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. 2016 May;186(5):1375–1386. doi: 10.1016/j.ajpath.2016.01.011

Figure 1.

Figure 1

Blocking kinesin-1 function via microinjection of SUK4 in HUVECs inhibits TEM and TR. HUVECs were microinjected with SUK4 (kinesin-1 mAb) or isotype-matched mouse IgG control antibody. Antibodies were mixed with a fluorescent-conjugated dextran to label injected cells. Monocytes were allowed to settle on the monolayer and then transmigrate for 7.5 minutes. A: HUVEC monolayers (labeled by PECAM in red) remained intact after microinjection (microinjected cells labeled blue) and monocyte (labeled green) TEM. B and C: Confocal stacks were imaged, and the numbers of PBMCs that have attached and migrated to EC junctions were counted (B), and TEM was quantified (C). D: High-power images of confocal stacks to show differences in TEM and TR in SUK4- versus IgG control-microinjected cells. Constitutive recycling occurs evenly but spottily along the junctions; however, TR enhances LBRC fluorescence at sites of TEM. Orthogonal projections (xz) are depicted as the smaller images to the right of their corresponding images. The monocyte shown for the IgG control-microinjected cells is just starting TEM, as seen in the orthogonal projection. Arrowhead indicates site of leukocyte TEM. Dotted lines in the orthogonal projection indicate abluminal surface of endothelial cells. E: LBRC enrichment was measured around leukocytes at endothelial junctions. F: TR significantly diminishes after microinjection of SUK4 mAb against kinesin-1. G: TEM is significantly lower in SUK4-injected cells compared with cells injected with K2.4 (anti–kinesin-2). Data are expressed as means ± SEM. n = 3 experiments with two monolayers per condition for each experiment and at least 100 monocyte/EC interactions per monolayer (F); n = 2 experiments with one monolayer per condition for each experiment and at least 100 monocyte/EC interactions per monolayer (G). ∗P < 0.05, ∗∗P < 0.01. Scale bar = 10 μm. HUVEC, human umbilical vein endothelial cell; LBRC, lateral border recycling compartment; mAb, monoclonal antibody; PBMC, peripheral blood mononuclear cell; PECAM, platelet endothelial cell adhesion molecule; TEM, transendothelial migration; TR, targeted recycling.