Morphological changes of plasma membrane and protein assembly during clathrin-mediated endocytosis

Clathrin-mediated endocytosis (CME) proceeds through a series of morphological changes of the plasma membrane induced by a number of protein components. Although the spatiotemporal assembly of these proteins has been elucidated by fluorescence-based techniques, the protein-induced morphological changes of the plasma membrane have not been fully clarified in living cells. Here, we visualize membrane morphology together with protein localizations during CME by utilizing high-speed atomic force microscopy (HS-AFM) combined with a confocal laser scanning unit. The plasma membrane starts to invaginate approximately 30 s after clathrin starts to assemble, and the aperture diameter increases as clathrin accumulates. Actin rapidly accumulates around the pit and induces a small membrane swelling, which, within 30 s, rapidly covers the pit irreversibly. Inhibition of actin turnover abolishes the swelling and induces a reversible open–close motion of the pit, indicating that actin dynamics are necessary for efficient and irreversible pit closure at the end of CME.网格蛋白介导的内吞作用（Clathrin-mediated endocytosis, CME）是一类通过多种蛋白质组分诱导的质膜形态变化序列完成的生理过程。尽管基于荧光成像的技术已阐明了这类蛋白质的时空组装机制，但活细胞内由蛋白质诱导的质膜形态变化仍未得到完全阐释。本研究借助结合共聚焦激光扫描单元的高速原子力显微镜（high-speed atomic force microscopy, HS-AFM），实现了对CME过程中膜形态与蛋白质定位的同步可视化观测。研究发现，在网格蛋白开始组装约30秒后，质膜即开始内陷，且随着网格蛋白不断积累，内陷孔的直径逐渐增大。肌动蛋白会快速在凹陷位点周围聚集，并诱导产生小型膜隆起；该隆起会在30秒内快速且不可逆地覆盖凹陷处。抑制肌动蛋白的周转过程会消除该膜隆起，并导致凹陷位点出现可逆的开合运动，这表明肌动蛋白动态变化对于CME末期实现高效且不可逆的凹陷闭合是必需的。