Profilin promotes lamellipodium protrusion by tuning the antagonistic activities of capping protein and VASP

Figures

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  Fig. 1. Generation and initial characterization of profilin 1 knockout (Pfn1-KO) cell lines. A Western blotting of B16-F1 cell lines before (B16) and after clonal isolation of cells treated with a Pfn1-specific guide construct, using two independent antibodies specific for the N- and C-terminus of profilin and GAPDH as loading control. B Summary of the sequencing results of the genomic locus encoding the Pfn1 gene in each cell line, as indicated. No WT or in-frame deletion was detected in at least 32 independent sequencing reactions per cell line. C Representative Western blot of whole-cell extracts of Pfn1-KO clones and B16-F1 cells next to a serial dilution of recombinant human profilin 2 (Pfn2). GAPDH was used to normalize for unequal sample loading. D Example standard curve that relates the immunoblot signal to the known quantity of recombinant Pfn2. The line is a linear fit to the data. E Calculated cellular concentrations of Pfn2 for WT (B16) and Pfn1-KO clones. Data from three independent experiments (pink, small symbols) were averaged to obtain mean (large, black symbols) concentrations. Error bars are standard deviations of means (SD). F Comparison of actin and tubulin…

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  Fig. 2. Characterization and quantification of lamellipodial features in Pfn1-KO cell lines. A Representative wildtype and Pfn1-deficient cells (derived from clones as indicated) growing on laminin and fixed and stained for the actin cytoskeleton using fluorescently-coupled phalloidin. The rhomb-shaped area in red marks an example region used to measure the lamellipodial actin network intensity as shown in B. Areas lacking microspike bundles were measured, as these were analyzed separately. For obtaining the data displayed in B, each area in red was subtracted by a close-by area outside the cell (yellow rhomb) representing extracellular background fluorescence. Scale bar in top left is valid for all panels: 10 µm. B Quantitation of lamellipodial actin filament (F-actin) intensities in cell lines as indicated and measured as illustrated in subpanel (A). The arithmetic means of fluorescence background-subtracted B16-F1 wildtype (B16) data were normalized to 1 for each of three independent experiments, and shown as so called superplots81, displaying measurements from individual cells (see “Methods”) and their arithmetic means as color-coded dots and triangles, respectively. Gray lines and…

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  Fig. 3. Consequences of profilin loss of function on lamellipodial accumulation of key actin assembly regulators. A Representative images of B16-F1 melanoma cells seeded on laminin and stained for Arp2/3 complex (ArpC5, blue), capping protein (CP, green), Ena/VASP family members (VASP and Mena, pink), lamellipodin (Lpd, yellow). White squares depict insets displayed as magnified and inverted images at top left corners of each individual panel. Note respective changes in lamellipodial intensities that can be appreciated as black lines in inverted insets for each lamellipodial regulator. B–F Quantitations of lamellipodial fluorescence intensities of respective components, color-coded as described in (A) and displayed as superplots with independent experiments shown in distinct shadings of respective colors. Gray lines and error bars display means and standard errors of means (SEMs). Intensities measured of all components were normalized to the arithmetic mean of the WT population for each individual experiment (colored rectangles), n = measured cells from at least three independent experiments (four in C). For all superplot data shown in (B–F), arithmetic means of replicates from each…

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  Fig. 4. Profilin 1 removal in Ena/VASP-deficient cells compromises lamellipodia protrusion with simultaneous, differential impact on distinct core actin machinery components. A Western blotting of WT (B16-F1) as control and Ena/VASP-deficient cells before (EVM-KO) and after clonal isolation of cells treated with a Pfn1-specific guide construct. Quadruple-KO (EVM+Pfn1-KO) clones #10, #19 and #27 were selected for further analysis, and confirmed for the absence of Pfn-1 using antibodies raised against the N- or C-terminus or full-length protein. Confirmatory VASP staining was employed for all lines and HSP70 as loading control. B Representative cell images of the actin cytoskeleton of B16-F1, EVM-KO and EVM+Pfn1-KO cell lines, as indicated. Note the incremental decrease of lamellipodial dimension (width) from WT to EVM-KO and EVM+Pfn1-KOs. The scale bar in the B16-F1 panel applies to all subpanels and equals 10 µm. C Quantitation of lamellipodial F-actin intensities for the cell lines, shown in (A, B). Data displayed as superplots as described for Fig. 2B. D Quantitation of lamellipodial widths in distinct cell lines, data as in Fig. 2C. Gray lines and error bars in (C, D) display means…

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  Fig. 5. Suppression of profilin expression in the absence of lamellipodin generates intermediate phenotypes for VASP up- and CP downregulation in the lamellipodium. A Western blot displaying Pfn1-staining in cell extracts from lamellipodin (Lpd)-deficient lines isolated upon additional CRISPR/Cas9 targeting of the Pfn1 gene, termed Lpd+Pfn1 double knockout (Lpd+Pfn1-DKO), as compared to B16-F1 control. Note that Pfn1 expression was reduced, but is still detectable in most (five out of six) DKO-clones. DKO cell lines thus constituted Lpd-deficient cells with very low Pfn1 expression. B Immunolabeling using VASP-specific antibodies in B16-F1 wildtype (B16), Lpd-KO (clone #8), Pfn1-KO (clone #20) and Lpd1+Pfn1-DKO clones, as indicated (for clone numbers see right bottom corners). White squares depict insets displayed as magnified and inverted images at top left corners of each individual panel. Scale bar: 10 µm. C Quantitation of lamellipodial VASP intensities performed in a fashion identical to Fig. 3, but for cell lines as indicated. D Comparative stainings for the actin cytoskeleton (phalloidin), Arp2/3 complex (ArpC5, blue) and capping protein (CP, green) in B16-F1 wildtype (B16),…

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  Fig. 6. Rescue of filopodial and lamellipodial actin assembly phenotypes by transient transfection with mPfn1. A Numbers of filopodia-like structures formed in live Pfn1-KO #20 cells without and with transient co-expression of constitutively active mDia2 (mDia2-ΔDAD) and Halo-tagged, murine Pfn1 (Halo-mPfn1) wildtype (WT) or polyproline (Y6D)- or actin binding (R88E)-deficient variants. Data were quantified from movies of 47 to 82 cells, as indicated. Gray lines and error bars are means and standard errors of means (SEMs), respectively. For statistics, all datasets were compared with each other using ANOVA with Tukey’s post hoc test; n.s., not significant, *p ≤ 0.05. B Width of club-shaped, individual, mDia2-induced filopodia known to correlate with the extent of actin filament packing in these structures61,82 plotted against their number per cell for each aforementioned, experimental condition, as indicated (color-code). C Representative images of fields of Pfn1-KO #20 cells with (orange squares) or without (green squares) expression of mPfn1 variants (see Halo-tag channel), as indicated, and analyzed for the lamellipodial accumulation of ArpC5 (blue), VASP (pink) or actin filaments…

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  Fig. 7. Essential assumptions defining the mathematical model and modeling results. A Summary of assumed consequences on Arp2/3-dependent actin networks of profilin suppression (Pfn1-KO). Molecules are not drawn to scale, and Pfn1 exchange activity (ADP to ATP-actin) is symbolized by binding to both actin monomers. Note reduced actin monomer, polymer as well as branch concentrations in Pfn1-KO. B Different stages in the lifecycle of Ena/VASP family members as assumed in the model. Individual, functional units of cytosolic VASP (or Evl or Mena; tetramerization not indicated) can go through distinct stages, with elongation being followed by dissociation of barbed end-bound VASP as dominant process in the model (black arrow pointing to the right), but other forms of dissociation are also possible (black arrows to the left). Note that the model does not specify molecular mechanisms of Ena/VASP recruitment14 C Summary of assumptions on CP dynamics. Note the absence of a filament elongation phase and the immediate removal from the membrane of the barbed end as direct consequence of capture by CP. D In the model, Ena/VASP and CP experience two separable types of competition, (i) for barbed ends…