Lymphocytes cross endothelial cells via large transcellular channels marked by rings of PV-1 and caveolin-1 and are embraced by a vimentin network. (A) Lymphocyte migrating through a HUVEC transfected with a PV-1–EGFP construct. A pore forms in front of the transmigrating cell and widens to a diameter of approximately 7 μm (white arrows). The time course is outlined in minutes in the upper left corner of the pictures. (B) Localization of endogenous PV-1 and caveolin-1 in an endpoint transmigration assay. Lymphocytes were overlaid on top of TNF-α–activated HUVECs, allowed to transmigrate, and fixed. A transendothelial channel with a migrating lymphocyte is depicted. The channel is outlined by colocalizing rings of PV-1 and caveolin-1 (white arrows). (C) Localization of PV-1 and vimentin. HDMECs were stained for vimentin and PV-1 after an endpoint transmigration assay. White arrows point to an accumulation of PV-1 around the migrating cell and to a vimentin network surrounding the lymphocyte. Nuclear counterstaining was performed with DAPI. Scale bars represent 10 μm.