁移极性结构的稳定至关重要。本课题拟通过探究SRC-1和PTP-3对受体蛋白MIG-13的时空调控，加深对迁移细胞极性结构建立与维持的理解。

定向细胞迁移的发生与维持，不仅需要信号分子与蛋白机器完成微丝细胞骨架在迁移细胞前导端的组装，而且需要未知分子机制抑制它们在迁移细胞的两侧和尾部发挥功能。我们先前的研究成果表明：线虫单次跨膜蛋白MIG-13与人源LRP12高度同源且功能保守，MIG-13通过活化微丝成核促进因子WAVE和WASP，从而激活Arp2/3依赖的微丝骨架在迁移细胞前导端的聚合，促进Q神经前体细胞的定向迁移。在本项目中，我们首先发现：非受体酪氨酸激酶SRC-1与MIG-13的胞内端直接结合，并且磷酸化MIG-13胞内端的关键酪氨酸位点，从而在Q细胞的前导端激活MIG-13下游信号通路；其次，采用CRISPR/Cas9基因组编辑和活细胞荧光成像技术，我们发现：绿色荧光蛋白标记的内源MIG-13和SRC-1均匀地分布于迁移细胞的细胞膜四周；再者，我们发现：受体酪氨酸磷酸酶PTP-3通过维持极性结构在迁移方向上的稳定，在Q细胞的方向性迁移中，发挥至关重要的作用；尤其值得注意，内源PTP-3定位于迁移细胞的两侧和尾部，具有去磷酸化MIG-13胞内端的能力。本项目的成果表明：酪氨酸激酶SRC-1和酪氨酸磷酸酶PTP-3的空间协同作用，决定了MIG-13/Lrp12在定向迁移细胞内的不对称活化。