Distinct Interaction Sites of Rac GTPase with WAVE Regulatory Complex Have Non-redundant Functions in Vivo

Abstract

Cell migration often involves the formation of sheet-like lamellipodia generated by branched actin filaments. The branches are initiated when Arp2/3 complex [1] is activated by WAVE regulatory complex (WRC) downstream of small GTPases of the Rac family [2]. Recent structural studies defined two independent Rac binding sites on WRC within the Sra-1/PIR121 subunit of the pentameric WRC [3, 4], but the functions of these sites in vivo have remained unknown. Here we dissect the mechanism of WRC activation and the in vivo relevance of distinct Rac binding sites on Sra-1, using CRISPR/Cas9-mediated gene disruption of Sra-1 and its paralog PIR121 in murine B16-F1 cells combined with Sra-1 mutant rescue. We show that the A site, positioned adjacent to the binding region of WAVE-WCA mediating actin and Arp2/3 complex binding, is the main site for allosteric activation of WRC. In contrast, the D site toward the C terminus is dispensable for WRC activation but required for optimal lamellipodium morphology and function. These results were confirmed in evolutionarily distant Dictyostelium cells. Moreover, the phenotype seen in D site mutants was recapitulated in Rac1 E31 and F37 mutants; we conclude these residues are important for Rac-D site interaction. Finally, constitutively activated WRC was able to induce lamellipodia even after both Rac interaction sites were lost, showing that Rac interaction is not essential for membrane recruitment. Our data establish that physical interaction with Rac is required for WRC activation, in particular through the A site, but is not mandatory for WRC accumulation in the lamellipodium.

Keywords: Arp2/3 complex; CRISPR/CAS9; Rho-GTPase; filopodium; lamellipodium; migration; protrusion.

Graphical abstract

Figure 1

Contribution of Distinct Rac Binding Sites in Sra-1 to Lamellipodia Formation (A) Cell morphologies and lamellipodial phenotypes of B16-F1 control versus Sra-1/PIR121 KO cells (clone 3) transfected with EGFP or EGFP-tagged Sra-1, and stained for the actin cytoskeleton with phalloidin (scale bars, 20 μm). (B) Cell lysates of B16-F1 cells, Sra-1/PIR121 KO cells (clone 3), as well as KO cells expressing EGFP-Sra-1 were subjected to western blotting to detect expression levels of WAVE complex components, as indicated. (C) B16-F1 control cells, Sra-1/PIR121 KO clone 3, and the latter forming lamellipodia upon transfection with EGFP-tagged Sra-1 were analyzed for random migration speed (^∗∗∗p ≤ 0.001; n.s. [not significant]: p > 0.05). Box and whisker plots represent data as follows: boxes correspond to 50% of data points (25%–75%), and whiskers correspond to 80% (10%–90%). Outliers are shown as dots, and lines and red numbers in boxes correspond to medians. (D) Crystal structure of the WAVE complex (PDB: 3P8C [4]). From the view chosen, only WAVE (magenta), Sra-1 (green), and Nap1 (blue) are visible. Sra-1 possesses two binding sites for Rac (termed A site and D site) and sequesters the WH2 and C regions of WAVE. Rac binding to Sra-1 is thought to release interactions with the WH2 and C regions, thereby activating the WCA domain of WAVE. (E) Sra-1/PIR121 KO cells (clone 3) were transfected with EGFP or various EGFP-Sra-1 constructs, lysed, and subjected to pull-downs with constitutively active Rac1 (Rac1-L61). Note strongly increased interaction of the WCA^∗ mutant with Rac1, which was strongly and virtually entirely diminished upon additional mutation of the A and D site, respectively. Combinatorial mutation of both Rac binding sites in the WCA^∗ background appeared to abolish detectable Rac1 interaction entirely. WCA^∗: disrupted WH2 and C region contact sites (L697D/Y704D/L841A/F844A/W845A). (F) Sra-1/PIR121 KO cells (clone 3) were transfected with the indicated EGFP-Sra-1 constructs and assayed for lamellipodia formation. Lamellipodial actin networks that were small, narrow, or displayed multiple ruffles were defined as “immature lamellipodia,” marked by arrowheads in cell images (right), as opposed to regular lamellipodia, marked by arrows (scale bar, 10 μm). Data in the bar chart are arithmetic means ± SEM from three independent experiments. Note that the A site mutation diminished lamellipodia formation in a fashion that could be restored by additional WCA^∗ mutation of Sra-1. In the case of the D site, lamellipodial morphology was compromised in a fashion mostly independent from the WCA^∗ mutation. The WIRS mutation had no detectable effect. To assess statistical significance of differences or confirm the absence of statistically relevant differences between experimental groups, a non-parametric, Mann-Whitney rank-sum test was performed in multiple, individual combinations of datasets. For each experimental group, we compared the number of cells with regular, i.e., “fully developed” lamellipodia, immature lamellipodia (see above), or the two groups combined, and hence all cells display either one of the lamellipodium-like structures. Selected combinations are as follows, with three p values representing aforementioned lamellipodial categories: WT-WIRS (n.s., n.s., n.s.); WT-C179R/R190D+WCA^∗ (n.s., n.s., n.s.); WT-Y967A (^∗∗, ^∗∗, n.s.); WT-G971W (^∗∗, ^∗, n.s.); WT-Y967A+WCA^∗ (^∗∗, ^∗∗, n.s.); Y967A-Y967A+WCA^∗ (^∗, n.s., n.s.); WT-WCA^∗ (n.s., n.s., ^∗∗). Statistical significance is expressed as ^∗∗p ≤ 0.01, ^∗p ≤ 0.05, and n.s. (not significant): p > 0.05. WIRS: Y923A/E1084A to mutate the WIRS-binding pocket; WCA^∗: disrupted WH2 and C region contact sites (L697D/Y704D/L841A/F844A/W845A). See also Figure S1 and Video S1.

Figure 2

Functional Comparison of Rac Binding Sites in Mouse and Dictyostelium Cells (A) Subcellular localization of Scar/WAVE and Arp2/3 complexes. Wild-type and mutated Pir121-EGFP were expressed in PirA knockouts and visualized by confocal microscopy while migrating under agarose up a folate gradient. Top: wild-type Pir121-EGFP; middle: A site mutant (K193D/R194D); bottom: D site mutant (Y961A). Arp2/3 complex (red) is recruited to sites of WT Scar/WAVE complex (green) localization, but not upon expression of either PIR121 mutant. The D site mutant allows its recruitment to pseudopods, but there is no detectable Arp2/3 complex activation. (B) Lifetimes of Scar/WAVE patches. Cells expressing PIR121-EGFP were allowed to migrate up folate gradients under agarose, and areas of local EGFP enrichment were observed by confocal microscopy. A site mutants showed no patches. Graph shows means ± SD; n > 25 cells; ^∗∗∗p ≤ 0.001, non-parametric t test, Mann-Whitney. (C) Frequency of Scar/WAVE patch generation. Cells were measured as in (B), but the rate of formation of patches was quantitated. Graph shows means ± SD; n > 25 cells; ^∗∗∗p ≤ 0.001, non-parametric t test, Mann-Whitney. (D) Reduced migration speed of cells expressing both A and D site mutants. The chemotactic speeds of cells expressing WT and mutant PIR121 were measured from the speed of cells allowed to migrate up folate gradients under agarose, and observed by differential interference contrast (DIC) microscopy. Data show means ± SD; n > 25 cells on 3 days; ^∗∗∗p ≤ 0.001, one-way ANOVA, Dunnett’s multiple-comparison test. (E) Random migration assay with B16-F1 Sra-1/PIR121 KO cells (clone 3) re-expressing the indicated Sra-1 variants, and analyzed as described in STAR Methods; cells with and without lamellipodia are displayed separately. n of cells analyzed was ≥130 per condition and specifically indicated for cells harboring lamellipodia. Lamellipodia-forming cells always migrated faster than respective controls, i.e., those cells lacking lamellipodia in each condition. Statistical significance is expressed as ^∗∗∗p ≤ 0.001, ^∗∗p ≤ 0.01, and ^∗p ≤ 0.05; n.s. (not significant): p > 0.05. (F and G) FRAP analysis of EGFP-Sra-1 variants expressed in Sra-1/PIR121 KO B16-F1 cells (clone 3). Data are arithmetic means with SEM of fluorescence intensities at acquired time points after bleaching, with intensities before each bleach individually normalized to 1. “n” equals the number of individual FRAP videos analyzed for each component (F). Half-times of recovery for each component were derived from curve fits (not shown) generated as described in STAR Methods. In (G), fitted data curves are displayed for comparison upon normalization of bleaching time point (0 s) to 0 and fluorescence recovery asymptote to 1. Constructs used were: WCA^∗: disrupted WH2 and C region contact sites (L697D/Y704D/L841A/F844A/W845A); A site: C179R/R190D; D site: Y967A. See also Figures S2 and S3 and Videos S2 and S4.

Figure 3

The D Site in Sra-1 Is Needed for Proper Lamellipodia Formation (A) Representative Sra-1/PIR121 KO cells expressing different EGFP-Sra-1 variants, as indicated, and stained with phalloidin (scale bar, 10 μm). (B) Quantification of lamellipodial width measurements. (C) Quantification of filopodia formed anterior to lamellipodia. (D) Representative kymographs of lamellipodial protrusion, induced by the indicated EGFP-Sra-1 variants. (E) Quantification of lamellipodial protrusion velocity mediated upon expression of the respective Sra-1 variants. (F) Representative Sra-1/PIR121 KO cells expressing different EGFP-Sra-1 variants and stained for the Arp2/3 complex subunit ArpC5A (scale bar, 10 μm). (G) Quantification of ArpC5A intensity at the lamellipodium. For quantifications in (B), (C), (E), and (G), data are displayed as described for Figure 1C, n = number of cells analyzed, and statistical significance is expressed as ^∗∗∗p ≤ 0.001 and ^∗∗p ≤ 0.01; n.s. (not significant): p > 0.05. See also Figure S3 and Video S3.

Figure 4

Further D Site-Associated Phenotypes and Analysis of the D Site Interaction Surface on Rac (A and B) Determination of lamellipodial actin assembly rates. Sra-1/PIR121 KO cells (clone 3) were co-transfected with mCherry-Sra-1 variants (WT, D site mutant, and D site+WCA^∗, as indicated) and EGFP-actin. Lamellipodial actin assembly rates were determined by photobleaching EGFP-actin within lamellipodial regions, and reading out network assembly rates as the sum of actin rearward flow during the fluorescence recovery period and simultaneous forward protrusion (A). Representative frames before and after bleaching (at 0 s) are shown in (B). Scale bar, 5 μm. (C) Quantification of F-actin intensity levels in the lamellipodium obtained from phalloidin stainings. Data and results of statistical analyses in (A) and (C) are displayed as described for quantifications in Figure 3. (D) Rac1 docking onto the D site of Sra-1 based on the cryo-EM structure of Rac1 occupying the D site [3]. Amino acids mutated in this study (i.e., E31 and F37 in Rac1, as well as Y967 and G971 in Sra-1) are shown as sticks, except for G971, the position of which in the green helix is shown in red. Additional amino acids interacting with either E31/F37 of Rac1 or Y967 of Sra-1 are shown as lines. (E) RAC1/2/3 genes were disrupted in B16-F1 cells using CRISPR/Cas9, and derived knockout clones were assayed for Rac expression. Antibodies employed are capable of detecting Rac1 or 3 and Rac2, respectively (see [25]). All lines including B16-F1 wild-type lack expression of hematopoietic Rac2, except for RAW macrophages (MΦ) used as control, and all KO clones except for clone 2 lack full-length Rac1/3 protein. (F) Representative Rac1/2/3 KO clone (clone 1) and B16-F1 control cells plated on laminin-coated coverslips for analysis of the actin cytoskeleton by phalloidin staining. Note the complete absence of lamellipodia or lamellipodia-like structures upon elimination of Rac expression. Scale bar, 20 μm. (G) Rac1/2/3 KO clone 1 was transfected with EGFP-tagged Rac1-L61 constructs as indicated, and plated on laminin-coated coverslips for analysis of cell morphology. Representative cells are shown. Note that mutation of E31 or F37 residues in Rac1-L61 resulted in the induction of compromised lamellipodia, highly reminiscent in phenotype of those observed upon expression of D site-mutated Sra-1 in Sra-1/PIR121 double KO cells. Scale bar, 10 μm.