Figure 8: PtdIns5P induces Rac1 activation in dorsal ruffles and vesicles.
From: Phosphatidylinositol 5-phosphate regulates invasion through binding and activation of Tiam1
(a) MEF cells transfected with the Raichu-Rac1 encoding vector were plated on micropatterns of fibronectin and serum-starved for 6 h. Exogenous PtdIns5P was added (0.5 μM) and cells were imaged every 4 min during 20 min. Rac1 localization was visualized by monitoring CFP fluorescence. Rac1-enriched ruffles are visible on the dorsal side of the cells. On the right side, a kymograph assembled along the lamellipodium shows the dynamic towards the front of the cell. (b) Rac1 activation is visualized by color-coded FRET. High ratio values (red) correlate with higher Rac1 activity in ruffles and at the cell periphery. The lower panels represent higher magnifications for areas at the indicated time points. Arrowheads represent circular ruffles moving towards the front of the lamellipodium. (c) Similar experiments as in (b) except that no micropattern was used. The right panels represent higher magnifications of boxed areas a and b depicted on the larger image. Arrowheads indicate vesicles (area a) and ruffles (area b) with active Rac1. (d) Similar experiment as in (c) except that no PtdIns5P was added to the cells. (e) Similar experiment as in (c) except that it was performed on Tiam1−/− MEFs challenged with PtdIns5P. Scale bar, 10 μm. See also Supplementary Movies 5–9.
