Morphological changes of the plasma membrane and protein assembly during CME.

<p>(A, B) Time-lapse hybrid imaging of COS-7 cells expressing EGFP-CLCa and mCherry-epsin. Fluorescence images for epsin and clathrin, as well as AFM images are shown every 10 s (panel A). Image size: 1.2 × 1.2 μm². The signal intensities of the fluorescence spots for clathrin (green) and epsin (red), and the diameter of the membrane invagination in the AFM image (dotted black) were plotted against time (panel B). The FL intensity is plotted relative to its maximum value. (C, D) Time-lapse hybrid imaging of COS-7 cells expressing EGFP-CLCa and Dyn2-mCherry. Fluorescence images for dynamin and clathrin, as well as AFM images are shown every 10 s (panel C). The signal intensities of the fluorescence spots for clathrin (green) and dynamin (red), and the diameter of the membrane invagination in the AFM image (dotted black), were plotted against time (panel D). The FL intensity is plotted relative to its maximum value. (E) A summary of protein assembly at CCPs. Based on the AFM images, the time when the plasma membrane started to invaginate (left half) and when the pit had completely closed (right half) were defined as t = 0. Arrowheads indicate the time when fluorescence signal was maximum. The results of hybrid imaging for clathrin, epsin, dynamin, and actin are summarized along this time scale. The timings when the fluorescence signal appeared on the CCP (left half) and disappeared (right half) are plotted. <i>n</i> = 35 for clathrin, <i>n</i> = 8 for epsin, <i>n</i> = 13 for dynamin, and <i>n</i> = 14 for actin. Error bars represent SD. Underlying data may be found in <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.2004786#pbio.2004786.s027" target="_blank">S1 Data</a>. AFM, atomic force microscopy; CCP, clathrin-coated pit; CME, clathrin-mediated endocytosis; COS-7, CV-1 in origin with SV40 gene line 7; EGFP, enhanced green fluorescent protein; EGFP-CLCa, EGFP-fused clathrin light chain a; FL, fluorescence; n.d., not determined.