A Study on Molecular Mechanisms of Endocytosis

体外无细胞体系的建立：为了阐明细胞内吞作用的分子机制，在体外重组了细胞内吞作用中的囊泡形成。将直径大于1μm的大脂质体与脑胞浆孵育，可形成大量直径小于10 0 nm的小囊泡。囊泡的形成既需要ATP，也需要GTP。当脂质体与动力蛋白1耗尽的胞浆孵育时，囊泡的形成显著减少，这表明在这个实验体系中囊泡的形成是内吞的囊泡形成。动态光散射法可以对孵育过程中形成的囊泡进行定性和定量的分析。膜脂的功能和动力学：通过分析不同组成的脂质体形成囊泡的情况，研究了膜脂的功能和动力学。随着脂质体中磷脂酰肌醇-4.5-二磷酸(PIP_2)浓度的增加，囊泡形成增加。此外，PIP_2被降解为磷脂酰肌醇-4-二磷酸，然后再降解为磷脂酰肌醇。其次，通过在反应混合物中加入PIP_2降解抑制剂新霉素，对反应过程中PIP_2的合成进行了分析。活性形式的ADP-核糖化因子6(Arf6)可促进PIP2的合成。结果提示，Arf6促进膜蛋白AP2的募集可能与增加PIP_2的合成有关。培养细胞膜脂的动力学：在细胞内吞的过程中，PTP_2的合成降解。代谢标记的HeLa细胞被刺激内吞，并分析PIP_2的含量。PIP2的降解与无细胞体系中观察到的类似。

Establishment of in vitro cell-free system: In order to elucidate molecular mechanisms involved in endocytosis, vesicle formation in endocytosis was reconstituted in vitro. Incubation of large liposomes, larger than 1 μm in diameter, with brain cytosol resulted in massive formation of small vesicles, smaller than 100 nm in diameter. The vesicle formation required both ATP and GTP. Vesicle formation was drastically reduced when liposomes were incubated with dynamin 1 -depleted cytosol, indicating that vesicle formation in this experimental system represents endocytic vesicle formation. The vesicle formed during the incubation can be analyzed quantitatively and qualitatively by dynamic light scattering.Functions and Kinetics of membrane lipids: Functions of membrane lipids were studied by analyzing vesicle formation from liposomes of with various compositions. Vesicle formation increased as phosphatidylinositol-4.5-bisphosphate (PIP_2) concentration in liposomes was increased. Furthermore, PIP_2 was degraded to phosphatidylinositol-4-bisphosphate, then to phosphatidylinositol. Next, PIP_2 synthesis during the reaction was analyzed by addition of neomycin, inhibitor for PIP_2 degradation, in the reaction mixture. PIP_2 synthesis was increased by active form of ADP-ribosylation factor 6 (Arf6). It was suggested that increase of membrane recruitment of AP2, clathrin adaptor protein, by Arf6 might attribute to the increase of PIP_2 synthesis.Kinetics of membrane lipids in culture cells: Degradation of PTP_2 synthesis upon endocytosis was examined in culture cells. Metabolically labeled HeLa cells were stimulated for endocytosis and the amount of PIP_2 was analyzed. Similar PIP_2 degradation as that observed in the cell-free system was observed.