A human genome-wide screen for regulators of clathrin-coated vesicle formation. Homo sapiens

Clathrin-mediated endocytosis is essential for a wide range of cellular functions. We used a multi- step siRNA-based screening strategy to identify novel regulators of the first step of clathrin- mediated endocytosis, formation of clathrin-coated vesicles (CCVs) at the plasma membrane. A primary genome-wide screen identified 334 hits that caused accumulation of CCV cargo on the cell surface. A secondary screen identified 92 hits that inhibited cargo uptake and/or altered the morphology of clathrin-coated structures. The hits include components of four functional complexes: coat proteins, V-ATPase subunits, spliceosome-associated proteins, and acetyltransferase subunits. Electron microscopy revealed that V-ATPase depletion caused the cell to form aberrant non-constricted clathrin-coated structures at the plasma membrane. The V- ATPase knockdown phenotype was rescued by addition of exogenous cholesterol, indicating that the knockdown blocks clathrin-mediated endocytosis by preventing cholesterol from recycling from endosomes back to the plasma membrane. The microarray analysis was performed to test whether the siRNA targets are expressed in the cell lines used in the screen. Overall design: For the microarray analysis of gene expression, the two cell lines used in the study were analyzed in duplicate: HeLa-YXXΦ and HeLa-FXNPXY (Hela-M cells expressing a CD8-YXXΦ and CD8-FXNPXY constructs respectively). YXXΦ and FXNPXY are motifs for clathrin-mediated endocytosis.

网格蛋白介导的内吞作用（clathrin-mediated endocytosis）对于众多细胞生理功能而言至关重要。本研究采用基于小干扰RNA（siRNA）的多阶段筛选策略，以鉴定网格蛋白介导的内吞作用第一步——质膜处网格蛋白包被囊泡（clathrin-coated vesicles, CCVs）形成过程的新型调控因子。

全基因组一级筛选共获得334个候选基因，其沉默后会导致CCV内吞货物在细胞表面蓄积。二级筛选则得到92个候选基因，其沉默后会抑制内吞货物摄取，或改变网格蛋白包被结构的形态。

上述候选基因涵盖四类功能复合物的组成蛋白：包被蛋白、液泡型ATP合酶（V-ATPase）亚基、剪接体相关蛋白以及乙酰转移酶亚基。电子显微镜观察显示，沉默V-ATPase会使细胞在质膜处形成异常的、未缢缩的网格蛋白包被结构。向细胞添加外源性胆固醇可挽救V-ATPase沉默引发的表型，这表明该沉默通过阻碍胆固醇从内吞体循环返回质膜，从而抑制网格蛋白介导的内吞作用。

本研究开展基因芯片分析，以验证筛选所用细胞系中是否表达上述siRNA的靶基因。

整体实验设计：针对基因表达的芯片分析，本研究采用两种细胞系各进行两次重复实验：分别表达CD8-YXXΦ与CD8-FXNPXY重组载体的HeLa-YXXΦ细胞、HeLa-FXNPXY细胞（即HeLa-M细胞）。其中YXXΦ与FXNPXY是网格蛋白介导的内吞作用的特征基序。