A genetically encoded photoactivatable Rac controls the motility of living cells

Abstract

The precise spatio-temporal dynamics of protein activity are often critical in determining cell behaviour, yet for most proteins they remain poorly understood; it remains difficult to manipulate protein activity at precise times and places within living cells. Protein activity has been controlled by light, through protein derivatization with photocleavable moieties or using photoreactive small-molecule ligands. However, this requires use of toxic ultraviolet wavelengths, activation is irreversible, and/or cell loading is accomplished via disruption of the cell membrane (for example, through microinjection). Here we have developed a new approach to produce genetically encoded photoactivatable derivatives of Rac1, a key GTPase regulating actin cytoskeletal dynamics in metazoan cells. Rac1 mutants were fused to the photoreactive LOV (light oxygen voltage) domain from phototropin, sterically blocking Rac1 interactions until irradiation unwound a helix linking LOV to Rac1. Photoactivatable Rac1 (PA-Rac1) could be reversibly and repeatedly activated using 458- or 473-nm light to generate precisely localized cell protrusions and ruffling. Localized Rac activation or inactivation was sufficient to produce cell motility and control the direction of cell movement. Myosin was involved in Rac control of directionality but not in Rac-induced protrusion, whereas PAK was required for Rac-induced protrusion. PA-Rac1 was used to elucidate Rac regulation of RhoA in cell motility. Rac and Rho coordinate cytoskeletal behaviours with seconds and submicrometre precision. Their mutual regulation remains controversial, with data indicating that Rac inhibits and/or activates Rho. Rac was shown to inhibit RhoA in mouse embryonic fibroblasts, with inhibition modulated at protrusions and ruffles. A PA-Rac crystal structure and modelling revealed LOV-Rac interactions that will facilitate extension of this photoactivation approach to other proteins.

Figure 1. Engineering and in vivo characterization of a photoactivatable Rac1 (PA-Rac1)

a, Cartoon representation of PA-Rac1 design. b, Pulldown of PA-Rac1 constructs with PAK in the dark. Truncations of LOV and Rac at their linkage point were tested: 539–547, in red = terminal amino acid of Jα; 2–4, in green = first residue of Rac1. 546−4 showed the strongest inhibition; PA-Rac1 = 546−4, Q61L/E91H/N92H; -C450A, light-insensitive mutant; -I539E, lit state mutant. Pulldown by constitutively active (Q61L) and dominant negative (T17N) mutants are included for comparison with PA-Rac1. c, Whole cell irradiation of a HeLa cell expressing PA-Rac1. (minutes after irradiation, DIC, short axis of box = 20 µm). d, Spatial control of Rac1 activity. A 20-µm circle (red) was irradiated every 60 seconds in serum-starved MEF cells. Solid line = cell border at time 0, dotted line = 10 minutes after initial light pulse. Little movement of the cell border was detected, except adjacent to the point of irradiation. The kymograph (taken using white line, 20 µm), shows the initial formation of ruffles after each pulse, followed by protrusion (arrowheads = irradiation pulses).

Figure 2. Localized activation or inactivation of PA-Rac1 induces myosin-dependent migration

a, Protrusion/retraction map after a single pulse of activating illumination. MEFs expressing PA-Rac1 (left) generated protrusions at the site of irradiation (red) and retraction at the opposite side of the cell (blue) (in all 50 cells studied). Irradiation of the dominant negativeT17N mutant of PA-Rac1 (right) produced retraction near the point of irradiation, with protrusion in area(s) other than the site of irradiation (in all 25 cells studied). b, Repeated activation of PA-Rac1 at the cell edge induces directional migration. (MEF, 2-minute intervals, avg. 0.8 microns/pulse, n = 6). c, Localized activation of PA-Rac1 in the presence of ML-7 (MLCK inhibitor, 1 µM), Blebbistatin (Myosin II ATPase inhibitor, 1 µM), or Y-27632 (ROCK inhibitor, 10 µM). Protrusions analyzed as in panel a. d, Effect of myosin or ROCK inhibition on the ability of Rac1 to specify the direction of movement. The cosine of the angle between two lines (from the irradiation spot to the cell centroid at time 0, from the centroid at time 0 to the centroid at the end of the experiment) indicated how much the cell deviates from the direction specified by local irradiation. (panels c and d, n > 25; means +/− 95% confidence intervals; throughout figure 3 irradiation at 458 nm, Ø = 10 µm).

Figure 3. Inhibition of RhoA by PA-Rac1

a, HeLa cells expressing RhoA biosensor and either PA-Rac1 or its C450M photo-inactive mutant, illuminated in a 10-µm circle with a single pulse of 473 nm light. Changes in the FRET efficiency (E_corr) of the RhoA biosensor, indicative of RhoA activation, are shown in pseudocolor and as plots of average FRET efficiency within the irradiated circle (blue) and a nearby circle (red). In the PA-Rac1 cells, the irradiated spot showed bleaching of the biosensor followed by a relatively constant level of reduced RhoA activity. The nearby spot showed no bleaching, but a gradual decrease in RhoA activity reaching the low level achieved in the irradiated spot (n = 3 cells). In the control cells (C450M), the biosensor returned to near initial activation readouts after bleaching, and no change was seen in the nearby spot (n =3 cells). b, RhoA activation in constitutive pseudopods, versus pseudopods induced by PA-Rac1 (473 nm, 20-µm circle shown, Supplemental Movie S15). The bar graph (c) shows the percent increase in biosensor FRET/CFP ratio in the region 1 micron from the cell edge versus the mean of the flat region at the left of the line scan. (means +/− 95% confidence intervals, 18 lines from 6 cells per bar).

Figure 4. Crystallization and structural modelling of PA-Rac1

a, Dark state crystal structure of PA-Rac1. Blue = LOV domain, red = Jα helix, and green = Rac1. b, Interacting residues at the LOV-Rac interface (arrow in panel a), including Trp56. c, Mutating Cdc42 to include the Trp involved in stabilizing the LOV2-Rac1 interaction substantially improved LOV inhibition of Cdc42. Lane 1, PA-Cdc42; linking LOV to Cdc42 using the same truncations that produced good inhibition for Rac does not inhibit Cdc42-PAK binding. Lane 2, PA-Cdc42-CRIB; covalently linking the CRIB domain of PAK to PA-Cdc42 blocks PAK binding. Lane 3, PA-Cdc42-F56W; introduction of the tryptophan substantially improves LOV inhibition of Cdc42 binding to PAK. Lane 4, lit state mutant of PA-Cdc42-F56W, showing that Cdc42 inhibition is sensitive to the lit/dark state of the LOV domain. Supplemental Movie S16 and Fig. S14 demonstrate the ability of PA-Cdc42-F56W to produce filopodia and protrusions in living cells.