Molecular mechanism of endocytosis that regulates membrane dynamics

为了阐明内吞蛋白在膜动力学中的作用，研究了动力蛋白和两栖动物蛋白在吞噬过程中的定位和细胞内的动态变化。为此，用含磷脂酰丝氨酸的脂质体刺激睾丸支持细胞吞噬功能，并检测胞内蛋白的定位和动态。通过免疫荧光和活细胞成像，Dynamin2和两栖动物蛋白都集中在片状脂肪和褶皱的前缘。在双体菌素siRNA处理的细胞中，褶皱形成、肌动蛋白形成和吞噬作用明显受到抑制，这表明双体菌素在这些过程中是必不可少的。此外，在两栖动物1被击倒的细胞中，这些效应被结构性活性Rac 1的共过表达所挽救，这表明Rac 1参与了这一过程。其次，体外观察了两栖动物1对肌动蛋白聚合活性的影响。为此，在小鼠睾丸胞浆中添加了芘标记的肌动蛋白，并进行了体外定量肌动蛋白聚合实验。肌动蛋白聚合也在荧光显微镜下观察到，使用胞浆添加罗丹明偶联肌动蛋白。肌动蛋白1基因敲除小鼠的细胞液中肌动蛋白组装活性显著降低，这种活性可以通过重新加入重组蛋白来恢复，因此，内吞蛋白1在肌动蛋白动力学的调节中发挥作用，从而刺激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.