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. 2010 Aug 23;190(4):553–563. doi: 10.1083/jcb.201002067

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© 2010 Heasman et al.

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Figure 3. — RhoA localizes to and is active at the leading edge and uropod during crawling and TEM. (a) RhoA (green) localization in CEM T cells or T lymphoblasts (T-LB) on EC. Pink, cell tracker dye. Maximum projections of 11–31 confocal z-stack images at 0.5-µm intervals. RhoA is enriched at the uropod (arrows) and leading edge (arrowheads). (right) Circles, uropods; dashed lines, lamellipodium under EC. (b and c) FLIM images of RhoA Raichu probe (wild type) were acquired every 45 s as cells migrated on (b) or transmigrated across (c) EC. RhoA activity localizes to the leading edge (white arrowheads), including filopodia during TEM (red arrowheads), the uropod during rear contraction (solid arrows), and in dynamic puncta in the lamellal region (dashed arrows). Probe intensity images identify uropod (asterisks) above and transmigrated lamella below EC. (d) The plasma membrane region of FLIM images from six videos was analyzed to quantify RhoA activity levels (mean FRET efficiency value) in each of four 90° segments (see Materials and methods). The center of the leading edge is center of the front segment. The sum of values in the front or back segment was compared with the two side segments. (e) F-actin localization in transmigrating CEM cell imaged by STED microscopy. (middle) Magnified image of boxed area shows filopodia extending from the leading edge (arrows). (right) Circle, uropod; dashed line, leading edge under EC. (f) Localization of Cd11a/αL integrin (green) and F-actin (red) during TEM 30 min after addition of CEM cells to EC. Bars, 10 µm.