Transcellular pore closure is initiated by novel VL. (a) Combined epifluorescence (red) and TIRF (green) imaging of diapedesis pore (yellow line) being closed by a lamellar structure in an MVEC expressing mDsRed. Arrowheads show a high TIRF signal indicating a VL. (b, i) Dynamic serial-section spinning disk confocal imaging of a mYFP-expressing endothelial cell during T cell diapedesis. Sections of dorsal/apical (red), intermediate (blue), and ventral/basal (green) planes were differentially pseudo-colored and overlaid. Dashed yellow lines indicate transcellular pores (1–5) and paracellular gaps (a–e) where individual T cells are transmigrating. (ii) Expanded view of the boxed region in panel i shows membrane protrusions (VL, arrowheads) formed in the ventral plane spreading under three pores. See Video 3. (c) Ultrastructural view of basal F-actin–rich protrusions putatively representing VL (arrows) during late stages of T cell (green) diapedesis across the endothelium (blue). (d) Schematic model of VL pore closure. (e) Representative images (i) and membrane-normalized quantitation (ii) of β3-GFP and α5-GFP integrin distribution within pore-closing VL. (iii–v) Anti-αvβ3 and -α5β1 function-blocking antibodies were added immediately after exit of a T cell (cyan outline) from a diapedesis pore. (iii) Representative images show VL that initially protrude and then retract. Green line (iii) traces location used for kymograph representation (iv). (v) Percentage of pores that closed in the 10 min after addition of blocking antibodies. Values represent mean ± SEM; n > 20. Bars, 5 µm.