Multi-monoubiquitylation controls VASP-mediated actin dynamics

Abstract

The actin cytoskeleton performs multiple cellular functions, and as such, actin polymerization must be tightly regulated. We previously demonstrated that reversible, non-degradative ubiquitylation regulates the function of the actin polymerase VASP in developing neurons. However, the underlying mechanism of how ubiquitylation impacts VASP activity was unknown. Here, we show that mimicking multi-monoubiquitylation of VASP at K240 and K286 negatively regulates VASP interactions with actin. Using in vitro biochemical assays, we demonstrate the reduced ability of multi-monoubiquitylated VASP to bind, bundle, and elongate actin filaments. However, multi-monoubiquitylated VASP maintained the ability to bind and protect barbed ends from capping protein. Finally, we demonstrate the electroporation of recombinant multi-monoubiquitylated VASP protein altered cell spreading morphology. Collectively, these results suggest a mechanism in which ubiquitylation controls VASP-mediated actin dynamics.

Keywords: Actin; Filopodia; Nondegradative; TRIM9; Ubiquitylation; VASP.

Fig. 1.

VASP is ubiquitylated at K240 and K286. (A) Western blot (IB) of ubiquitylated Myc–VASP. HEK293 cells were transfected with Myc–VASP and HA–ubiquitin, lysed and boiled in denaturing buffer before Myc immunoprecipitation (IP). Western blot is representative of two experimental repeats. (B) VASP domain architecture and proteomic detection of ubiquitylation sites on VASP by mass spectrometry. Purple residues were selected for follow-up study due to their abundance and replicability. Graph represents cumulative PSM counts from two experimental replicates. PSM, peptide-to-spectrum match. (C) Chemical ubiquitylation scheme, demonstrating the bismaleimidoethane-mediated crosslinking of ubiquitin G75C to cysteine-240 on VASP. (D) Coomassie-stained gel of purified, ubiquitylated VASP at indicated locations. Gel image is representative of two or three experimental replicates. (E) Mass photometry distributions of unmodified and ubiquitylated VASP. The estimated molecular mass was calculated with a Gaussian curve fit. These graphs show one representative trace for each construct (n=2).

Fig. 2.

Multi-monoubiquitylation of VASP impairs actin bundling and binding. (A) High-speed actin co-sedimentation assay utilizing 1 µM actin and various concentrations of VASP. (B) Coomassie gels of supernatant and pellet fractions following centrifugation. (C) Quantification of VASP localized to the pellet fraction, determined by densitometry of Coomassie gels. Data points represent the mean±s.e.m. from three independent experiments. *P<0.0500; **P<0.0100 (two-way ANOVA with a Geisser-Greenhouse correction and the Dunnett's multiple comparisons test; unmodified VASP versus mUb-K240/K286: 400 nM=0.0315, 600 nM=0.0466; unmodified VASP versus mUb-K240: 600 nM=0.0089). (D) Low-speed actin co-sedimentation assay utilizing 2 µM actin and various concentrations of VASP. (E) Coomassie gels of the pellet fraction following centrifugation. (F) Quantification of actin localized to the pellet fraction, determined by densitometry of Coomassie gels. Data points represent the mean±s.e.m. from three independent experiments. *P<0.0500 (mixed effects model with a Geisser–Greenhouse correction and the Dunnett's multiple comparisons test; unmodified VASP versus mmUb-K240/K286: 200 nM=0.06399, 400 nM=0.0399).

Fig. 3.

Monoubiquitylation and multi-monoubiquitylation of VASP impairs elongation of actin filaments. (A) Schematic of pyrene actin assays. (B) Fluorescent traces of 0.5 µM (20% pyrene labeled) actin monomer elongation from preformed actin seeds (0.5 µM) in the presence of 80 nM VASP. AU, arbitrary units. (C) The initial elongation rate from seeded pyrene elongation assays (calculated from the first 300 s of the assay) plotted against VASP concentration. A one-site binding curve was fit to each data set. The elongation rate of actin seeds alone was subtracted from each data point. Each data point represents the mean of 1–4 measurements collected across three independent experiments. Error bars represent standard deviation. (D) Polymerization of 1.5 µM actin (10% Alexa Fluor 488 labeled) visualized with TIRF microscopy for 10 min in the presence of 25 nM TAMRA–VASP. Inverted images in this panel only show actin at 0, 125 and 250 s of polymerization (full time-lapse imaging shown in Movie 1). (E) Quantification of actin filament elongation rates from the time-lapse images. n=23–32 filaments per condition from two or three experiments. Lines represent median elongation rate. Elongation rate was calculated assuming 375 subunits were added per µm. Statistical significance calculated with the Kruskal–Wallis test and Dunn's multiple comparison test. (F) Quantification of actin filament elongation rates at a range of VASP and mmUb-K240/K286 concentrations. n=17–36 filaments from two to four experiments for each data point. Lines represent median elongation rate. Indicated P-values were calculated with the Kruskal–Wallis test and Dunn's multiple comparison test.

Fig. 4.

Multi-monoubiquitylation of VASP does not change barbed end binding or anti-capping activity. (A) Schematic of pyrene actin assays in the presence of capping protein. (B) Fluorescent traces of 0.5 µM (20% pyrene labeled) actin monomer elongation from preformed actin seeds (0.5 µM) in the presence of 5 nM capping protein (CP) and 50 nM VASP. AU, arbitrary units. (C) The initial elongation rate from seeded pyrene elongation assays in the presence of 5 nM CP (calculated from the first 300 s of the assay) plotted against VASP concentration. A one-site binding curve was fit to each data set. The dotted line represents the average elongation rate of actin seeds alone. Each data point represents the mean of 3–5 measurements collected across four independent experiments. Error bars represent standard deviation. (D) TIRFM demonstrating localization of 1 nM TAMRA–VASP to the barbed end of a trailing actin filament (1.5 µM actin, 10% Alexa Fluor 488 labeled) in a fascin-mediated actin bundle (673 nM fascin). Arrows indicate VASP bound to the trailing barbed end. (E) The growth of the trailing actin filament and the dynamic localization of VASP to the barbed end in D was visualized with kymograph analysis. Arrows denote two processive VASP binding events at the barbed end of two-filament bundles. These highly processive binding events are rare, with the majority of events lasting <1 s. (F) Frequency distribution of VASP barbed end binding events in D and E. Only VASP-binding events at the trailing barbed end of a two-filament bundle were quantified. Graph insets demonstrate [1−cumulative frequency (C.F.)] versus dwell time fit with a one-phase decay curve. n=145 events (unmodified VASP) and 131 events (mUb-K240/K286) from two experiments. τ represents time constant; r², goodness of fit.

Fig. 5.

Multi-monoubiquitylation of VASP enhances actin ruffling during cell spreading. (A) Representative widefield images of fixed MV^D7 cells with filopodial spreading morphology. Cells were electroporated with TAMRA–VASP (or buffer control) and allowed to spread for 30 min on fibronectin. (B) Magnified views of areas highlighted in A showing VASP localization to the tips of filopodia. (C) Classification of cell spreading into smooth-edged, filopodial or ruffled phenotypes. The plot indicates mean±s.d. percentage of each classification across three experiments. A Chi-square test for the goodness of fit was used to compare outcomes with a Bonferroni correction. 92–104 cells were classified across the experiments.

Fig. 6.

Multi-monoubiquitylation of VASP reduces enrichment at both filopodial tips and lamellipodia. (A) Representative maximum projection images of fixed MV^D7 cells stably expressing GFP–VASP and electroporated with TAMRA–VASP. (B) Quantification of VASP enrichment at focal adhesions (FA) normalized to cytoplasmic intensity. Line represents the median VASP enrichment at focal adhesions. The enrichment of TAMRA–VASP or TAMRA–mmUb VASP at focal adhesions was then normalized to the GFP–VASP localization in each cell. Line represents the mean enrichment of TAMRA-labeled protein normalized to GFP–VASP. Statistics were calculated with a one-way ANOVA with Sidak's multiple comparisons or a paired two-tailed t-test, respectively. n=32 (unmodified) and 31 (mmUb-K240/K286 VASP) cells across three experiments for all focal adhesion measurements. (C) Raw intensity values of average filopodial tip intensity from linescans. The filopodial tip was defined as the first four pixels (0.255 µm) in a linescan drawn from tip to base. Each large data point represents the average from one independent experiment with smaller data points representing individual filopodia. The line represents the mean of three independent experiments. Statistics were calculated with a paired two-tailed t-test. n=34 (unmodified) and 35 (mmUb-K240/K286 VASP) cells across three experiments for all filopodia measurements. (D) Heatmap localization of proteins from throughout the length of the filopodia. The fluorescence intensity of each protein with a filopodium was normalized and the length binned. (E) Quantification of normalized VASP intensity at the filopodial tip (first three bins). Each data point represents the mean of one independent experiment and the bar graph extends to the mean of three experiments. Statistics were calculated with an unpaired two-tailed t-test. (F) Heatmap localization of proteins at the lamellipodia. Line scans were drawn from the cell periphery and extended approximately 1.5 µm into the cell. The fluorescence intensity of each linescan was normalized and the length binned. (G) Quantification of normalized VASP intensity at the lamellipodia (first three bins). Each data point represents the mean of one independent experiment and the bar graph extends to the mean of three experiments. Lamellipodia enrichment was calculated as the intensity at the first three bins normalized to the intensity and the subsequent fourteen bins. Statistics were calculated with a paired two-tailed t-test. n=31 (unmodified) and 33 (mmUb-K240/K286 VASP) cells across three experiments for all lamellipodia measurements.