微丝细胞骨架调控凋亡细胞吞噬的机制研究

The efficient removal of apoptotic cells by phagocytes is essential for tissue homeostasis in metazoans. Engulfment of apoptotic cells in Caenorhabditis elegans is controlled by two conserved genetic pathways, which activate CED-10/Rac to reorganize the actin cytoskeleton during phagocytosis; however, little is known about how CED-10/Rac is linked to actin reorganization. Phagocytosis requires highly dynamic branched actin filament networks, which are nucleated by the ARP2/3 (actin-related protein 2/3) complex and regulated by actin nucleation promoting factor WASP (Wiskott–Aldrich syndrome protein) and WAVE (WASP-family verprolin homologous protein) family proteins. The proposed study aims to elucidate the mechanism underlying actin reorganization when the C. elegans apoptotic Q cells are engulfed by the hypodermal hyp 7 cells. Our preliminary results have shown that WASP and ARP2/3 were recruited to apoptotic Q cells and that the clearance of apoptotic Q cells was delayed in WASP mutants. Our biochemical analysis has revealed that CED-10 interacted with WASP. In addition, we have uncovered that a WASP-associated protein SEM-5/GRB2 promoted the formation of branched F-actin in vitro and was required for the removal of apoptotic Q cell in vivo. We will combine the CRISPR-Cas9-based genome editing, fluorescence live imaging and biochemistry to identify actin polymerization regulators required for the clearance of apoptotic cells. We will further investigate how CED-10 regulates these actin regulators. We expect that our results will advance our understanding of actin regulators in corpse removal and provide new insights into the related diseases.

凋亡细胞清除对维持多细胞生物器官动态稳定有重要意义。在吞噬细胞内，两条保守信号通路共同激活小G蛋白CED-10，重排微丝细胞骨架，实现对凋亡细胞的吞噬。然而CED-10调控下游微丝骨架组装的机制尚不清楚。凋亡细胞的吞噬通常依赖于高度动态的分枝状微丝骨架，其组装由ARP2/3复合物和成核促进因子WASP和WAVE家族蛋白激活。本课题拟利用线虫凋亡Q细胞和吞噬细胞hyp 7为模型，研究CED-10调控微丝骨架重排机制。申请人发现CED-10通过与WASP结合调控其在凋亡Q细胞表面的富集；且WASP结合蛋白SEM-5通过促进分叉状微丝形成参与调控凋亡Q细胞清除。以此为基础，本课题将结合CRISPR-Cas9基因编辑技术，显微成像技术和生化分析，鉴定凋亡Q细胞清除过程中微丝调控因子，深入研究CED-10与这些因子的关系，拓展对微丝调控因子在凋亡细胞清除中的认识，为理解细胞凋亡相关疾病提供新思路。

凋亡细胞的有效清除对多细胞生物体的器官发育和维持器官动态稳定有重要意义。在秀丽隐杆线虫(Caenorhabdifis elegans)中的遗传研究发现两条保守的信号通路激活吞噬细胞的CED-10/Rac1 GTPase，促进微丝细胞骨架的重排，驱动吞噬细胞形成围绕凋亡细胞的板状伪足，最终实现对凋亡细胞的包裹和内吞。本课题以线虫吞噬细胞hyp7吞噬凋亡Q神经前体细胞为研究系统，研究被激活的CED-10调控下游微丝骨架组装的机制。我们发现CED-10结合微丝成核促进因子WASP，CED-10在吞噬细胞中把WASP和Arp2/3招募到凋亡Q细胞周围，WASP和Arp2/3的功能缺失破坏吞噬细胞对凋亡细胞的吞噬；然而另一个微丝成核促进因子WAVE复合物不在凋亡Q细胞周围定位，破坏WAVE复合物的功能不影响大多数凋亡Q细胞的清除。WASP激活蛋白SEM-5/GRB2也在吞噬细胞中促进凋亡细胞的吞噬。综上所述，本课题研究发现CED-10通过与WASP结合介导WASP-Arp2/3微丝成核复合物在凋亡细胞表面的富集，促进凋亡细胞的有效清除。本课题的研究成果阐明参与调控凋亡细胞清除的微丝细胞骨架蛋白，为设计治疗凋亡细胞清除相关疾病的药物靶点提供新思路。