Movies

Time lapse recordings of neutrophils interacting with PAF-presenting HUVEC under shear stress conditions.

A representative neutrophil spread and crossing the endothelial barrier is highlighted in the brightened square.

Effector T cells crawling on and transmigrating through TNF-activated HUVEC under shear flow.

The ECs were transiently transfected with the membranal marker tH-EGFP (a plasmid encoding the C’ terminal 9 amino acids of H-Ras which targets the fused EGFP to the plasma membrane) prior to stimulation with TNF-α. Fluorescence (green) and DIC (grey) images were taken at 15 s intervals and the time lapse videos are displayed at 4 frames/s. Scale bar= 15 μm.

Effector lymphocytes can cross the endothelium through paracellular and transcellular TEM routes

Effector T cells crawling on and transmigrating through TNF-activated ICAM-1-EGFP expressing HUVEC under shear flow. Left panels: merged fluorescence (green) and DIC (grey) videos taken at 15 s intervals and displayed at 2 frames/s. Right panels depict the fluorescence channel of these video segments. The route of TEM (paracellular or transcellular) is indicated for individual T cells. Scale bar= 15 μm.

T cells migrate towards and over autologous DCs in the presence of immobilized CCL21.

Monocyte-differentiated DCs were spread on immobilized CCL21. Autologous BCECF-labeled CD3+ PBL were added and tracked for 15 min.

T cells migrate towards autologous DCs in the presence of immobilized CCL21.

Monocyte-differentiated DCs were spread on immobilized CCL21. BCECF labeled CD3+ PBL were added and tracked for 15 min.

Alpha4 integrins are confined to the uropod of T lymphocytes locomoting on immobilized CCL21

Human CD3+ PBL were pre-labeled with Alexa488-tagged B5G10 (a non-blocking α4-specific antibody) and allowed to migrate on CCL21 co-immobilized with collagen and fibronectin. 1 sec movie = 0.5 min.

CCL2 vesicles are mobilized on microtubules and docked on actin fibers in inflamed endothelial cells

TNF-activated HUVEC transiently transfected with the CCL2-EGFP (green) and the actin marker, Lifeact-RFP (red) or α-tubulin-mCherry (red) were imaged by time lapse fluorescence microscopy at 5 s intervals. Time lapse videos are displayed at 2 frames/s. Scale bar= 15 μm.

The basolateral leading edge of effector lymphocytes consumes vesicle stored CCL2 during productive TEM

A lymphocyte transmigrating through TNF-activated HUVEC transiently transfected with the CCL2-EGFP (green) and the actin marker, Lifeact-RFP (red). Fluorescence and DIC (blue) images were taken at 6 s intervals and are displayed at 2 frames/s. The yellow arrowhead points the rear of the transmigrating lymphocyte. Individual yellow circles mark multiple CCL2 vesicles that come in direct contact with the basolateral leading edge of the actively transmigrating lymphocyte. Scale bar= 15 μm.