Grassart Alexandre, Cheng Aaron T, Hong Sun Hae, Zhang Fan, Zenzer Nathan, Feng Yongmei, Briner David M, Davis Gregory D, Malkov Dmitry, Drubin David G
Department of Molecular and Cell Biology, University of California, Berkeley, Berkeley, CA 94720.
Cell-Based Assays/Reporter Cell Lines, Sigma-Aldrich Research Biotech, St. Louis, MO 63103.
J Cell Biol. 2014 Jun 9;205(5):721-35. doi: 10.1083/jcb.201403041. Epub 2014 Jun 2.
Clathrin-mediated endocytosis (CME) involves the recruitment of numerous proteins to sites on the plasma membrane with prescribed timing to mediate specific stages of the process. However, how choreographed recruitment and function of specific proteins during CME is achieved remains unclear. Using genome editing to express fluorescent fusion proteins at native levels and live-cell imaging with single-molecule sensitivity, we explored dynamin2 stoichiometry, dynamics, and functional interdependency with actin. Our quantitative analyses revealed heterogeneity in the timing of the early phase of CME, with transient recruitment of 2-4 molecules of dynamin2. In contrast, considerable regularity characterized the final 20 s of CME, during which ∼26 molecules of dynamin2, sufficient to make one ring around the vesicle neck, were typically recruited. Actin assembly generally preceded dynamin2 recruitment during the late phases of CME, and promoted dynamin recruitment. Collectively, our results demonstrate precise temporal and quantitative regulation of the dynamin2 recruitment influenced by actin polymerization.
网格蛋白介导的内吞作用（CME）涉及众多蛋白质在规定时间被募集到质膜上的位点，以介导该过程的特定阶段。然而，在CME过程中，特定蛋白质是如何进行编排募集和发挥功能的仍不清楚。我们利用基因组编辑在天然水平表达荧光融合蛋白，并通过具有单分子灵敏度的活细胞成像，探究了发动蛋白2的化学计量、动力学以及与肌动蛋白的功能相互依赖性。我们的定量分析揭示了CME早期阶段时间上的异质性，即有2 - 4个发动蛋白2分子短暂募集。相比之下，CME最后20秒具有相当的规律性，在此期间通常会募集约26个发动蛋白2分子，足以在囊泡颈部形成一圈。在CME后期，肌动蛋白组装通常先于发动蛋白2募集，并促进发动蛋白的募集。总体而言，我们的结果表明肌动蛋白聚合作用影响了发动蛋白2募集的精确时间和定量调控。
