Transcellular endothelial micro-wounds are closed efficiently after diapedesis through bursts of lamellar activity. Live-cell imaging of lymphocyte diapedesis through MVECs. (a) DIC and fluorescence imaging of migrating T cells (cyan outlines) on an ICAM-1-GFP–expressing endothelial cell (green). Open diapedesis pores (1–3) and gaps (a and b) associated with T cells in the process of diapedesis are outlined in yellow. The subsequent time points show opening of additional pores (4–7) and gaps (c and d) formed by other T cells initiating diapedesis. The progressive closure of the pores and gaps after completion of each diapedesis event is denoted by a switch from yellow to red outlines. (b) Representative time series of a pore closure event in MVECs coexpressing actin-GFP and mDsRed. Yellow outline shows an open transcellular pore associated with a T cell (cyan outline) that has nearly completed diapedesis. A pore closing structure (CL; white arrows and dashed line) is evident as a burst of actin and membrane traveling across the pore to close it. See Video 1, Part I. (c) Analysis of width and dynamics of >68 individual pore closing structures. (d) Percentage of three classes (Fig. S1 a) of pore closing types (i) and quantitative analysis of CL travel distances (ii and iii). n > 100. (e) Doubling/Folding of membrane during pore closure. (i). Arrows indicate actin protrusions initiated at the edge of the pore (blue) and at distant sites (white). White line shows location of a fluorescence intensity line scan analysis (ii) with regions that are part of (CL) and outside of (non-CL) the closure structure indicated. (iii) Membrane fluorescence analysis in CL and non-CL structures. n = 15. Error bars represent SEM. See Video 1, Part II. Bars, 5 µm.