Picco A, Kaksonen M
University of Geneva, Geneva, Switzerland.
Methods Cell Biol. 2017;139:51-68. doi: 10.1016/bs.mcb.2016.11.002. Epub 2016 Dec 29.
Endocytosis is a complex and dynamic process that involves dozens of different proteins to define the site of endocytosis, form a membrane invagination, and pinch off a membrane vesicle into the cytoplasm. Fluorescent light microscopy is a powerful tool to visualize the dynamic behaviors of the proteins taking part in the endocytic process. The resolution of light microscopy is, however, a serious limitation. Here, we detail a fluorescence microscope method that we have developed to visualize the dynamics of the clathrin-mediated endocytic protein machinery in yeast cells. This method is based on subpixel centroid tracking of endocytic proteins. For each endocytic protein, the centroid trajectories obtained from multiple endocytic events are used to compute an average trajectory that describes, at nanometer scale, the assembly and movement of the protein during endocytosis. The average trajectories of the different endocytic proteins are then aligned together in space and time to reconstruct how the different proteins behave relative to each other during the endocytic process.
内吞作用是一个复杂且动态的过程，涉及数十种不同的蛋白质来确定内吞作用的位点、形成膜内陷，并将膜囊泡 pinched off 到细胞质中。荧光显微镜是一种强大的工具，可用于可视化参与内吞过程的蛋白质的动态行为。然而，光学显微镜的分辨率是一个严重的限制。在这里，我们详细介绍一种我们开发的荧光显微镜方法，用于可视化酵母细胞中网格蛋白介导的内吞蛋白机制的动态。该方法基于对内吞蛋白的亚像素质心跟踪。对于每个内吞蛋白，从多个内吞事件获得的质心轨迹用于计算平均轨迹，该轨迹在纳米尺度上描述了内吞过程中蛋白质的组装和运动。然后将不同内吞蛋白的平均轨迹在空间和时间上对齐，以重建在整个内吞过程中不同蛋白质相对于彼此的行为方式。 （注：“pinched off”此处暂未准确对应中文，可根据上下文理解为“掐断、脱离”等意思）
