Article |
Address correspondence to Danijela Vignjevic, Northwestern University Medical School, Department of Cell and Molecular Biology, 303 E. Chicago Ave., Ward 8-063, Chicago, IL 60611. Tel.: (312) 503-2854. Fax: (312) 501-7912. E-mail: nele{at}northwestern.edu
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Abstract |
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Key Words: filopodia; actin; Arp2/3; capping protein; fascin
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Introduction |
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Whereas lamellipodia seem designed for protrusion over a smooth surface, filopodia seem designed for exploring the extracellular matrix and surfaces of other cells. Filopodia, in contrast to the branched network of lamellipodia, contain an unbranched bundle of actin filaments that are aligned axially, packed tightly together, and of uniform polarity (Small et al., 1978; Lewis and Bridgman, 1992). Unlike lamellipodia, the Arp2/3 complex is excluded from filopodia (Svitkina and Borisy, 1999), and filaments in filopodia are relatively long and do not turn over rapidly (Mallavarapu and Mitchison, 1999). The roots of filopodia extend well into the cell lamellipodium. As proposed by the filament treadmilling model (Small et al., 1994), filaments elongate with their barbed ends oriented toward the leading edge, pushing the membrane and at the same time continuously depolymerizing from the pointed ends.
A major question in understanding filopodial formation is how they are initiated. One member of the WASP family, N-WASP, facilitates Cdc42-induced filopodia formation in cells (Miki et al., 1998), suggesting that Arp2/3-mediated nucleation of actin filaments plays a role in generating parallel bundles. How might Arp2/3 complex be involved in the formation of unbranched actin structures? One possibility is that the nucleation and branching activities of Arp2/3 complex can be separated, resulting in the production of dendritic or parallel actin structures, depending on the way in which it is activated. Another possibility is that the dendritic array initially produced by Arp2/3-mediated nucleation is subsequently transformed into parallel bundles of actin filaments.
Valuable insights into the mechanism of lamellipodial formation and protrusion have been obtained using bacterial- and bead-based in vitro motility systems (Theriot et al., 1994; Loisel et al., 1999; Cameron et al., 2000). Filopodial formation is less understood, one reason being that a similar in vitro approach is lacking. In this study, we report the development of in vitro systems for producing filopodia-like bundles, one of which employs cytoplasmic extracts and another that reconstitutes filopodia-like bundles from purified proteins. Using these systems, we provide evidence that filopodia-like bundles are formed by reorganization of the dendritic array.
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Results |
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One possible explanation for the formation of stars instead of comet tails was depletion of some protein(s) by adsorption to the glass during sample spreading. We tested this idea by blocking and preadsorption experiments. Pretreatment of the coverslip with 1% BSA blocked star formation, whereas other actin structures (tails and clouds) did form (unpublished data), suggesting that protein adsorption to glass plays a critical role in star formation. Preadsorption was performed by mixing extract with ground glass to simulate protein depletion during sample spreading, followed by centrifugation to collect the unadsorbed fraction. Glass-depleted extracts supported star formation throughout the entire coverslip, not only at the edges. Further, glass-depleted extracts also supported the formation of stars on WASP-coated beads in plastic tubes. If absorption to glass reduced the concentration of some star-inhibiting factor(s), then simple dilution of the extract might be sufficient to induce stars. Indeed, as shown in Fig. 1 C, when extracts were diluted fivefold with buffer, stars formed across the entire coverslip. The percentage of stars at the center of the coverslip increased from 0% (n = 1603), for undiluted extracts, to 73 ± 6% (n = 542), for extracts diluted fivefold. Thus, the formation of stars could be induced by lowering the concentration of some factor(s) in the extract. Star formation was not limited to rat brain extracts. Extracts of Xenopus oocytes and rat embryo fibroblasts (REFs) also supported star formation (unpublished data), but required greater dilution than rat brain extracts. 10% Xenopus oocyte extracts and 50% REF extracts were comparable to full-strength brain extracts in their ability to produce stars.
Star formation in extracts is an Arp2/3-dependent process
The formation of bundles in association with WASP-coated beads suggested the involvement of the Arp2/3 complex. To investigate the role of Arp2/3 complex in star assembly, it was depleted from brain extracts by GSTverprolin-homology/connecting/acidic domain of WASP (VCA) sepharose beads. At least 90% depletion was achieved, as assayed by immunoblotting (Fig. 2 A). In control and mock-depleted extracts, stars were present throughout the entire coverslip, 96% (n = 126) and 90% (n = 138), respectively (Fig. 2, B and C). In Arp2/3-depleted extracts (Fig. 2 D), actin assembly around the beads was completely abolished (0%, n = 213). Only spontaneously polymerized filaments could be observed in the background. Add-back of pure Arp2/3 complex to the depleted brain extracts restored star formation (Fig. 2 E) (84%, n = 170), although stars were slightly smaller than in control samples. Based on our immunoblotting experiments, the concentration of Arp2/3 complex necessary to rescue star formation (0.5 µM) was similar to that calculated to be present in glass-depleted extracts (0.45 µM). Lower concentrations of added Arp2/3 complex induced the formation of branched filaments on the background or actin clouds on the beads. We conclude that star formation is mediated by the Arp2/3 complex.
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Stars display both lamellipodial and filopodial types of actin organization
The radial bundles comprising stars bear a superficial similarity to filopodia in cells. To test whether these two kinds of structures have a deeper similarity, we analyzed the kinetics of star formation, their structural organization, sites of actin incorporation, and protein composition. Star development was observed by time-lapse microscopy. Initially, a diffuse actin cloud was formed around the bead (Fig. 3 A, 15 min). As time progressed, radial actin bundles appeared and began to elongate, with an average rate of 0.15 µm/min. Finally, we observed long stable actin bundles radiating from the bead-associated cloud. Some bundles in the course of star formation fused together by zippering in a proximaldistal direction, producing thicker bundles (Fig. 3 B). Zippering in a distalproximal direction was also observed (unpublished data).
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Lamellipodia and comet tails, on the one hand, and filopodia in cells, on the other hand, have distinct protein composition (Goldberg, 2001; Small et al., 2002). For example, the Arp2/3 complex and capping protein are present in lamellipodia and comet tails but have not been found in filopodia (Svitkina and Borisy, 1999; Svitkina et al., 2003), whereas fascin is enriched in filopodia and less abundant in lamellipodia (Kureishy et al., 2002). -Actinin has been found in lamellipodia (Langanger et al., 1984) but only in the roots of filopodia (Svitkina et al., 2003). We determined the localization of these proteins in stars by immunofluorescence staining or by incorporation of the labeled protein (Fig. 5). Arp2/3 complex and capping protein were found in the dendritic network proximal to the bead but not in actin bundles.
-Actinin was clearly enriched around the bead but could be faintly detected in bundles when a high amount of exogenous protein was added. In contrast, fascin was strongly localized to actin bundles but was diminished in the network surrounding the bead. Thus, by structural, kinetic, and biochemical criteria, our data demonstrate that the proximal dendritic network and radial bundles of stars are similar to the actin organization of lamellipodia and filopodia, respectively.
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Capping protein concentration was estimated in terms of a chicken capping protein standard by immunoblotting. Rat brain extracts contained 3.8 ng/µl (58 nM) and REF extracts contained 8 ng/µl (122 nM) capping protein (Fig. 6 A). The twofold higher concentration of capping protein in REF extracts was consistent with the twofold dilution of this extract required to produce stars. We were unable to estimate the concentration of capping protein in Xenopus oocyte extracts because of a lack of cross-reactivity of the antibodies used. The initial total protein concentration for all tested extracts was similar: brain extract, 16 mg/ml; REF, 21 mg/ml; and Xenopus oocyte, 24 mg/ml. Next, we examined how much capping protein was depleted from brain extract by its adsorption on ground glass. As assayed by immunoblotting, 35% of capping protein was depleted (Fig. 6 B), whereas actin was not depleted (0%) (Fig. 6 B), Arp2/3 was 18% depleted (Fig. 2 A), and fascin was only 8% depleted (unpublished data). These results demonstrate that glass absorbs motility proteins differentially and that capping protein is preferentially depleted.
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Reconstitution of filopodia-like bundles using pure proteins
We tried to define a minimal set of components necessary for star assembly. Because data obtained with extracts indicated that Arp2/3 complex was necessary and capping protein had to be depleted, initial reconstitution experiments were performed with WASP-coated beads, actin, and Arp2/3 complex. Concentrations of proteins were based on published data for reconstitution of comet tails (Loisel et al., 1999). Beads coated with WASP were put into physiological ionic strength buffer solution, pH 7, containing rhodamine-labeled actin (6.9 µM) and increasing amounts of Arp2/3 complex over the range 0.10.9 µM. At 0.7 µM Arp2/3 complex and above, actin polymerization at the bead surface resulted in clouds within 15 min (Fig. 7 A). Clouds were 3.2 mm in diameter (measured as full width at 1/e of maximum fluorescence) and were azimuthally homogeneous in intensity except for apparently stochastic fluctuations. Lower concentrations of Arp2/3 did not generate clouds or did so more slowly.
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Discussion |
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One possibility for how the action of the Arp2/3 complex can be explained is that it promotes dendritic or filopodial initiation depending upon the specific Arp activator. This idea is consistent with findings that different members of the WASP family vary in their ability to activate the Arp2/3 complex (Zalevsky et al., 2001). However, we found no significant differences in the process of star formation when they were induced by a variety of Arp2/3 activators, including ActA, WASP, Scar, N-WASP, and the pVCA COOH-terminal domains of WASP and Scar. ActA was active both as endogenous bacterial protein and as a recombinant protein at the bead surface. Thus, our data do not support a model that Arp2/3 complex produces different arrays depending on the specific activator.
Our data are in agreement with a model in which the Arp2/3 complex, irrespective of its activator, produces a normal dendritic array, which subsequently becomes reorganized into bundles. Time-lapse observations showed that diffuse clouds with dendritic organization preceded the formation of bundles. Structural studies demonstrated a gradual transition from dendritic arrays around the bead to distal radial bundles. Both results suggest that a normal dendritic network serves as a precursor for bundles. These results are in close agreement with recent results elucidating the in vivo mechanism of filopodial initiation (Svitkina et al., 2003). We propose that the role of the Arp2/3 complex in the in vitro system is to supply barbed ends. The high local concentration of barbed ends created by activator-coated beads and the high concentration of Arp2/3 in solution were essential for bundle formation. A similar method may be used by cells if Arp2/3 activators (or molecules recruiting them) are not evenly distributed along the leading edge, but exist in clusters. A high local concentration of barbed ends could also be created by the clustering of some barbed endbinding molecules at the membrane. Possible candidates for such a role are members of the enabled/vasodilator-stimulated phosphoprotein (Ena/VASP) family (Bear et al., 2002; Svitkina et al., 2003) and formins (Pruyne et al., 2002), which have recently been shown to bind barbed ends but allow for filament elongation. Formins can also nucleate unbranched actin filaments (Pruyne et al., 2002; Sagot et al., 2002b) and thus are candidates for an alternative, Arp2/3-independent pathway of bundle initiation, similar to how actin cables in yeast are formed (Evangelista et al., 2002; Sagot et al., 2002a). However, this mechanism does not account for the N-WASP induction of filopodia.
Elongation
After filaments are nucleated, they have to elongate and encounter each other before they can form a bundle. A priori, elongation of filaments could be facilitated by increasing the concentration of an "elongation" factor or by decreasing the concentration of a "termination" factor. The main arguments in favor of the latter possibility are that star formation was induced in extracts by depletion of capping protein and was antagonized specifically by add-back of capping protein. A key point here is that add-back of capping protein to depleted extracts did not simply block all actin polymerization. Rather, it induced the formation of clouds as opposed to stars. Our interpretation is that cloud formation was the result of the termination of filament elongation shortly after branch nucleation under conditions when active Arp2/3 complex continuously nucleates new filaments. This process results in short filaments and a dense dendritic network. The fact that stars could be reconstituted in a pure protein system lacking barbed-end capping proteins but allowing for filament nucleation, elongation, and bundling is consistent with the interpretation that star formation was facilitated by decreasing a termination factor.
In the cellular context, the cytoplasmic concentration of capping protein is high (Huang et al., 1999). In lamellipodia where barbed ends are constantly being produced, the high concentration of capping protein can be understood as necessary to cap unproductive barbed ends. In filopodia where filaments elongate continuously, their barbed ends need to be protected from capping. Protection may be provided by Ena/VASP family proteins because they are present at the extreme leading edge, bind to barbed ends, and can antagonize capping in vitro and in vivo (Bear et al., 2002). Ena/VASP proteins are also enriched at the tips of filopodia (Lanier et al., 1999; Rottner et al., 1999) and become gradually accumulated at the tips of filopodial precursors during the filopodial initiation (Svitkina et al., 2003). It is attractive to speculate that the presence of Ena/VASP at the filopodial tips in the cellular context prevents filament termination and allows filopodial elongation. Consistent with this idea, our results showing that the level of capping protein controls the transition between two types of actin arrays in vitro match the observations that the level of Ena/VASP proteins performs analogous control in vivo, but in the opposite direction. That is, a low level of Ena/VASP induced short branched filaments, and an excess of Ena/VASP promoted the formation of long filaments (Bear et al., 2002).
In our in vitro system, the rates of bundle elongation in stars were 0.15 µm/min, similar to the reported rates of comet tail motility in undiluted brain extracts (0.20.3 µm/min) (Laurent et al., 1999; Yarar et al., 1999). Whereas in Xenopus oocyte extract or during leading edge protrusion in cells, the rates of actin array assembly are at least an order of magnitude higher (Mallavarapu and Mitchison, 1999). These observations indicate that additional factors may contribute to the overall performance of actin machinery, but balance between nucleation and elongation seems to be critical for the determination of supramolecular organization of the actin array.
Bundling
Bundling is necessary to allow long filaments to push efficiently without buckling under the cell surface. The leading candidate for filament bundling in filopodia is fascin. It shows the greatest enrichment in filopodial bundles in cells (Kureishy et al., 2002), where it significantly prevails over -actinin (Svitkina et al., 2003), and it is essential for the maintenance of filopodia (Yamashiro et al., 1998; Adams et al., 1999; Cohan et al., 2001).
Consistent with in vivo data, we found that fascin was the major bundling protein present in stars assembled in cytoplasmic extracts. Fascin was also sufficient to form filopodia-like bundles in a reconstitution system in which fascin represented the only bundling protein. The straightness of the fascin-induced star bundles suggests that they were quite rigid, similar to filopodia. In contrast to fascin, the other actin filament cross-linker, -actinin, was more abundant in the dendritic network surrounding beads and was absent from star bundles. Although
-actinin was also able to drive star assembly in a reconstitution system, the resulting star bundles had wavy rays and a dendritic, not parallel bundled, organization. Thus,
-actinin did not recapitulate filopodia formation in vitro. These results suggest that the two cross-linkers may be specialized with respect to the particular actin filament array that they stabilize. Segregation of fascin and
-actinin to bundles and the dendritic network, respectively, correlates with the biochemical properties of these two cross-linkers: fascin is a short cross-linker that makes tight parallel bundles (Yamashiro-Matsumura and Matsumura, 1985) and
-actinin is a longer molecule that, when combined with actin, makes loose bundles with both parallel and antiparallel filament orientation. The absence of
-actinin in filopodial bundles in more complex systems, such as extracts or cells, when both proteins are present may be explained as fascin outcompeting
-actinin because of a slightly higher affinity or, possibly, specific recruitment to areas of filopodial assembly.
In the course of star formation, we frequently observed zippering of radial bundles, an effect that superficially resembles fusion of filopodia during their normal dynamics in cells. However, distinctions between these two phenomena should be noted. In cells, the fusion site moves backward because of treadmilling and retrograde flow of the whole assembly, but no actual displacement of bundles, with respect to each other, occurs. In stars, we did not find independent indications of retrograde flow, suggesting that zippering in vitro actually brings distant bundles together. A likely reason for the difference between the two systems is that the movement of bundles in vivo is precluded by the more crowded conditions and cross-linking occurring in the cytoplasm, compared with extracts. Nevertheless, the similarity of the systems suggests that cross-linking molecules, presumably fascin, have a potential for zippering and may accomplish this function under permissive circumstances, for formation of bundles in vitro and for filopodia in vivo.
In summary, our results suggest that an Arp2/3-mediated pathway is compatible with filopodia formation, and that it is not necessary to postulate unusual properties of WASP family members to stimulate a nonbranching mode of actin filament formation. Filopodia-like structures can be accounted for as a transformation from a dendritic organization by a combination of elongation and bundling.
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Materials and methods |
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DNA encoding WASP tagged at its NH2 terminus with both Met, Arg, Gly, Ser (MRGS) 6xHis and FLAG epitopes was amplified by PCR from a human WASP cDNA (a gift of Arie Abo, PPD Discovery, Menlo Park, CA) and subcloned into pFastBac1 (Life Technologies; Amersham Biosciences). DNA encoding human Scar1 was amplified by PCR and subcloned into the WASP-pFastbac1 vector in the place of the WASP DNA. Recombinant WASP and Scar1 proteins were expressed in Sf9 cells using the baculovirus system. Baculovirus strains were generated and used for infections according to the Bac-to-Bac baculovirus expression system (Life Technologies). After 72 h of infection, cells were harvested by centrifugation at 500 g for 10 min at 25°C, resuspended in lysis buffer (50 mM NaH2PO4, pH 8.0, 300 mM KCl) with protease inhibitors (1 mM PMSF and 10 µg/ml leupeptin, pepstatin, and chymostatin [LPC]), and frozen in liquid N2. To prepare the lysate, cells were thawed and centrifuged at 200,000 g for 15 min at 4°C.
To purify the recombinant proteins, Sf9 lysates were supplemented with 20 mM imidazole, incubated with Ni-NTA agarose (QIAGEN) resin for 45 min at 4°C, washed with 50 mM NaH2PO4, pH 8.0, 300 mM KCl, 20 mM imidazole, and eluted with 200 mM imidazole, 50 mM NaH2PO4, pH 8.0, 300 mM KCl, and protease inhibitors. Eluted proteins were further purified by gel filtration chromatography on a Superdex-200 column (Amersham Biosciences) equilibrated with 20 mM MOPS, pH 7.0, 100 mM KCl, 2 mM MgCl2, 5 mM EGTA, 1 mM EDTA, 0.2 mM ATP, 0.5 mM DTT, 10% vol/vol glycerol. Full-length recombinant WASP has constitutive ability to activate Arp2/3 complex (Higgs and Pollard, 2001).
DNA encoding human fascin was amplified by PCR and sublcloned into the pGEX-4T-3 vector (Amersham Biosciences) using BamH1/Xho I sites. Recombinant human fascin was prepared by a modification of the method of Ono et al. (1997). E. coli carrying the plasmid was grown at 37°C until the A600 reached 0.6. Protein expression was induced by adding 0.1 mM IPTG at 20°C for 4 h. Cells were harvested by centrifugation and extracted with B-PER in phosphate buffer (Pierce Chemical Co.) plus 1 mM PMSF and 1 mM DTT. The lysate was centrifuged at 20,000 g for 20 min, and the supernatant was mixed for 1 h at RT with 2 ml glutathioneSepharose 4B (Amersham Biosciences) equilibrated with PBS plus 1 mM DTT. The glutathione-Sepharose was poured into a column and washed with 20 ml of PBS plus 1 mM DTT. 80 µl of thrombin (Amersham Biosciences) was added, and digestion was allowed to proceed overnight at 4°C. Flowthrough fractions were collected in 2 mM PMSF and concentrated by Centricon 10 (Amicon).
Arp2/3 was purified from bovine brain as described by Laurent et al. (1999). Recombinant chicken CapZ (Soeno et al., 1998; Palmgren et al., 2001) was provided by John Cooper (Washington University School of Medicine, St. Louis, MO). FITC-labeled -actinin was provided by Marion Greaser (University of Wisconsin, Madison, WI). ActA protein (Cameron et al., 1999) was provided by Julie Theriot (Stanford University School of Medicine, Stanford, CA). Recombinant human cofilin and human profilin were purchased from Cytoskeleton, Inc.
Cytoplasmic extracts
Rat brain extract was prepared as previously described (Laurent et al., 1999). Metaphase Xenopus oocyte extract was prepared as previously described (Murray, 1991) and kept frozen at -80°C. Before use, it was centrifuged at 100,000 g for 1 h at 4°C, and the supernatant was used for experiments. Rat embryonic fibroblast extract was prepared as described by Saoudi et al. (1998).
Actin polymerization bead assay
Carboxylated polystyrene beads (Polysciences) were coated with ActA as previously described (Cameron et al., 1999). For coating beads with WASP/Scar proteins, we took 15 µl of 0.5 µM WASP or Scar proteins, mixed up with 15 µl of brain buffer (BB) (Laurent et al., 1999) or Xenopus buffer (Murray, 1991) and 0.5 µl of 0.5-µm carboxylated polystyrene beads. This mixture was incubated at RT for 1 h. Beads were washed twice with the appropriate buffer and resuspended in 10 µl of buffer. For longer storage, beads were supplemented with 50% glycerol and placed at -80°C.
Coated beads (0.5 µl) were introduced into 10 µl of cell extract supplemented with energy mix (15 mM creatine phosphate, 2 mM ATP, and 2 mM MgCl2) and 1.25 µM rhodamine-labeled actin. In experiments to evaluate the effect of capping protein concentration, the assay mix was supplemented with increasing amounts of capping protein. The assay mix was incubated on ice for 1 h before preparation for observation.
For reconstitution system, 0.5 µl of coated beads was introduced in 1x KME buffer (50 mM KCl, 1 mM MgCl2, 1 mM EGTA, 10 mM imidazole, pH 7) supplemented with Arp2/3 complex. After incubation for 5 min at RT, 6.9 µM actin was added, and the mixture was incubated on ice for 20 min before the addition of fascin and allowing for actin assembly at RT.
Microscopy
For light microscopy, a 1-µl sample was removed and pressed tightly between a microscope slide and a 22-mm square glass to create a chamber 5 µm thick, which was then sealed with vaseline/lanolin/paraffin (at 1:1:1). Samples were incubated at RT for 15 min and then observed with a Nikon Eclipse inverted microscope equipped with phase contrast and epifluorescence optics. Time-lapse images were acquired with a back-thinned CCD camera (CH250; Photometrics) using METAMORPH (Universal Imaging Corp.) software. Fluorescence images were recorded every 5 min for 2 h.
For EM, samples were prepared as described by Cameron et al. (2001). After incubation for 2 h at RT in a humid environment, chambers were opened into solution containing 0.2% Triton X-100 and 2 µM phalloidin in BB. Although some stars were washed out during this procedure, some remained attached to the coverslip. Coverslips were washed with 2 µM phalloidin in BB, fixed with 2% glutaraldehyde, and processed for EM. Procedures for S1 decoration and EM were as previously described (Svitkina and Borisy, 1998). Polarity of filaments, determined in blind experiments by two independent observers, gave similar results.
Immunostaining
Rabbit polyclonal antibody to the Arc p16 subunit of the Arp2/3 complex was prepared against the RFRKVDVDEYDENKFVDEED peptide of the human sequence. By Western blotting, the affinity-purified antibody recognized in rat brain extract a doublet of closely spaced bands in the16-kD range, supposedly corresponding to two isoforms of Arc p16 (Millard et al., 2003). Polyclonal rabbit antibody against capping protein (R22) was provided by Dorothy A. Schafer (University of Virginia, Charlottesville, VA). Mouse monoclonal -actinin antibody was from Sigma-Aldrich, and mouse monoclonal fascin antibody was from DakoCytomation. Immunostaining was performed in perfusion chambers. Solutions were applied on one side of the chamber with a pipet and withdrawn from the other side with filter paper. A 4-µl assay sample was sandwiched between a glass slide and a coverslip (22 x 22 mm) separated by two strips of teflon tape and sealed. Stars were allowed to form for 2 h at RT in humid conditions. 10 µl of BB containing 0.2% Triton X-100 and 2 µM phalloidin was perfused through the chamber, followed by 10 µl of 2 µM phalloidin in BB. For most immunostaining experiments, stars were fixed with 0.2% glutaraldehyde for 20 min at RT, washed with PBS, and quenched for 20 min with 2 mg/ml of NaBH4 in PBS supplemented with 0.1% Tween 20. For fascin staining, samples were fixed with methanol for 10 min at -20°C.
Pulse-labeling assay
Stars were allowed to form for 1 h in a perfusion chamber containing 4 µl of assay sample without labeled actin. Then 1 µl of 1.8 µM rhodamineactin was added by diffusion from the edge. After 10 min, FITCphalloidin was added in the same way to label all actin filaments. The chamber was sealed, and observations were made after 10 min of incubation at RT.
Glass depletion experiment
30 µl of brain extract was applied to 70 mg of ground glass coverslip in a mini filtration tube (0.65 µm; UltraFree-MC; Millipore), and the filtrate was collected by centrifugation twice for 1 min at 11,000 g.
Depletion of Arp2/3 complex
Arp2/3 complex was depleted from glass-depleted brain extract using a variation of the method described by Egile et al. (1999). 15 µl of glutathione-Sepharosecoupled GSTVCA beads was incubated with 40 µl of brain extract for 30 min at 4°C on a rotating wheel. Beads were pelleted at 10,000 g for 2 min. Mock depletion is performed by the same amount of glutathione-Sepharose beads. Depletion of the Arp2/3 complex was monitored by Western blotting of aliquots of extracts and beads. The add-back experiment was performed using the Arp2/3 complex purified from bovine brain. For the microscopy assay, we scored the percentage of stars assembled on all beads.
Quantification of capping protein in cell extracts by Western blotting
Different amounts of rat brain and rat embryonic fibroblast extracts were subjected to SDS-PAGE (420% polyacrylamide) and immunoblotting with anti-CapZ. The amount of capping protein present was evaluated by comparing the intensity of the bands of each sample with the chicken CapZ standard by densitometry using NIH image software.
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Acknowledgments |
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This work was supported by United States Army USAMRMC grant DAMD 17-00-1-0386 (D. Vignjevic), National Institutes of Health (NIH) grant GM 62431 (G.G. Borisy), and NIH Glue Grant on Cell Migration IU 54 GM 63126.
Submitted: 12 August 2002
Revised: 24 January 2003
Accepted: 24 January 2003
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References |
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