FIG. 5.

FA-CRKL induced cytoskeletal reorganization. (A) Expression of FA-CRKL in NIH 3T3 cells induced multiple membrane protrusions decorated by lamellipodia (arrows). The part highlighted by a white rectangle was enlarged and shown at right. Blue arrowheads indicate FA-CRKL localized to focal adhesions. Green arrowheads indicate FA-CRKL localization at the membrane cortex where F-actin (red arrowheads) also colocalized. Merged color images use red and green to indicate localization of F-actin and FA-CRKL, respectively. (B) Cytoskeletal reorganization induced by transient expression of mutants of FA-CRKL. Localization of F-actin (red) and GFP (green) signals for fusion proteins is shown. Arrowheads indicate membrane protrusions decorated by lamellipodia. (C) Subcellular targeting of CRKL to the outer membrane of mitochondria. Localization of F-actin (red), CRKL-GFP-mito (green), and paxillin (blue) is shown. (D) The incidence of lamellipodial formation was counted after transient expression of each transgene indicated. Percent values indicate the number of NIH 3T3 cells with more than two membrane protrusions decorated by lamellipodia per total transfected (GFP-positive) cells. To assist observation of lamellipodia, cells were stained with phalloidin. Error bars indicate the standard deviation of the sample data collected in three independent sets of samples, each of which was scored with at least 60 cells in random fields under a 40× objective lens. (E) The number of protrusions containing lamellipodia per cell (x axis) was counted in each cell and plotted for occurrence (y axis) in NIH 3T3 cells transiently expressing FA-CRKL, FA-CRKL(ΔSH3n), or FA-GFP. For example, four cells had seven lamellipodium-containing protrusions in the FA-CRKL group. A total of 65 cells was counted for each group. Transient expression shown in panels B to E was performed at a dose of 0.1 μg/well of the expression plasmid, except that the experiments for panel D were performed at a dose of 0.3 μg/well.