Medical Research Council Laboratory of Molecular Biology, Cambridge, United Kingdom.
PLoS Biol. 2011 Mar;9(3):e1000604. doi: 10.1371/journal.pbio.1000604. Epub 2011 Mar 22.
Dual colour total internal reflection fluorescence microscopy is a powerful tool for decoding the molecular dynamics of clathrin-mediated endocytosis (CME). Typically, the recruitment of a fluorescent protein-tagged endocytic protein was referenced to the disappearance of spot-like clathrin-coated structure (CCS), but the precision of spot-like CCS disappearance as a marker for canonical CME remained unknown. Here we have used an imaging assay based on total internal reflection fluorescence microscopy to detect scission events with a resolution of ∼ 2 s. We found that scission events engulfed comparable amounts of transferrin receptor cargo at CCSs of different sizes and CCS did not always disappear following scission. We measured the recruitment dynamics of 34 types of endocytic protein to scission events: Abp1, ACK1, amphiphysin1, APPL1, Arp3, BIN1, CALM, CIP4, clathrin light chain (Clc), cofilin, coronin1B, cortactin, dynamin1/2, endophilin2, Eps15, Eps8, epsin2, FBP17, FCHo1/2, GAK, Hip1R, lifeAct, mu2 subunit of the AP2 complex, myosin1E, myosin6, NECAP, N-WASP, OCRL1, Rab5, SNX9, synaptojanin2β1, and syndapin2. For each protein we aligned ∼ 1,000 recruitment profiles to their respective scission events and constructed characteristic "recruitment signatures" that were grouped, as for yeast, to reveal the modular organization of mammalian CME. A detailed analysis revealed the unanticipated recruitment dynamics of SNX9, FBP17, and CIP4 and showed that the same set of proteins was recruited, in the same order, to scission events at CCSs of different sizes and lifetimes. Collectively these data reveal the fine-grained temporal structure of CME and suggest a simplified canonical model of mammalian CME in which the same core mechanism of CME, involving actin, operates at CCSs of diverse sizes and lifetimes.
双色全内反射荧光显微镜是解码网格蛋白介导内吞作用(CME)分子动力学的有力工具。通常,荧光蛋白标记的内吞蛋白的募集被参考为点状网格蛋白包被结构(CCS)的消失,但作为经典 CME 标志物的点状 CCS 消失的精确性仍然未知。在这里,我们使用基于全内反射荧光显微镜的成像测定法,以约 2 秒的分辨率检测断裂事件。我们发现,断裂事件在不同大小的 CCS 上吞噬了相当数量的转铁蛋白受体货物,并且 CCS 并不总是在断裂后消失。我们测量了 34 种内吞蛋白向断裂事件的募集动力学:Abp1、ACK1、 amphiphysin1、APPL1、Arp3、BIN1、CALM、CIP4、网格蛋白轻链(Clc)、丝切蛋白、 coronin1B、cortactin、dynamin1/2、内收蛋白 2、Eps15、Eps8、epsin2、FBP17、FCHo1/2、GAK、Hip1R、lifeAct、mu2 亚基的 AP2 复合物、肌球蛋白 1E、肌球蛋白 6、NECAP、N-WASP、OCRL1、Rab5、SNX9、synaptojanin2β1 和 syndapin2。对于每种蛋白,我们将大约 1000 个募集曲线与各自的断裂事件对齐,并构建了特征性的“募集特征”,这些特征与酵母一样进行分组,以揭示哺乳动物 CME 的模块化组织。详细分析揭示了 SNX9、FBP17 和 CIP4 的出乎意料的募集动力学,并表明相同的蛋白组以相同的顺序被募集到不同大小和寿命的 CCS 的断裂事件中。总的来说,这些数据揭示了 CME 的细粒度时间结构,并提出了一个简化的哺乳动物 CME 经典模型,其中涉及肌动蛋白的相同核心 CME 机制在不同大小和寿命的 CCS 上起作用。