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Figure 2: PMN transmigration at a bicellular junction. Real-time DIC microscopy images of a PMN in the process of transmigration (top) are paired with simultaneous two-color fluorescence (bottom) that show the leukocyte stained with an intravital dye (red channel) and VEcadGFP (green channel). The time in minutes is shown at the upper right corner of the DIC images. At t = 3:00, a de novo visible gap is easily detected (arrow). At t = 4:15, the PMN has partially transmigrated and is visible as a bulge under the endothelium (arrow). Transmigration is complete at t = 4:45. Note that the leukocyte is visible in the red channel during the entire process of transmigration, indicating that the entire thickness of the monolayer was visualized. Thus, gap formation in VEcadGFP did not reflect its movement out of the focal plane.

Figure 3: Monocyte transmigration at a bicellular junction. Under DIC, stably arrested monocytes flatten on the apical HUVEC surface to a greater degree than do PMN, with the edge of the leukocyte barely visible at t = 0:45 (arrow). At t = 3:00, a de novo gap is easily detected in the junction. In the next six panels, the monocyte is shown in the process of transmigration. After transmigration, at t = 9:15, the junction has resealed.

Fig. 4B: QuickTime Video

Figure 4: PMN transmigration at a tricellular corner or through preexisting gaps. A, In this series, a PMN approaches an intact tricellular junction (t = 0 min, arrow), but after a prolonged duration caused a de novo gap to appear in VEcadGFP (t = 9:00), transmigrated through (t = 11:30, arrow), after which the gap partially recloses (t = 16:15). B, Occasional gaps in VEcadGFP were present at bicellular junctions and at tricellular corners, and leukocytes frequently migrated through these. In this example, a (preexisting) gap is present before the PMN arrival (t = 0, arrow). The PMN transmigrates through this gap in the next three panels, and at t = 16:15, the gap has almost completely reclosed (arrow), and another PMN has arrested and spread, apparently ready to transmigrate.

Figure 5: Curtain effect: VEcadGFP moves aside like a curtain, during gap formation or widening. Here, a monocyte approaches an almost unbroken wall of VEcadGFP (t = 0 min). In the next few panels, the gap gradually widens. A bunching effect of VEcadGFP is visible adjacent to the gap (t = 1:00, arrows). After the monocytes has completed transmigration, this bunched up material gradually diffuses back to fill the gap. A t = 5:00 and subsequent panels, a clump of green fluorescence (arrow) is seen to move into the vacant area, which is once more mostly resealed at t = 8:30.

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