Picco Andrea, Mund Markus, Ries Jonas, Nédélec François, Kaksonen Marko
Cell Biology and Biophysics Unit, European Molecular Biology Laboratory, Heidelberg, Germany.
Elife. 2015 Feb 12;4:e04535. doi: 10.7554/eLife.04535.
Clathrin-mediated endocytosis is an essential process that forms vesicles from the plasma membrane. Although most of the protein components of the endocytic protein machinery have been thoroughly characterized, their organization at the endocytic site is poorly understood. We developed a fluorescence microscopy method to track the average positions of yeast endocytic proteins in relation to each other with a time precision below 1 s and with a spatial precision of ~10 nm. With these data, integrated with shapes of endocytic membrane intermediates and with superresolution imaging, we could visualize the dynamic architecture of the endocytic machinery. We showed how different coat proteins are distributed within the coat structure and how the assembly dynamics of N-BAR proteins relate to membrane shape changes. Moreover, we found that the region of actin polymerization is located at the base of the endocytic invagination, with the growing ends of filaments pointing toward the plasma membrane.
网格蛋白介导的内吞作用是一个从质膜形成囊泡的重要过程。尽管内吞蛋白机制的大多数蛋白质成分已得到充分表征，但它们在内吞位点的组织情况却知之甚少。我们开发了一种荧光显微镜方法，以低于1秒的时间精度和约10纳米的空间精度来追踪酵母内吞蛋白彼此之间的平均位置。利用这些数据，结合内吞膜中间体的形状和超分辨率成像，我们能够可视化内吞机制的动态结构。我们展示了不同的包被蛋白如何分布在包被结构内，以及N-BAR蛋白的组装动力学如何与膜形状变化相关。此外，我们发现肌动蛋白聚合区域位于内吞凹陷的底部，细丝的生长端指向质膜。
