Formation of filopodia-like bundles in vitro from a dendritic network

Abstract

We report the development and characterization of an in vitro system for the formation of filopodia-like bundles. Beads coated with actin-related protein 2/3 (Arp2/3)-activating proteins can induce two distinct types of actin organization in cytoplasmic extracts: (1) comet tails or clouds displaying a dendritic array of actin filaments and (2) stars with filament bundles radiating from the bead. Actin filaments in these bundles, like those in filopodia, are long, unbranched, aligned, uniformly polar, and grow at the barbed end. Like filopodia, star bundles are enriched in fascin and lack Arp2/3 complex and capping protein. Transition from dendritic to bundled organization was induced by depletion of capping protein, and add-back of this protein restored the dendritic mode. Depletion experiments demonstrated that star formation is dependent on Arp2/3 complex. This poses the paradox of how Arp2/3 complex can be involved in the formation of both branched (lamellipodia-like) and unbranched (filopodia-like) actin structures. Using purified proteins, we showed that a small number of components are sufficient for the assembly of filopodia-like bundles: Wiskott-Aldrich syndrome protein (WASP)-coated beads, actin, Arp2/3 complex, and fascin. We propose a model for filopodial formation in which actin filaments of a preexisting dendritic network are elongated by inhibition of capping and subsequently cross-linked into bundles by fascin.

Figure 1.

Different actin structures are assembled on the beads. Actin assembly was assayed on Arp2/3 activator-coated beads in brain extract supplemented with rhodamine-labeled actin. (A) The pattern of actin assembly depends on the location of the bead on the coverslip. In the center of the coverslip, beads induce the formation of tails. Halfway between the center and edge of the coverslip, actin clouds and chimeras are formed. At the edge of the coverslip, star-like structures are associated with the beads. Beads are shown in yellow. Bar, 5 μm. (B) Stars are the dominant actin structure at the edge of the sample. Low magnification view of a field at the edge of a coverslip. Bar, 50 μm. (C) Percentage of stars increases with extract dilution. 1, center of the coverslip; 2, transitional zone; 3, edge of the coverslip. (D) Percentage of stars produced at the edge of the coverslip in undiluted rat brain extract by beads coated with different Arp2/3-activating proteins.

Figure 2.

Arp2/3 complex is essential for star formation. (A) Arp2/3 complex depletion in brain extract. Glutathione-Sepharose or glutathione-Sepharose–coupled GST–VCA beads were incubated with 40 μl of brain extract. Arp2/3 depletion was monitored by immunoblotting using anti-p16 polyclonal antibody. Lane 1, 10 μl of untreated extract; lane 2, 10 μl of glass-depleted extract; lane 3, 10 μl of mock-depleted extract; lane 4, 10 μl of Arp2/3-depleted extract; lane 5, Arp2/3 associated with glutathione-Sepharose beads; lane 6, Arp2/3 associated with GST–VCA beads; lane 7, pure Arp2/3 from bovine brain, 4 μg. (B–E) Star assembly in Arp2/3-depleted brain extract. (B) Control, glass-depleted extract. (C) Mock-depleted extract. (D) Arp2/3-depleted extract; stars do not form. (E) Arp2/3-depleted extract rescued by add-back of 0.64 μM pure Arp2/3 complex; star formation is restored. Left panels, phase contrast. Individual 0.5-μm beads are visible. Right panels, fluorescence. Bright stars are evident on a background of faint individual filaments in B, C, and E. Only faint filaments are seen in D. Bar, 10 μm.

Figure 3.

Kinetics of star formation. (A) Time-lapse sequence of star assembly in rat brain extract. Bar, 5 μm. (B) Actin bundle zippering. Two bundles zipper together in a centrifugal direction. Time shown in min.

Figure 4.

Structural organization of stars and polarity of actin assembly. (A) Actin filaments form a dendritic network around the bead and filament bundles away from the bead. Platinum replica EM. Bar, 0.5 μm. Overview of the star is shown in the left inset. Examples of branched filaments are highlighted in yellow and enlarged in the small panels at right. (B) Actin filaments in bundles are oriented with their barbed end away from the bead. Polarity of actin filaments was determined by myosin S1 decoration and is indicated by arrowheads. The bead is on the left. Bar, 0.1 μm. (C) Actin assembly occurs at tips of bundles and around the bead. Pulse labeling of actin incorporation. Rhodamine–actin (red) marks sites of new actin incorporation. All actin filaments were labeled with FITC–phalloidin (green). Bar, 5 μm.

Figure 5.

Localization of actin binding proteins in stars. Arp2/3 complex, capping protein, and α-actinin are enriched near the beads but not in bundles, whereas fascin is enriched in bundles but is not prominent at beads. Stars were labeled with rhodamine–actin (green) and FITC–α-actinin (red) during assembly. Immunostaining for Arp2/3 (p16 Arc), capping protein (β2), or fascin (red) was performed after fixation of stars. Bar, 5 μm.

Figure 6.

Formation of stars depends on the concentration of capping protein. (A) Capping protein concentration in rat brain and REF extracts (7.5 μl per lane) was determined by Western blotting. Purified CapZ was used as a standard at concentrations shown above the respective lanes. (B) Capping protein and actin concentration in control and glass-depleted brain extract. (C) Addition of capping protein inhibits star formation. Percentage of the indicated bead-associated structure (Y axis) is shown versus concentration of added capping protein (X axis) to the 50% diluted rat brain extract. (D) Addition of capping protein induces growth of clouds. Time-lapse sequence of actin assembly around a WASP-coated bead in 50% brain extract supplemented with 50 nM capping protein. Bar, 5 μm. (E) Dendritic organization of clouds formed after the addition of 50 nM capping protein to the 50% brain extract. Bar, 0.2 μm. Overview of the cloud is shown in the left panel. Bar, 1 μm. Examples of branched filaments are highlighted in boxes and enlarged in small right panels.

Figure 7.

Reconstitution of filopodia-like bundles from pure proteins. Samples of WASP-coated beads preincubated on ice with 6.9 μM rhodamine-labeled actin and 0.7 μM Arp2/3 complex were brought to RT to allow for actin assembly. (A) Actin clouds formed in the absence of fascin. (B) Addition of 3.1 μM fascin to the sample before incubation at RT produced stars. Low magnification panels (top row) show distinctive pattern of actin assembly under each condition. (C) EM of star bundles formed in the presence of fascin as in B. Bars: (top row) 50 μM; (bottom row) 5 μm; (C) 100 nm.

Figure 8.

Model for the formation of filopodial bundles. We propose that filopodia are formed from a preexisting dendritic network by barbed-end elongation of actin filaments and their subsequent cross-linking into bundles. At normal levels of capping activity, clouds and tails are formed around the bead in the in vitro system (top left), and lamellipodia are formed in cells (top right). If the concentration of capping protein is lowered in the in vitro system, filaments elongate and become bundled by cross-linking proteins, e.g., fascin (bottom left). Two examples of cross-linking are presented. Thin bundles may further zipper into the thicker bundles (arrows). In the cell, some filament barbed ends at the membrane become protected from capping, perhaps by Ena/VASP proteins, so that they can elongate and be cross-linked to form bundles in filopodia (bottom right).