Centlein蛋白新亚型参与网格蛋白介导的内吞作用和囊泡运输的分子机制

我们已经证明centlein为微管成核和锚定在细胞中心体所必需，且centlein在细胞中心体的定位取决于微管负端导向驱动蛋白KIFC3。KIFC3既结合centlein又与dynactin最大亚基p150Glued相互作用。我们最近分离得到一个centlein蛋白新亚型，含有1410个氨基酸，故命名为centlein(1410aa)。该亚型在细胞中呈点状分布，且部分与内吞作用的标记蛋白clathrin、AP2和早期胞内体标记蛋白EEA1共定位。进一步实验证实centlein(1410aa)分别与clathrin、AP2、EEA1免疫共沉淀。本申请旨在阐明（1）Centlein(1410aa)如何参与clathrin介导的内吞作用；（2）KIFC3/dynactin复合体如何通过centlein调控囊泡运输。最终提出KIFC3/dynactin复合体驱动细胞内囊泡朝微管负端运输的新模式。

Our previous study revealed that centlein was required for microtubule nucleation and anchoring at centrosomes, while centlein required KIFC3 for its centrosomal localization. The microtubule minus-end directed motor KIFC3 interacted with both centlein and the dynactin's largest subunit p150Glued. .Recently, we have unexpectedly found that a new monoclonal anti-centlein antibody displayed a punctate pattern in cultured human cells, which was diminished following siRNA centlein treatment. The authenticity of the centlein punctum was thus verified. The punctate vesicular-like pattern represented a novel protein isoform because overexpression of the isoform showed a punctate staining similar to that labeled by the monoclonal antibody. The novel protein isoform is composed of 1410 amino acids, named centlein(1410aa). We further found that the centlein(1410aa) punctum partially colocalized with markers of endocytosis AP2 and clathrin, and the early endosomal marker EEA1, of which all were co-immunoprecipitated with centlein(1410aa), respectively..The proposal described here aims to address （1）how centlein(1410aa) is involved in clathrin-mediated endocytosis; （2）how the KIFC3/dynactin mediates the centlein-containing vesicle transport. To this end, we are able to propose a model for cenltein in clathrin-mediated endocytosis, endosomal motility and vesicle transport driven by KIFC3/dynactin complex toward the minus end of the microtubule.