【作者】 陈丹；

【导师】 史岸冰；

【作者基本信息】 华中科技大学 ， 生物化学与分子生物学， 2019， 博士

【摘要】 在囊泡循环运输过程中,小G蛋白Arf6/ARF-6是调节细胞内体上磷脂酰肌醇4,5-二磷酸的一个决定性因子。为了进一步了解ARF-6的分子调节过程,我们在秀丽隐杆线虫中展开了对ARF-6结合蛋白的筛选,并从中获得了一个新的ARF-6结合蛋白SAC-1。SAC-1是酵母磷脂酰肌醇磷酸酶Sac1p在线虫中的同源物。在线虫极性肠上皮细胞中,ARF-6的缺乏会导致基底端内体丧失SAC-1的标记。货物蛋白的稳态分布实验显示,SAC-1缺失会特异性地影响顶膜端的分泌运输途径和基底端的循环运输途径。在sac-1突变动物中,PI(4,5)P2的水平以及ARF-6效应因子UNC-16标记的膜结构都显著增加,提示SAC-1可能是ARF-6的一个负调控因子。进一步研究发现,SAC-1能够与ARF-6的GEF蛋白BRIS-1相互作用,并且BRIS-1与SAC-1的互作能够以浓度依赖的方式减弱BRIS-1与无活性态ARF-6(GDP)的结合。因此,SAC-1缺失会促进细胞内ARF-6(GDP)与GEF蛋白BRIS-1的相互作用。而在sac-1突变动物中,敲降BRIS-1确实会降低PI(4,5)P2水平。值得注意的是,SAC-1禁锢BRIS-1使其与ARF-6隔离的这一功能并不受SAC-1自身磷酸酶活性的影响。我们的结果提示,SAC-1与BRIS-1的相互作用限制了ARF-6(GDP)与其GEF蛋白BRIS-1的接近,从而防止ARF-6的活性异常增高。目的:研究磷脂酰肌醇磷酸酶Sac1p/SAC-1参与调控秀丽线虫肠上皮细胞网格蛋白非依赖型内吞(clathrin-independent endocytic,CIE)循环运输的机制。方法:已知小G蛋白Rab10/RAB-10是调控CIE循环运输的关键因子,线虫rab-10突变体动物的肠上皮细胞内产生了异常增大的内体膜结构,并且CIE货物蛋白h TAC-GFP会在其中大量累积。据此,我们以rab-10(RNAi)作为阳性对照,对线虫全基因组进行RNAi筛选,检测哪一种基因表达被RNAi敲降后能够导致h TAC-GFP在肠细胞异常累积。通过筛选我们发现SAC-1缺失能引起h TAC-GFP的异常积累,说明SAC-1参与调控CIE循环运输。利用GST pull-down实验进一步发现SAC-1能特异性结合小G蛋白Arf6/ARF-6。已知在线虫肠细胞内,ARF-6对于CIE循环运输至关重要。随后,我们使用体细胞特异性CRISPR/Cas9基因编辑技术获得肠细胞特异性敲除的sac-1(ycx18)突变体,通过遗传学和细胞生物学的方法获得大量转基因荧光标记动物,并利用激光共聚焦显微成像技术对sac-1突变背景下线虫肠细胞内的ARF-6相关蛋白进行检测分析,探究SAC-1是否参与调控ARF-6活性及其调控机制。结果:在秀丽线虫肠上皮细胞内,我们发现SAC-1缺失导致CIE循环运输受阻,PI(4,5)P2水平异常增高,顶膜端细胞连接复合体错误定位以及ARF-6潜在效应因子JIP3/UNC-16在内体上大量积累。而ARF-6缺失导致SAC-1丧失其基底侧的内体定位。同时SAC-1可以特异性结合ARF-6-GEF蛋白BRIS-1,二者之间的相互作用不受SAC-1磷酸酶催化活性的影响。并且SAC-1与BRIS-1的结合以浓度依赖方式抑制了BRIS-1与ARF-6(GDP)的相互作用,从而限制ARF-6(GDP)的鸟苷酸交换防止ARF-6过度激活。另一方面,SAC-1缺失也导致网格蛋白依赖型内吞(clathrin-dependent endocytic,CDE)循环运输和顶膜端分泌运输出现异常。结论:在秀丽线虫肠上皮细胞内,SAC-1作为一个新发现的ARF-6结合蛋白,可能通过ARF-6-GEF蛋白BRIS-1介导了对ARF-6的负反馈调节。更多还原

【Abstract】 Arf6/ARF-6 is a crucial regulator of the endosomal PI(4,5)P2 pool in endocytic recycling.To further characterize ARF-6 regulation,we performed an ARF-6 interactor screen in C.elegans and identified SAC-1,the homolog of the phosphoinositide phosphatase Sac1 p in yeast,as a novel ARF-6 partner.In the absence of ARF-6,basolateral endosomes show a loss of SAC-1 staining in epithelial cells.Steady-state cargo distribution assays revealed that loss of SAC-1 specifically affected apical secretory delivery and basolateral recycling.PI(4,5)P2 levels and the endosomal labeling of ARF-6 effector UNC-16 were significantly elevated in sac-1 mutants,suggesting that SAC-1 functions as a negative regulator of ARF-6.Further analyses revealed an interaction between SAC-1 and the ARF-6-GEF BRIS-1.This interaction outcompeted ARF-6(GDP)for binding to BRIS-1 in a concentration-dependent manner.Consequently,loss of SAC-1 promotes the intracellular overlap between ARF-6 and BRIS-1.BRIS-1 knockdown resulted in a significant reduction in PI(4,5)P2 levels in SAC-1-depleted cells.Interestingly,the action of SAC-1 in sequestering BRIS-1 is independent of SAC-1’s catalytic activity.Our results suggest that the interaction of SAC-1 with BRIS-1 curbs ARF-6 activity by limiting the access of ARF-6(GDP)to its GEF BRIS-1.Objective: To study the regulatory mechanisms of phosphatidylinositol phosphatase Sac1p/SAC-1 involved in the clathrin-independent endocytic(CIE)recycling transport in C.elegans intestinal epithelial cells.Methods: It is known that the small GTPase Rab10/RAB-10 is a key regulatory factor in the clathrin-independent endocytic recycling pathway.In particular,rab-10 mutants overaccumulate h TAC-GFP in enlarged structures in the deep cytosol of intestinal epithelia.Thus,we performed a genome-wide RNAi screen using rab-10(RNAi)as the positive control to identify candidate genes whose expression knockdown lead to the h TAC-GFP overaccumulation in the intestine.After initial screen and the follow-up validation,we noted that RNAi-mediated knockdown of SAC-1 resulted in intracellular h TAC-GFP aggregation.This observation suggested that SAC-1 is required for the clathrin-independent endocytic recycling.Further screening via GST pull-down assays revealed that SAC-1 specifically interacts with the small GTPase Arf6/ARF-6,which is essential for the basolateral recycling flow of clathrin-independent cargo.We then developed a transgenic strain sac-1(ycx18),an intestine-specific CRISPR/Cas9 somatic mutant,and a large number of transgenic fluorescent-labeled animals by genetic and cell biology methods.With the utilization of laser confocal microscopy,we analyzed the ARF-6-related proteins in the intestinal cells of sac-1(ycx18)mutant animals to investigate whether and how ARF-6 is regulated.Results: In the intestinal epithelial of C.elegans,we observed that loss of SAC-1 resulted in the block of clathrin-independent recycling cargos,abnormally elevated PI(4,5)P2 levels,mislocalization of apical junctional complex and significantly accumulation of ARF-6 putative effector JIP3/UNC-16.In arf-6(tm1447)mutants,the labeling of GFP-SAC-1 in basolateral puncta was significantly diminished.Specially,we found that SAC-1 displayed a robust protein-protein interaction with BRIS-1/ARF-6-GEF through its SAC domain,whereas the catalytic activity of SAC domain is not implicated in the interaction with BRIS-1.Thus far,our experiments demonstrated that SAC-1 competes with ARF-6(GDP)for interaction with BRIS-1/ARF-GEF in a concentration-dependent manner,thereby interfering with the interaction of BRIS-1 and ARF-6(GDP)and restricting ARF-6 activity.On the other hand,SAC-1 deletion also led to defects in clathrin-dependent endocytic recycling transport and apical secretory transport in intestinal epithelia.Conclusions: Our experiments identify SAC-1 as a novel ARF-6 interactor that mediates negative feedback regulation of ARF-6 in C.elegans intestinal cells,likely with the involvement of ARF-6 GFE protein BRIS-1.更多还原