Ena/VASP proteins enhance actin polymerization in the presence of barbed end capping proteins

Abstract

Ena/VASP proteins influence the organization of actin filament networks within lamellipodia and filopodia of migrating cells and in actin comet tails. The molecular mechanisms by which Ena/VASP proteins control actin dynamics are unknown. We investigated how Ena/VASP proteins regulate actin polymerization at actin filament barbed ends in vitro in the presence and absence of barbed end capping proteins. Recombinant His-tagged VASP increased the rate of actin polymerization in the presence of the barbed end cappers, heterodimeric capping protein (CP), CapG, and gelsolin-actin complex. Profilin enhanced the ability of VASP to protect barbed ends from capping by CP, and this required interactions of profilin with G-actin and VASP. The VASP EVH2 domain was sufficient to protect barbed ends from capping, and the F-actin and G-actin binding motifs within EVH2 were required. Phosphorylation by protein kinase A at sites within the VASP EVH2 domain regulated anti-capping and F-actin bundling by VASP. We propose that Ena/VASP proteins associate at or near actin filament barbed ends, promote actin assembly, and restrict the access of barbed end capping proteins.

Figure 1

VASP inhibits barbed end capping by capping protein (CP). (A) VASP increases the rate of actin polymerization in the presence of CP in a dose-dependent manner. Plotted are actin polymer concentrations vs. time in reactions containing 1μM actin, 4nM CP, 0.2nM spectrin F-actin seeds (SAS) and VASP as indicated beside each curve: no VASP, red; 12nM, orange; 25nM, green; 50nM, blue; 100nM, pink; 150nM, dark blue; 200nM, violet. The curve labeled “SAS alone” (black) contained neither VASP nor CP. (B) Dependence of the initial rate of actin polymerization in the presence of 4nM CP on the concentration of VASP. Initial rates were calculated from a linear fit to the first 60s of each time course in panel A and normalized to the initial rate in reactions containing SAS alone. (C) Anti-capping activity is dependent on the concentrations of both CP and VASP. Reactions contained 1μM actin, varying amounts of CP and VASP as indicated: no VASP, filled circles; 16nM, open circles; 50nM, filled squares; 82nM, open diamonds. Initial rates were calculated from a linear fit to the first 15–60s (depending on the [CP]) of each time course and normalized to that obtained in reactions containing no CP. (D) VASP increased the rate of actin polymerization from SAS. Plotted are actin polymer concentrations vs. time in reactions containing 1μM actin, 0.2nM SAS and VASP as indicated: no VASP, black; 25nM, orange; 100nM, green. (E) Spontaneous actin polymerization by VASP was negligible under the conditions used for anti-capping assays. Plotted is the actin polymer concentration vs. time in reactions containing 1μM actin and VASP as indicated: 50nM,blue; 100nM, pink; 150nM, dark blue; 200nM, violet. (F) VASP did not promote de novo formation of new actin filament barbed ends. Reactions containing 1μM actin, 0.2nM SAS, and VASP (50nM VASP, green; 100nM VASP, blue; 200nM, violet) and with 10μM profilin (25nM VASP, orange; 50nM VASP, red) as indicated were incubated for 90s at room temperature followed by 25-fold dilution in 0.5μM actin (5% pyrenyl-actin). Plotted is the actin polymer concentration vs. time after dilution.

Figure 2

VASP inhibits barbed end capping by gelsolin and CapG. (A) VASP inhibits capping by 2nM gelsolin:actin. Plotted is the fluorescence of pyrenyl-actin vs. time in reactions containing 0.4μM actin (5% pyrene-labeled), 2nM gelsolin:actin, 0.2nM SAS, 0.2mM CaCl₂ and VASP as indicated: none, red; 8nM, green; 16nM, blue. The reaction labeled “SAS alone” (black) contained no gelsolin:actin or VASP. (B) VASP inhibits capping by 4nM CapG. Plotted is the fluorescence of pyrenyl-actin vs. time in reactions containing 1μM actin (5% pyrene-labeled), 0.2nM SAS, 4nM CapG, 0.5mM CaCl₂ and VASP as indicated: none, red; 50nM, green; 100nM, blue. The reaction labeled “SAS alone” (black) contained no CapG or VASP. (C) VASP decreases the amount of G-actin present at steady-state in reactions containing CP. Plotted is the amount of G-actin detected in supernatant fractions of reactions containing 1μM actin and varying amounts of VASP in MKEI-100 buffer. Reactions were incubated with (open circles) or without 10nM CP (filled circles) for 16h at room temperature to reach equilibrium. The amount of G-actin present in the supernatant fraction was quantified from the Coomassie-stained SDS gels using NIH ImageJ. (D) VASP competes with CP for binding to pre-formed actin filaments. F-actin (1μM, 50% pyrene-labeled) was incubated for 5 min with and without 17nM CP (black) and varying amounts of VASP as indicated; none, red; 26nM, green; 53nM, blue; 67nM, orange. Plotted is the pyrenyl-actin fluorescence vs. time after 20-fold dilution into MKEI-100 buffer.

Figure 3

The VASP EVH2 domain is sufficient to protect barbed ends from CP. (A) EVH2 promotes actin polymerization in the presence of CP in a dose-dependent manner. Plotted is the actin polymer concentration vs. time in reactions containing 0.5μM actin, 0.2nM SAS, 4nM CP, and EVH2 as indicated: no EVH2, red; 2.7nM, orange; 5.4nM, green; 14nM, blue; 28nM, pink; 55nM, violet. (B) Anti-capping activity of VASP EVH2 is higher than that of full-length VASP. Plotted are the initial rates calculated from linear fits to the first 60s of each time course in panel A (or for reactions with VASP obtained under identical conditions) normalized to that obtained in reactions with SAS alone. EVH2: filled circles, VASP: open squares. (C) Spontaneous actin polymerization by VASP EVH2 is negligible under the conditions used for anti-capping assays. Plotted is the actin polymer concentration vs. time in reactions containing 0.5μM actin and EVH2 as indicated: none, black; 28nM, orange; 55nM, violet; 110nM, blue; 220nM, green. (D) EVH2 does not promote de novo formation of actin filament barbed ends. Reactions contained 0.5μM actin, 0.2nM SAS, and EVH2 as indicated: no EVH2, black; 55nM, red; 110nM, blue. Reactions were incubated 90s followed by 25-fold dilution into 0.5μM actin (5% pyrene-labeled). Plotted is the actin polymer concentration vs. time after dilution.

Figure 4

Profilin enhances actin polymerization in the presence of VASP and CP. (A) Profilin increases VASP anti-capping in a dose-dependent manner. Plotted is the time course of actin polymerization in the presence of 1μM actin, 4nM CP, 0.2nM SAS, 25nM VASP, and varying concentrations of profilin as indicated: none, blue; 1μM, light blue; 5μM, orange; 7.5μM, green; 10μM, pink; 15nM, violet. The reaction labeled “SAS alone” (black) contained no CP, profilin or VASP. (B) Dependence of the initial rate of actin polymerization in the presence of CP and VASP on profilin. The initial rates were calculated from a linear fit to the first 60s of each time course in panel A and normalized to the initial rate obtained in the reaction with SAS alone. (C) Profilin does not promote spontaneous actin polymerization in the presence of VASP. Plotted is the actin polymer concentration vs. time in reactions containing 1μM actin without (black) and with VASP (25nM, blue; 50nM, red) or VASP and 10μM profilin (25nM, light blue; 50nM, orange) as indicated. (D) Binding of G-actin and the proline-rich region of VASP is required for profilin to enhance actin polymerization in the presence of VASP and CP. Plotted is the time course for reactions containing 2μM actin, 0.2nM SAS, 4nM CP, with 97nM VASP (violet), and either 10μM profilin (orange) or mutant profilin (R88E, blue; Y6D, green) as indicated. The reaction depicted in blue contained no VASP. (E) Profilin decreases actin assembly in the presence of CP and EVH2. Plotted is the time course for actin polymerization in reactions containing 1μM actin, 0.2nM SAS, 4nM CP, with 15nM EVH2 (green), or 15nM EVH2 and 10μM profilin (blue) as indicated. (F) Profilin and VASP increase the rate of actin assembly from SAS. Plotted is the time course for actin polymerization in reactions containing 1μM actin, 0.2nM SAS with 25nM VASP (red), 10μM profilin (green) or 25nM VASP and 10μM profilin (blue) as indicated.

Figure 5

Phosphorylation by Protein kinase A (PKA) regulates VASP anti-capping activity. Wild-type VASP (Panel A) or VASP triple mutant (S153A,S235A,T274A) (Panel B) was phosphorylated by PKA or mock-treated. VASP proteins were tested in anti-capping assays containing 4nM CP, 0.2nM SAS, 2μM actin and no VASP (red), 50nM mock-treated VASP (blue) or PKA-treated VASP (orange), without and with 10μM profilin (mock-VASP+profilin, violet; PKA-VASP+profilin, green) as indicated. Plotted is fluorescence of pyrenyl-actin vs. time.

Figure 6

VASP F-actin bundling activity is regulated by PKA. Actin filaments formed from 0.1nM SAS and 1μM actin in the presence of 30nM VASP for 5min (A) were incubated for an additional 5 min in the presence (PKA-treated, C) or absence (mock-treated, B) of PKA. Aliquots of each reaction were mixed with rhodamine-phalloidin and prepared for microscopy. Scale bar is 10μm.

Figure 7

The structural organization of VASP-induced actin filament bundles is salt dependent. Actin filaments in the presence of VASP formed highly ordered bundles in 15mM (Panel B) and 50mM KCl (Panels A and D). Characteristic actin layer lines are apparent in the Fourier transform obtained from one 500x500 box (Panel C, left). The 1/375, the equator, and the 1/59 and 1/51 layer lines are visible in the 1000x1000 box transform (Panel C, right) suggesting long range order in the actin-VASP bundles. The meridian reflection indicates the presence of a crosslinker, its height (1/125Å) suggests three crosslinkers per actin filament crossover. Filaments are loosely bundled at higher KCl concentrations (100mM, Panel E; 150mM, Panel F). Bar = 300nm for Panel A and 450nm for Panels B, D, E and F.

Supplementary Figure 1

Schematic representation of VASP and VASP mutant proteins. Murine VASP (amino acids 1–375) was expressed as N-terminal His₆-tagged fusion protein in E. coli. The following deletions or point mutations were introduced: VASP-EVH2 (amino acids 221–375), VASP-ΔPRO (deletion of amino acids 156–207), VASP-ΔFAB (deletion of amino acids 256–273), VASP-GAB (point mutations R232E and K233E), and VASP-ΔCoCo (deletion of amino acids 331–375). The EVH1 domain is indicated in green, the proline-rich central domain in red, and the EVH2 domain in light orange and dark orange.

Supplementary Figure 2

F-actin and G-actin binding sites are required for VASP anti-capping activity. (A) A VASP mutant lacking the F-actin binding site does not exhibit anti-capping activity. Time course of actin polymerization in the presence of 1μM actin, 0.2nM SAS, 4nM CP and VASP-ΔFAB as indicated (none, red; 50nM, green; 75nM, blue). (B) Mutation of the G-actin binding site abolishes VASP anti-capping activity. Time course of actin polymerization in the presence of 1μM actin, 0.2nM SAS, 4nM CP, and either 50nM VASP (blue) or VASP-GAB (50nM, orange; 75nM, green). (C) Anti-capping by VASP is independent of the concentration of G-actin. Plotted are the initial rates of actin polymerization from SAS in reactions containing varying amounts of G-actin and 4nM CP (circles) with VASP as indicated (12.5nM, squares; 25nM, triangles). Initial rates were normalized to those obtained with SAS alone at each actin concentration.

Supplementary Figure 3

VASP is a tetramer and VASP tetramerization is required for anti-capping activity. (A) VASP is a stable tetramer. Sedimentation velocity analytical ultracentrifugation of 4μM VASP in 150mM NaCl, 0.5mM EGTA, 10mM NaH₂PO₄, pH 7.0, 0.5mM DTT. The major species (>95%) sedimented at 4.7S. (B) A mutant form of VASP lacking the C-terminal tetramerization domain does not exhibit anti-capping activity. Plotted is the fluorescence of pyrenyl-actin vs. time in reactions containing 2μM actin, 0.2nM SAS, 4nM CP and VASP-ΔCoCo as indicated (none, red; 100nM, blue; 200nM, green).