Induced Arp2/3 Complex Depletion Increases FMNL2/3 Formin Expression and Filopodia Formation

Abstract

The Arp2/3 complex generates branched actin filament networks operating in cell edge protrusion and vesicle trafficking. Here we employ a conditional knockout mouse model permitting tissue- or cell-type specific deletion of the murine Actr3 gene (encoding Arp3). A functional Actr3 gene appeared essential for fibroblast viability and growth. Thus, we developed cell lines for exploring the consequences of acute, tamoxifen-induced Actr3 deletion causing near-complete loss of functional Arp2/3 complex expression as well as abolished lamellipodia formation and membrane ruffling, as expected. Interestingly, Arp3-depleted cells displayed enhanced rather than reduced cell spreading, employing numerous filopodia, and showed little defects in the rates of random cell migration. However, both exploration of new space by individual cells and collective migration were clearly compromised by the incapability to efficiently maintain directionality of migration, while the principal ability to chemotax was only moderately affected. Examination of actin remodeling at the cell periphery revealed reduced actin turnover rates in Arp2/3-deficient cells, clearly deviating from previous sequestration approaches. Most surprisingly, induced removal of Arp2/3 complexes reproducibly increased FMNL formin expression, which correlated with the explosive induction of filopodia formation. Our results thus highlight both direct and indirect effects of acute Arp2/3 complex removal on actin cytoskeleton regulation.

Keywords: F-actin branching; F-actin turnover; cell division; chemotaxis; filopodium; lamellipodium; migration; nuclear envelope breakdown.

Figure 1

Tamoxifen treatment suppresses expression of Arp2/3 complex subunits. (A,B) Western blot analysis of Arp2/3 complex subunit levels in individual Actr3^fl/fl clones after tamoxifen treatment (72, 96, or 120 h) or DMSO/EtOH used as vehicle control. GAPDH and vinculin served as loading controls, dependent on the molecular weight of explored Arp2/3 complex subunit. (A) Although the extent of Arp3 protein decrease correlated with prolonged tamoxifen treatment, a treatment of 96 h (red box) was determined as best compromise between efficient protein run down and cell viability, which was significantly compromised upon 120 h (not shown). The 96 h-time point was thus used for all future experiments. (B) Tamoxifen-induced removal of Arp3 (96 h) concomitantly reduced all additional Arp2/3 complex subunits tested, as indicated.

Figure 2

Arp3-depleted MEFs are devoid of lamellipodia. (A) Phalloidin-stained examples of distinct Actr3^fl/fl clones (Arp3.5, Arp3.7, and Arp3.19) stably expressing tamoxifen-inducible Cre recombinase, with (tamoxifen) or without (DMSO/EtOH) tamoxifen induction (96 h) causing disruption of the Actr3 gene. Tamoxifen treatment causes near to complete elimination of lamellipodia in representative cells shown. Scale bars as indicated in individual panels. Note the strong increase of spread cell area upon Arp3 disruption. (B) Quantification of lamellipodia formation efficiency upon treatments as shown in A. Data are arithmetic means and standard errors of means from three independent experiments; n = total number of cells analyzed; data were statistically compared using two-sided, two-sample t-test (***p < 0.001).

Figure 3

Loss of the Arp2/3 complex interferes with treadmilling and F-actin turnover at the cell periphery. (A) Time-lapse images of representative FRAP experiments in Actr3^fl/fl cells with or without tamoxifen treatment (clone Arp3.19) expressing EGFP-actin. Regions of interest (ROI) are marked with white rectangles in overview images on the left. Different time points of enlarged ROI on right-hand panels display fluorescence signals of EGFP-actin immediately pre and after bleach as well as at different time points of fluorescence recovery. White polygons highlight photobleached regions at the cell periphery. Changes in signal intensities over time for calculations of treadmilling factors (TMF) were quantified within a region of 0.5 μm at the front part (blue rectangle) as well as back part (red rectangle) of the lamellipodium in the Actr3^fl/fl cell or the corresponding periphery of the cell following Arp3 depletion. (B) Average fitted recovery curves of front (blue) and back (red) regions of Arp3-expressing (left) and Arp3-depleted cells (right) used to calculate the TMF utilizing the illustrated equation. Data were collected from 9 control and 11 tamoxifen-treated cells acquired in three independent experimental days. The treadmilling factor is defined as average difference in fluorescence recovery in the distal vs. proximal half of the lamellipodium. Note that this difference is absent in tamoxifen-treated cells, demonstrating that presence of a functional Arp2/3 complex is essential for bona fide treadmilling at the cell periphery. (C) EGFP-actin FRAP recovery curves within a 1 μm deep peripheral region of DMSO/EtOH (blue) vs. tamoxifen-treated (red) Arp3.19 cells. Analysis was performed on the same time-lapse movies as used for the analysis in (B); data are arithmetic means and SEMs from movies the pre-bleach intensities of which were normalized to 1. Right panel shows fitted curves derived from raw data depicted on the left. Bar chart displays half times of fluorescent recovery as calculated from fitted curves. Note the marked decrease of F-actin turnover (increase of half-time of EGFP-actin fluorescence recovery) upon Arp3 depletion.

Figure 4

The Arp2/3 complex is not required for cell spreading. (A) Phalloidin stainings of Actr3^fl/fl cells (clone Arp3.19) with or without tamoxifen treatment (96 h) and subjected to cell spreading for different time points (0, 15, 60 min, and 24 h). Except for the 0-min time point, for which poly-L-lysine was used (see Methods), cells were allowed to spread on 25 μg/ml fibronectin. (B) Box and whiskers plots displaying quantification of spreading area using images as shown in (A). Boxes include 50% (25–75%) and whiskers 80% (10–90%) of all measurements. Outliers are shown as dots. Median values are given in red. n = total number of cells analyzed from three independent experiments. Differences in average cell area of control and tamoxifen-treated cells at different time points of spreading were confirmed to be statistically significant using non-parametric Mann-Whitney rank sum test (***p < 0.001). (C) Spreading kinetics of Actr3^fl/fl (EtOH/DMSO, blue) and tamoxifen-treated cells (red) reported as fold change after normalization to cell size at time point 0. Data taken from (B). Data are arithmetic means and error bars represent SEMs. Note that Arp3 knockout cells spread with kinetics highly similar to corresponding control cells, but adopt a much larger area 24 h after seeding, due perhaps to continuous increase in cell size effected by Arp3 removal.

Figure 5

Acute Arp2/3 complex removal reduces wound closure capacity. (A) Selected frames from wound healing, time-lapse microscopy (24 h) of control- and tamoxifen-treated Actr3^fl/fl clones. Wounds closed on average after roughly 18 h in controls, but failed to do so in Arp3 null cells. (B) Quantification of collective migration velocity during wound closure utilizing movies as displayed in (A). Bar charts show arithmetic means and SEMs. n = number of movies analyzed, generated from three independent experiments. Differences in wound closure speed between individual control- and tamoxifen-treated clones were confirmed to be statistically significant using two-sided two-sample t-test (***p < 0.001).

Figure 6

Arp2/3 complex depletion has little effect on the speed of random cell migration, but diminishes efficiency by compromising migration directionality. (A) Random migration speed of Actr3^fl/fl clones (Arp3.5, Arp3.7, and Arp3.19, as indicated) with or without treatment with tamoxifen causing acute gene disruption. Data obtained from time-lapse microscopy (20 h) followed by manual tracking of individual cells (for individual cell tracks see Supplementary Figure 9). Box and whiskers plots were as described for Figure 4B, and data derived from three independent experiments. n = total number of cells analyzed. Non-parametric Mann-Whitney rank sum test was used for statistical comparison of respective groups (***p < 0.001). (B) Mean square displacement analysis of the same data as shown in (A), revealing the strong decrease in migration efficiency upon Arp3 depletion in spite of the limited impact on speed. (C) Analysis of directional migration utilizing trajectories from (A) and shown in Supplementary Figure 9. An autocorrelation of 1 corresponds to strictly directed movement whereas an autocorrelation of 0 corresponds to pure random walk. Note the rapid and consistent loss of directional migration upon Arp2/3 complex removal (tamoxifen) in these experimental conditions in all three cell clones. Data are displayed as direction autocorrelation curves with error bars representing standard errors of means.

Figure 7

Arp2/3 complex contributes to, but is not essential for chemotaxis. (A) Trajectory plots of control (DMSO/EtOH)- and tamoxifen-treated MEFs (Actr3^fl/fl clone Arp3.19) migrating toward a gradient of 2.5% serum and 100 ng/ml HGF. Data obtained from time-lapse microscopy (20 h) followed by manual tracking of cells in three independent experiments. (B) Rose plots with each 10° segment showing the frequency of migratory tracks in that particular direction. Note that both DMSO/EtOH- and tamoxifen-treated fibroblasts migrate toward the given gradient with increased frequencies. (C) Forward migration index (FMI) obtained from the two experimental conditions. The FMI is defined as displacement of the cell in the direction of the gradient divided by the total distance migrated. Again, Arp3-depleted fibroblasts display a slightly impaired directional persistence. (D) Quantification of rates of chemotactic migration. Velocity of chemotactic migration of Arp3-depleted cells is reduced to an extent similar to that observed in directed, wound healing migration assays. Box and whiskers plots in (C,D) were as described for Figure 4B. Statistics: Non-parametric Mann-Whitney rank sum test (***p < 0.001).

Figure 8

Arp2/3 complex removal alters focal adhesion patterns. (A) Representative images of fibroblasts (clone Arp3.19) with or without tamoxifen treatment and stained with anti-vinculin antibodies. A binarized threshold was applied to these stainings in order to facilitate automated analysis of adhesion numbers and sizes within distinct regions of interest (yellow outlines, defined manually). Note the exclusion of the cell center and perinuclear area due to the lack of staining resolution in these areas. (B) Calculation of adhesion number/cell, (C) adhesion number/cell area and (D) adhesion size were all based on data obtained from three independent experiments as described in (A). Box and whiskers plots were as described for Figure 4B. n = total number of cells analyzed. Statistics were performed utilizing the non-parametric Mann-Whitney rank sum test (***p < 0.001). Note that Arp3-deficient cells display more adhesions that are also bigger in size than Arp3-expressing controls. However, the adhesion number per cell area is reduced upon acute Arp2/3 complex removal as compared to control cells.

Figure 9

Acute Arp3 removal up-regulates FMNL formin expression enhancing filopodia formation and cell spreading. (A) Representative Western blot of Arp3 as well as FMNL2 and FMNL3 protein levels in Actr3^fl/fl clones with and without tamoxifen treatment, as indicated. GAPDH served as loading control. (B) Quantification of FMNL2 and FMNL3 protein levels from Western blots as shown in (A). Bar charts display arithmetic means of FMNL2 and−3 protein levels normalized to GAPDH, with each FMNL variant (shades of red) being presented as fold-change relative to its respective control (blue). Error bars represent SEMs; data obtained from at least five independently generated cell extracts. Statistics were performed utilizing non-parametric Mann-Whitney rank sum test (***p < 0.001; ** < 0.01). Note the significant elevation of FMNL2 and−3 protein levels upon Arp3 suppression in all three clones. (C) Representative Western blot analysis of FMNL2 and FMNL3 protein amounts in detergent-soluble extracts from DMSO/EtOH- and tamoxifen-treated Actr3^fl/fl cells (clone Arp3.19) after co-transfection with RNAi plasmids individually downregulating FMNL2 and FMNL3 expression or mock-plasmid as control, as indicated. Note the moderate but consistent reduction of FMNL2 and−3 protein levels with and without tamoxifen-induced Arp3 depletion, as indicated (for quantification results see main text). (D) Representative phalloidin stainings of Actr3^fl/fl cells (clone Arp3.19) control or tamoxifen-treated, and combined with mock- or FMNL2/3-RNAi after 15 min of spreading on fibronectin-coated coverslips. (E,F) Quantification of filopodia formation (E) and spreading area (F) of cells as presented in (D); box and whiskers plots as described for Figure 4B. n = total number of cells analyzed from three independent experiments. Statistics were performed utilizing non-parametric, Mann-Whitney rank sum test (***p < 0.001; ** < 0.01; n.s. not significant). Note that Arp3 knockout is accompanied by a pronounced increase in filopodia numbers that are reduced even below Arp3-treatment control levels upon FMNL2/3 RNAi. In addition, the increase in tamoxifen-induced cell area upon 15 min of spreading is also suppressed significantly by FMNL2/3 knockdown.

Figure 10

Arp3 depletion causes an increase of centrosome and nucleus numbers as well as nuclear deformation. (A,B) Representative fluorescence microscopy images of Actr3^fl/fl cells (clone Arp3.19) stained for the F-actin cytoskeleton with phalloidin (a), nuclei using DAPI (b) and γ-tubulin for centrosomes (c). Merged images (d) display actin filaments in red, nuclei in blue and centrosomes in green. White rectangles in (d) mark regions of interest enlarged in (e) displaying centrosomes, the quantification of which is illustrated in (f). (C,D) Box and whiskers plots (as described for Figure 4B) displaying numbers of centrosomes (C) or of nuclei (D). For statistics, non-parametric, Mann-Whitney rank sum tests were used (***p < 0.001). (E) Analysis of nuclear morphologies. Cells were categorized according to the morphological integrity of their nuclei (intact nuclei in gray; deformed nuclei in green), represented as a fraction of all cells analyzed in individual cell populations. Results are depicted as stacked columns representing arithmetic means and SEMs from three independent experiments; non-parametric, Mann-Whitney rank sum test for statistics (***p < 0.001), n = total number of cells analyzed in (C–E).