Mettlen Marcel, Stoeber Miriam, Loerke Dinah, Antonescu Costin N, Danuser Gaudenz, Schmid Sandra L
Department of Cell Biology, The Scripps Research Institute, La Jolla, CA 92037, USA.
Mol Biol Cell. 2009 Jul;20(14):3251-60. doi: 10.1091/mbc.e09-03-0256. Epub 2009 May 20.
Diverse cargo molecules (i.e., receptors and ligand/receptor complexes) are taken into the cell by clathrin-mediated endocytosis (CME) utilizing a core machinery consisting of cargo-specific adaptors, clathrin and the GTPase dynamin. Numerous endocytic accessory proteins are also required, but their differential roles and functional hierarchy during CME are not yet understood. Here, we used a combination of quantitative live-cell imaging by total internal reflection fluorescence microscopy (TIR-FM), and decomposition of the lifetime distributions of clathrin-coated pits (CCPs) to measure independent aspects of CCP dynamics, including the turnover of abortive and productive CCP species and their relative contributions. Capitalizing on the sensitivity of this assay, we have examined the effects of specific siRNA-mediated depletion of endocytic accessory proteins on CME progression. Of the 12 endocytic accessory proteins examined, we observed seven qualitatively different phenotypes upon protein depletion. From this data we derive a temporal hierarchy of protein function during early steps of CME. Our results support the idea that a subset of accessory proteins, which mediate coat assembly, membrane curvature, and cargo selection, can provide input into an endocytic restriction point/checkpoint mechanism that monitors CCP maturation.
多种货物分子（即受体以及配体/受体复合物）通过网格蛋白介导的内吞作用（CME）进入细胞，这一过程利用了由货物特异性衔接蛋白、网格蛋白和GTP酶发动蛋白组成的核心机制。还需要众多内吞辅助蛋白，但其在CME过程中的不同作用和功能层次仍不清楚。在这里，我们结合了全内反射荧光显微镜（TIR-FM）进行定量活细胞成像，并对网格蛋白包被小窝（CCP）的寿命分布进行分解，以测量CCP动力学的独立方面，包括流产型和成熟型CCP种类的周转及其相对贡献。利用该检测方法的敏感性，我们研究了特定的小干扰RNA（siRNA）介导的内吞辅助蛋白缺失对CME进程的影响。在所检测的12种内吞辅助蛋白中，我们观察到蛋白缺失后出现了7种性质不同的表型。根据这些数据，我们得出了CME早期步骤中蛋白质功能的时间层次。我们的结果支持这样一种观点，即一部分介导包被组装、膜曲率和货物选择的辅助蛋白可以为监测CCP成熟的内吞限制点/检查点机制提供输入。
