Figure 2. Local Ral activation induces protrusiveness.

(A) Cell edge dynamics inside the illuminated area were measured using an automated method. (B) Edge dynamics velocity map of a representative OptoRal cell before and after illumination (dotted blue line). See Video 3 for the entire sequence. The color-coded map shows the velocity measurements for each sampled edge window (space, horizontal axis) and for each time point (time, vertical axis). Positive velocities (i.e. protrusion) are represented as warm color, negative velocities (i.e. retraction) as cold colors. As example of velocity profile over time, the velocity measurements are shown for a selected point in space (window 71) in the lower panel; the stimulation of edge velocity is visible immediately after light illumination. As examples of velocity profiles over the space dimension, the velocity measurements are shown in the right panels using the color-code along the cell edge for two selected time points, 1.4 min and 6.5 min, dark and light conditions, respectively. (C) Edge dynamics velocity map of a representative OptoControl cell before and after illumination. See Video 4 for the entire sequence. The velocity profiles over time (window 77, lower panel) or over space (1.3 min and 5 min, right panels) show that illumination did not induce any change of edge velocity. (D) Quantification of edge velocities before and after illumination. Positive velocities (i.e. protrusion) and negative velocities (i.e. retraction) are analysed separately. Each white dot represents the mean of all velocity measurements (all time and space points) before illumination. Each blue dot represents the mean of all velocity measurements (all time and space points) after illumination. Measurements from a same cell are connected by lines. Illumination stimulates positive edge velocities in OptoRal cells (left) but not in OptoControl cells (centre). Inhibition of Rac with NSC23766 (100 µM, treatment started 1 hr before experiment) did not impair stimulation by light of positive edge velocities in OptoRal cells (right). Bars represent mean of n = 18 cells per condition ±SEM from three independent experiments. *** indicates p<0.001, ** indicates p<0.01 and ns indicates not-significant, using Wilcoxon signed-rank test for paired measurements (same cell pre- and post-illumination). There was no significant difference in the delta velocities pre- and post-illumination when comparing presence and absence of the NSC23766 inhibitor (using Student t-test, not shown). There was no significant difference in the pre-illumination edge velocities when comparing OptoRal and OptoControl cells (using Student t-test, not shown). (E) RalB depletion, but not RalA depletion, impairs stimulation of positive edge velocities in OptoRal cells. OptoRal cells were transfected with the indicated siRNAs and analysed as in panel D. n = 10–11 cells per condition.

Figure 2—figure supplement 1. Additional evidences that light stimulates RalB activity in OptoRal cells.

(A) Pull-down assay of RalB-GTP in dark and after blue light stimulation (5 min), in OptoControl and OptoRal cells, using Sec5GBD beads. The quantifications express the RalB-GTP/total RalB ratios, normalized for OptoControl dark condition. Cells were starved overnight in DMEM supplemented 0.2% FBS before the assay. For dark conditions, cells were kept in a dark box to avoid daylight exposure and lysed in a dark room. For light conditions, cells were exposed to 488 nm excitation illumination under an epifluorescence microscope for 5 min prior lysis. (B) Visualization of RalB activity by FRET. OptoRal cells, expressing a RalB FRET-based biosensor, were imaged by FRET miscoscopy; in such setting, since the CFP stimulation results in optogenetics activation, the first time point is taken as dark condition. The graph shows the average ±SD whole cell FRET measurements from n = 3 cells. Scale bar, 10 µm.

Figure 2—figure supplement 2. Reversibility and repeatability of edge velocity stimulation by light. Validation of Rac1 inhibition and of RalA and RalB depletion. .

(A) Edge dynamics velocity maps of OptoRal cells upon alternation of dark and light periods. Pulsed light stimulation was alternated with periods of dark as following: 3 min dark, 10 min light, 20 min dark, and 10 min light. On the left, the velocity measurements over the time are shown for a selected space point (window 52). The stimulation of edge velocity repeatedly occurs immediately after light illumination. Upon switch off to dark, the reversion to base-line edge velocity occurs within 10 min. (B) Edge dynamics velocity maps of OptoControl cells upon alternation of dark and light periods. No matter light or dark, only small stochastic fluctuations of edge velocity are observed. (C) Validation of Rac1 inhibition by NSC23766 treatment. OptoRal cells were incubated 1 hr with 100 µM NSC23766 prior to cell lysis. Cell lysates were subjected to Rac1-GTP pull-down assay using Pak1GBD beads. The quantifications express the Rac1-GTP/total Rac1 ratios, normalized for control condition without treatment. (D) Validation of Ral depletions for cell edge dynamics in OptoRal cells. Representative western blots from cell lysates prepared 72 hr after transfection with the indicated siRNA. GAPDH was used as loading control. Quantifications of Ral depletion efficiencies, calculated as Ral/GAPDH and normalized for siControl, are shown below the WBs.