SNX3调控膜管介导的clathrin不依赖型内吞途径循环运输的功能与机理研究

Early endosome is the sorting hub on the endocytic pathway and consists of vacuolar sorting and tubular recycling domains that segregate components fated for degradation in lysosomes or reuse by recycling to the plasma membrane or Golgi. Mechanisms underlying tubule generation from vacuolar sorting endosomes remain less known. Sorting Nexins (SNXs) are vital for formation of the distinct membranous microdomains with different sorting functions. Studies on the intracelluar transport and regulation of nonclathrin-endocytic pathway haven’t been developed as well as those on the clathrin-endocytic pathway. The open questions include: What’s the distinction of the protein molecules and lipids decorated on the different endosomal tubules emanated from the early endosomes? How does this distinction underlie the sorting of cargoes and fate of the transport intermediates, for example, to be vesiculated or not? Particularly, how do the SNX members play their roles for the endosomal transport along the nonclathrin-endocytic pathways? Our recent published work of C. elegans intestine confirmed that, the transport intermediates of Arf6-associated clathrin-indpendent recycling cargoes were membranous tubules interconnected between early endosomes and recycling endosomes, disctinct from the classical vesicular carriers. We further found that depletion of SNX-3 resulted in disruption of the tubular intermediates and the cargoes were possibly switched from recycling into degradation in lysosomes. Based on these previous work, we propose this project, by using in vivo real-time high-resolution fluorescent microscopy technology with conventional biochemistry and molecular coloning methods, to investigate the novel functional roles and molecular mechanisms of SNX-3 underlying the formation of specific membranous transport intermediates emanating from the early endosomes. Results will help to clarify the detailed regulation of sorting and transportation of nonclathrin-endocytic cargo proteins.

早胞内体是细胞内吞途径上的分选枢纽，由空泡分选和管状循环微区构成。细胞分选蛋白SNXs对早胞内体上不同分选功能的膜微区的形成有着举足轻重的作用。与clathrin内吞途径相比，非clathrin途径（CIE）内吞的物质在胞内的运输与调控的研究在近年才有较快发展。早胞内体上不同的内涵体管具有怎样不同的蛋白分子与脂质特征，导致其在货物分选及剪切囊泡化的命运等方面不同？SNXs家族成员在CIE途径的运输中又扮演怎样的角色？我们近期在线虫肠细胞上研究证实，Arf6调控的CIE循环途径主要通过内涵体间的管网运输实现；SNX-3功能缺失导致膜管运输载体破坏，货物蛋白有被降解的趋势。基于此，本项目将综合运用活体高分辨的动态荧光显微成像技术，结合生化、分子等手段，阐释线虫SNX-3在早胞内体上特定膜运输载体形成中的新功能与分子机理，研究将有助于揭示CIE途径货物蛋白在早胞内体上的分选和运输如何被精密调控。

早胞内体是细胞内吞途径上的分选枢纽，由空泡分选和管状循环微区构成。细胞分选蛋白SNXs对早胞内体上不同分选功能的膜微区的形成有着举足轻重的作用。与clathrin内吞途径相比，非clathrin途径（CIE）内吞的物质在胞内的运输与调控的研究在近年才有较快发展。早胞内体上不同的内涵体管具有怎样不同的蛋白分子与脂质特征，导致其在货物分选及剪切囊泡化的命运等方面不同？SNXs家族成员在CIE途径的运输中又扮演怎样的角色？.围绕细胞内吞后在早期内吞体上如何分选、组织蛋白质运输这一基础科学问题，我们的结果首次揭示了SNXs家族成员中结构最简单、仅含PX结构域的SNX-3对一类clathrin不依赖内吞途径的货物蛋白通过特殊膜管结构进行的循环运输具有重要作用，SNX-3功能缺失直接导致这类CIE途径内吞的膜蛋白错误进入溶酶体中被降解。研究成果突破了关于蛋白质分选及膜运输调控机制的现有认知，即SNX-3执行上述功能无需VPS-26/29/35复合物辅助识别货物蛋白，且参与调控管状运输载体的生成。我们解释了SNX-3缺失所导致的早期内吞体分选平台上微结构域的变化对蛋白分选进入不同运输终点的影响。.F-actin可以与Dynamin共同促进哺乳动物细胞clathrin内吞囊泡的生成，然而有关二者时空调控关系的认识尚不清晰。此外还有哪些核促进因子以及调控蛋白促成了F-actin在CCPs剪切过程的组装也不清楚。借助于线虫的遗传优势，结合高分辨的荧光显微成像技术，我们首次鉴定出两个actin结合蛋白WIP-1和DBN-1调控F-actin的募集进而与DYN-1协同参与CCPs剪切过程，并解释了CCVs生成过程中多分子互作界面的关系。