【作者】 唐立春；

【导师】 刘笔锋； 杜伟；

【作者基本信息】 华中科技大学 ， 生物化学与分子生物学， 2012， 博士

【摘要】 胞吞作用是一种囊泡介导的生物途径。细胞无论是从外界环境中吸收营养物质还是更新膜脂膜蛋白,都要依赖于胞吞作用。虽然已经明确了很多蛋白,例如RabGTP酶,在胞吞作用中起到了至关重要的作用,但是对于胞吞作用调控的详细机制研究的并不透彻。大部分研究都集中在胞吞的早期进程,对于胞吞的晚期进程,尤其是溶酶体相关进程还鲜有研究。溶酶体命运的研究不仅对揭示溶酶体相关动力学的调控机制,更对揭示胞吞作用的调控网络具有非常重要的意义。本文利用模式生物秀丽隐杆线虫(Caenorhabditis elegans)对受体介导胞吞作用中的重要调控蛋白AGEF-1的作用机制做了进一步研究,克服了AGEF-1缺失后线虫表现为胚胎致死以及发育停滞不能用于深入研究的难题,利用胚胎后RNAi的方法,在腔胞胞吞作用研究模型中深入研究了AGEF-1对胞吞途径的影响,揭示了AGEF-1在胞吞途径中的新作用。在线虫中AGEF-1调控卵母细胞对卵黄蛋白的吸收并通过其GEF活性,调控ARF-1/3的活性,影响卵母细胞发育过程中caveolin-1的形成。但迄今为止,AGEF-1的亚细胞定位以及调控胞吞作用的详细机制并不清楚。我们应用含有大量标记的腔胞胞吞作用研究模型,发现AGEF-1的缺失不仅严重影响腔胞对肌肉细胞分泌到假体腔中的可溶性蛋白GFP的吸收,而且腔胞中溶酶体的体积明显增大。进一步研究发现,溶酶体的酸性环境并未改变,水溶性物质胞吞后运输至溶酶体的途径正常,溶酶体的降解功能依然存在,揭示了AGEF-1调控溶酶体的分离/融合过程,但并不调控溶酶体的功能。基因上位分析表明,AGEF-1于ARL-8蛋白的上游调控内吞的早期过程,又处于ARL-8的下游调控物质向溶酶体的运输以及溶酶体的融合/分离过程。细胞亚定位结果显示AGEF-1定位于溶酶体膜上,表明AGEF-1直接调控着溶酶体相关动力学。对内吞作用早期步骤标记的研究发现AGEF-1在质膜附近调控Clathrin的分布直接调控腔胞对液相物质的内吞。并通过蛋白过表达以及分段蛋白过表达进一步证明了AGEF-1通过结合不同的蛋白或者蛋白复合物调控腔胞内吞液相物质的能力以及腔胞溶酶体形态两个独立事件,且AGEF-1并不依赖ARF-1/3活性调控溶酶体的形态。总而言之,本论文发现了调控溶酶体融合/分离动力学过程的第一个GTP酶调控因子,对进一步全面深入了解溶酶体相关调控的详细机制以及胞吞作用的整合调控网络具有重要的推动作用。此外AGEF-1在其他组织器官中的功能研究发现,AGEF-1在神经系统中通过调节Clathrin的分布调控神经系统的活性。行为学分析发现AGEF-1缺失后,线虫的爬行速率变慢,泳动速率却变快,揭示了这两种运动行为很可能是由不同的神经环路所控制,为研究Clathrin介导的胞吞作用在神经活动中的功能提供了良好的模型。更多还原

【Abstract】 The endocytic pathway is a vesicle-mediated process required for various essential cellular functions, including processing fluid and macromolecules from the outside environment, and internalizing plasma membrane lipids and proteins. Although much is known about how some cellular proteins, such as Rab GTPases, regulate some steps of the endocytic pathway, the precise molecular mechanisms are not fully understood, which is especially true for lysosomal transport and dynamics.Because the endocytic machinery is highly conserved from yeast to mammals, several studies have utilized the worm Caenorhabditis elegans to reveal mechanisms associated with endocytosis in a multicellular organism. We analysed AGEF-1, which is thought to activate classⅠArfs(ARF1-3, small G proteins with long lipid tails function on vecicle traffic and cytoskeleton), endocytic functions in Caenorhabditis elegans coelomocytes. Knockdown of AGEF-1 levels results in two apparent trafficking defects in coelomocytes of Caenorhabditis elegans. First, there is a delay in the uptake of solutes from the extracellular medium. Second, there is a dramatic enlargement of the sizes of lysosomes which is likely the products of homotypic lysosome fusion events, even though lysosomal acidification is normal and degradation still occurs. Our epistasis analysis showed that reduced ARL-8 function abrogates the lysosome defect due to reduced AGEF-1 function, but not the uptake defect. Indeed, early steps of endocytosis analysis showed that after reduced of AGEF-1 levels, the distribution of clathrin at plasma membrane was obviously changed, suggesting that AGEF-1 has a direct role in regulating uptake. Full-length and truncated AGEF-1 proteins overexpressed experiment indicates that AGEF-1 associates with distinct complexs at surface and in endosome/lysosomes.In conclusion, AGEF-1 is the first identified GTPase regulator that functions at lysosomes fusion/fission stage of the endocytic pathway. Our study sheds light on lysosomal dynamics and endocytosis regulation network. What’s more, after reduced of AGEF-1 levels the distribution of clathrin at nervous system was obviously changed. Crawl speed was seriously suppressed, while swimming speed was increased, indicating these two types of locomotion were regulated by different neural circuits. It provides a good model in studing Clathrin-dependent endocytosis functions in maintenance of synaptic vesicle pools.更多还原