Molecular mechanism of endocytosis that regulates membrane dynamics

为了阐明内吞蛋白在细胞膜动力学中的作用，我们研究了动力蛋白和两性蛋白在吞噬过程中的定位和细胞内动力学。为了这个目的，睾丸支持细胞的吞噬作用刺激磷脂酰丝氨酸含有脂质体，胞内定位和动态的内吞蛋白进行了检查。通过免疫荧光和活细胞成像，动力蛋白2和两栖蛋白都集中在片状伪足和皱褶的前缘。皱褶形成，肌动蛋白形成，和吞噬作用显着抑制在Amphiphysin siRNA处理的细胞表明，Amphiphysin是必不可少的这些过程。此外，这些影响在两型生理素1敲除的细胞中被组成型活性Rac 1的共过表达所拯救，表明Rac 1参与了两型生理素下游的过程。为了这个目的，小鼠睾丸细胞质溶胶补充芘共轭肌动蛋白，进行定量肌动蛋白在体外聚合测定。肌动蛋白聚合也观察到在荧光显微镜下使用补充有罗丹明共轭肌动蛋白的胞质溶胶。敲除Amphiphysin 1基因的小鼠胞浆中肌动蛋白的组装活性显著降低，通过加入重组蛋白可以恢复，因此，Amphiphysin 1作为一种内吞蛋白，在肌动蛋白动力学的调节中发挥作用，从而刺激细胞膜动力学和吞噬功能。

In order to elucidate the role of endocytic proteins in membrane dynamics, localization and intracellular dynamics of dynamin and amphiphysin during phagocytosis were examined. For this purpose, testicular Sertoli cell phagocytosis was stimulated by phosphatidylserine containing liposomes, and intracellular localization and dynamics of the endocytic proteins were examined. By immunofluorescence and by live cell imaging, both dynamin2 and amphiphysin were concentrated at the leading edge of lamellipodia and ruffles. Ruffle formation, actin formation, and phagocytosis were markedly inhibited in Amphiphysin siRNA treated cells indicating that amphiphysin is essential for these processes. Furthermore, these effects in the amphiphysin 1-knocked down cells were rescued by co-overexpression of constitutive active Rac 1, suggesting that Rac 1 is involved in the process at the downstream of amphiphysin.Next, effect of amphiphysin 1 on actin polymerization activity was examined in vitro. For this purpose, mouse testis cytosol supplemented with pyrene-conjugated actin, were subjected to quantitative actin in vitro polymerization assay. Actin polymerization was also observed under fluorescent microscopy using cytosol supplemented with rhodamine-conjugated actin. Actin assembly activity was considerably reduced in cytosol from amphiphysin 1 knock out mice, which can be recovered by adding back recombinant proteins.Thus, amphiphysin 1, an endocytic protein, play a role in the regulation of actin dynamics, by which it stimulate in membrane dynamics and phagocytosis.