A Study on Molecular Mechanisms of Endocytosis

体外无细胞体系的建立：为了阐明内吞作用的分子机制，在体外重建了内吞作用中囊泡的形成。将直径大于1 μm的大脂质体与脑胞质液孵育，导致大量形成直径小于100 nm的小囊泡。囊泡的形成需要ATP和GTP。当脂质体与动力蛋白1耗尽的胞质溶胶一起孵育时，囊泡形成急剧减少，表明在该实验系统中囊泡形成代表内吞囊泡形成。在孵育过程中形成的囊泡可以通过动态光散射进行定量和定性分析。膜脂质的功能和动力学：通过分析不同组成的脂质体形成囊泡来研究膜脂质的功能。脂质体中磷脂酰肌醇-4.5-二磷酸（PIP_2）浓度增加，囊泡形成增加。此外，PIP_2被降解为磷脂酰肌醇-4-二磷酸，然后为磷脂酰肌醇。接下来，通过在反应混合物中添加新霉素（PIP_2降解抑制剂）来分析反应期间PIP_2的合成。ADP-核糖基化因子6（Arf 6）的活性形式可促进PIP_2的合成。细胞膜脂动力学：在培养细胞中，观察到胞吞作用导致PTP_2合成的降解。刺激代谢标记的HeLa细胞的内吞作用并分析PIP_2的量。观察到与在无细胞系统中观察到的类似的PIP_2降解。

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.