Fig. 5. Cdc42 facilitates the reversal of migration direction by controlling NHE1 repolarization.

a Migration velocity and b fraction of vehicle control and ML141-treated MDA-MB-231 cells displaying negative velocity before (gray) and after (red) hypotonic shock (165 mOsm/l) at the leading edge in confinement. Data represent the a mean ± SD for the indicated number of cells or b mean ± SEM from 3 (a) or 4 (b) independent experiments. c Representative images and d quantification of NHE1 polarization in vehicle control and ML141-treated MDA-MB-231 cells inside confining channels before (340 mOsm/l) and after (165 mOsm/l) application of hypotonic shock at the leading edge. NHE1 is polarized at the cell leading edge in control and ML141-treated cells before osmotic shock (yellow arrowheads) and repolarizes to the “new” leading edge in response to osmotic shock only in control (red arrowhead), but not ML141-treated (white arrowhead), cells. Data represent the mean ± SD from three independent experiments. e Time-lapse montage of an MDA-MB-231 cell expressing OptoSWELL1 and CAAX-CIBN-GFP treated with ML141. Optogenetic SWELL1 enrichment at the cell leading edge was initiated in a region enclosed by the yellow box just after t = 250 sec. Gradual SWELL1 accumulation at the cell front (yellow arrowhead) is accompanied by SWELL1 reduction at the cell rear (white arrowhead). At t = t₁ = 850 sec, SWELL1 is equally distributed at both cell poles. f Migration velocity (black line) and front to rear ratio of SWELL1 intensity (red line) of the cell shown in e. g Migration velocity (black dots) and front to rear ratio of SWELL1 intensity (red) of MDA-MB-231 cells expressing OptoSWELL1 and CAAX-CIBN-GFP that were treated with ML141 before stimulation, at t = t₁ and t ≥ 1,200 sec after stimulation. Data represent the mean ± SD for 12 cells from 3 independent experiments. *p < 0.05 and ***p < 0.001 relative to control data after shock assessed by two-tailed unpaired t-test.