VASP localization to lipid bilayers induces polymerization driven actin bundle formation

Abstract

Actin bundles constitute important cytoskeleton structures and enable a scaffold for force transmission inside cells. Actin bundles are formed by proteins, with multiple F-actin binding domains cross-linking actin filaments to each other. Vasodilator-stimulated phosphoprotein (VASP) has mostly been reported as an actin elongator, but it has been shown to be a bundling protein as well and is found in bundled actin structures at filopodia and adhesion sites. Based on in vitro experiments, it remains unclear when and how VASP can act as an actin bundler or elongator. Here we demonstrate that VASP bound to membranes facilitates the formation of large actin bundles during polymerization. The alignment by polymerization requires the fluidity of the lipid bilayers. The mobility within the bilayer enables VASP to bind to filaments and capture and track growing barbed ends. VASP itself phase separates into a protein-enriched phase on the bilayer. This VASP-rich phase nucleates and accumulates at bundles during polymerization, which in turn leads to a reorganization of the underlying lipid bilayer. Our findings demonstrate that the nature of VASP localization is decisive for its function. The up-concentration based on VASP's affinity to actin during polymerization enables it to simultaneously fulfill the function of an elongator and a bundler.

FIGURE 1:

Alignment during polymerization of actin elongated by VASP. (A) Experimental setup of actin polymerized by VASP on supported lipid bilayers. Actin is visualized with 12.5% of actin labeled with Atto488. (B) TIRFM images show actin filament alignment to bundles by 50 nM VASP (scale bar 2 µm). The triangles indicate the direction of polymerization and the arrows display alignment events to a bundle. (C) Kymograph (right) of a newly formed bundle marked by the dashed line at 5 min of polymerization in the left panel (scale bar 5 µm) shows mixed orientation of filaments during polymerization. (D) The first frame (yellow) and the direction of polymerization indicated by the triangles, overlaid over the network at 5 min (red) on the left and the temporal color-coded actin intensity increase on the right, illustrate the bundle network formation from actin seeds and subsequent elongation at the existing filaments (scale bar 5 µm).

FIGURE 2:

Bundle thickening by elongation at the bundle borders. (A) On supported lipid bilayers, actin bundles increase in size in a VASP concentration (0.125, 0.25, 0.5 µM)-dependent manner. Continuous growth of filaments at existing bundles is visible, with swelling of bundles at 0.25 µM VASP (B) and the corresponding intensity profile (C) indicated by the dashed line in (B). (D) Histogram of the bundle width distribution at different VASP concentration with a log-normal fit of the distribution. (E) Box plot of bundle width and pore size showing the median, 25–75th percentile range as boxes and 1–99th percentile range as whiskers. (F) At 1 µM VASP, the exchange of the actin-Atto488 to actin-Atto647 (0.5 µM) after 3 min exhibits the incorporation at the sides of big bundles (indicated by arrows). All scale bars are 5 µm.

FIGURE 3:

Actin network by different elongators and cross-linkers. (A) With monomeric VASPΔTetra (0.25 µM) the network formed is more heterogeneous with filaments more clustered to single vortices or bundles. (B) The nonelongating bundler α-actinin1b only pulls down filaments to the surface without bundling. To be able to visualize and analyze filaments, a concentration of 0.05 µM of α-actinin1b is shown. CP and profilin are emitted from α-actinin1b experiments to enable actin polymerization. C) The nonbundling elongator mDia1 (0.25 µM) is unable to form large bundles or an actin network. (D) mDia1 (0.25 µM) and α-actinin1b (0.25 µM) together on a bilayer form a high-density nematic field. (E) Kinetics of actin polymerization shown by measured actin intensity with TIRFM. To compare α-actinin1b with VASP and mDia1, different concentrations with or without profilin and CP are shown. (F) Box plot of bundle width and pore size showing the median, 25–75th percentile range as boxes, and 1–99th percentile range as whiskers. All scale bars are 5 µm.

FIGURE 4:

Colocalization of VASP to actin filaments. (A) Images of actin (12.5% Actin-Atto488) and 0.25 µM VASP-Alexa532 or VASPΔTetra-Alexa532 on SLBs or monolayers (ML) exhibit their colocalization to actin. (B) Colocalization score of labeled VASP or VASPΔTetra to actin during polymerization on SLBs or MLs. (C) Fluorescence recovery after photobleaching curves of VASP on SLBs or MLs show a reduced mobility of VASP after polymerization (30 min Pol) compared with before actin polymerization (prePol). (D) VASP forms a pattern of VASP enriched phase before actin addition. After start of polymerization (0 min) the VASP phase accumulates under the actin bundles. All scale bars are 5 µm.

FIGURE 5:

Reorganization of lipid bilayer through VASP accumulation. (A) Lipid fluorescence (Texas Red-DHPE) inversely correlates with actin bundles formed with 0.125 µM VASP. (B) Intensity profile of the bundle cross-section indicated in by the dashed line in (A). (C) Lipid intensity (Texas Red-DHPE) changes reciprocally to the actin intensity during polymerization at 0.25 µM VASP. Afterward it normalizes more toward the values at the start of polymerization inversely to the way the VASP-Alexa532 intensity behaves under bundles. (D) Schematic of the bundle formation mechanism. On the fluid bilayer, patches of VASP can freely diffuse to actin bundles and alongside the bundles. F-actin bundles have more sites to bind VASP and therefore a higher dynamic VASP concentration than single filaments. Barbed ends growing toward bundles are captured and aligned by localized VASP. Filaments are most likely to be continuously polymerized by the higher VASP concentration on the larger bundles, whereas actin filaments polymerizing away from VASP are inhibited by CP. This leads to an enhanced thickening of larger bundles. All scale bars are 5 µm.