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Fig. 5. ßPIX accumulates at the front of the cell to confer localized Rac activation. (A) ßPIX controls the formation of protrusions. Representative cells injected with pEGFP-F (Membrane) along with pXJ40-HA::wt ßPIX. Expression of constructs was confirmed by immunofluorescence (not shown). Protrusions are found at the front (open arrows) and also at the back (filled arrows). Expression of wt ßPIX has no effect on Golgi reorientation (asterisk). (B) Interfering with the endogenous Pak/ßPIX interaction blocks polarization of protrusions. Effects of various GFP-fusion constructs on Golgi reorientation (black bars) or protrusion polarization (white bars). (C) Down-regulation of ßPIX affects formation of the protrusions. 4 days after nucleofection with siRNA, monolayers were scratched and front row cells injected with pGFP-F to determine the localization of protrusions. siRNA-mediated down-regulation of ßPIX was visualized by western blotting (bottom left). The representative control cell extends protrusions at the front (open arrows) and has ßPIX localized in front focal-complex-like puncta (arrowheads). Down-regulation of ßPIX increases the proportion of cells having no detectable protrusions (bottom panel). Instead, cells have elongated, thick filopodia-like extensions (small open arrows). In A and B, error bars represent standard deviation and asterisks mark a highly significant (***P<0.0001) difference compared with control. Bars, 20 µm.
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