BUG 968 strain and BUG 896 strain expressing the membrane form of IcsA (BUG 968 harboring pHS3199, a pUC8 plasmid containing IcsA
gene; Kocks et al. 1995
) were grown at 37°C in 2YT in the presence of 100 μg/ml ampicillin until midexponential phase was reached. Listeria
strain Lut12 (pActA3) overexpressing ActA (Kocks et al. 1992
) was grown at 37°C in brain/heart infusion medium in the presence of 7 μg/ml chloramphenicol and 5 μg/ml erythromycin until midexponential phase was reached. Bacteria were kept frozen in 30% glycerol at −80°C.
Polyclonal rabbit antibodies were raised against human N-WASP protein residues 473-EVMQKRSKAIHSSDED-488 (C412 antibodies) and against recombinant human N-WASP protein obtained from baculovirus (NW011 antibodies). Anti-Arp3 polyclonal antibodies (AR3) were raised in rabbits against Arp3 residues YEEIGPSIVRHNPVFGVMS. Affinity-purified C421, NW011, and AR3 antibodies were used for Western blot and immunofluorescence, respectively, at 10 μg/ml concentration. Antibovine profilin polyclonal antibodies were raised in rabbits and used in Western blots at a 1:5,000 dilution.
Platelet and Brain Extracts
Human platelet extracts were prepared from outdated unstimulated preparations as previously described (Laurent and Carlier 1997
). Bovine brain extracts were prepared as follows. Brain was homogenized in 1 vol of 0.1 M MES, pH 6.8, 1 mM EGTA, 0.5 mM MgCl2
, 0.1 mM EDTA, 1 mM DTT, and clarified by centrifugation at 10,000 g
for 30 min at 4°C. Tubulin and neurofilaments were removed by centrifugation for 1 h at 150,000 g
after one cycle of microtubule assembly at 37°C for 30 min in the presence 0.5 mM GTP, 4 M glycerol, and 0.25 mM MgCl2
. The supernatant (S2
) was stored at −80°C.
Recombinant Proteins Expression and Purification
Histidine-tagged human N-WASP was expressed in High Five insect cells. Human cDNA was cloned in pFast Bac Htb (Life Technologies, Inc.) and recombinant baculovirus constructed using standard protocol of transfection–recombination in insect cell lines. High Five insect cells were infected at a multiplicity of infection of 2. Cell culture was performed in suspension, in High Five serum-free medium, under vigorous shaking at 27°C for 40 h. Protein purification was performed by DEAE cellulose at pH 7.8 and cobalt affinity chromatography, as described by Laurent et al. 1999
. Recombinant histidine-tagged human VASP was expressed and purified as described previously (Laurent et al. 1999
-terminal (Nt) and COOH-terminal domains (VCA) of human N-WASP were expressed and purified as GST fusion proteins in E
. Human cDNA regions encoding N-WASP Nt (residues 1-276) and VCA (residues 392-505) were amplified by PCR from human brain cDNA using oligonucleotides phNW1 (5′-ccggaattcATGAGCTCCGTCCAGCAGCAG-3′), phNW276 (5′-ccgctcgagTCACTTGCCTCCGCAGTTCATTTTTAAC-3′), and oligonucleotides phNW392 (5′-ccggaattcCCTTCTGATGGGGACCATCAG-3′), phNW505 (5′-ccgctcgagTCAGTC-TTCCCACTCATCATC-3′), respectively. N-WASP Nt and VCA PCR fragments were cloned in EcoRI and XhoI sites in pGEX4T-1 plasmid, respectively, to generate pCENHN and pCECHN plasmids. GST-Nt and GST-VCA protein expressions were obtained using BL21 E
strain according to standard induction and purification procedures. GST-Nt and GST-VCA glutathione Sepharose beads were kept at 4°C until use. GST-Nt protein was eluted from beads and VCA protein was obtained by thrombin cleavage. Both proteins were dialyzed against 10 mM Tris-Cl−
, pH 7.5, 1 mM DTT, 50 mM KCl, and 0.01% NaN3
, frozen on liquid nitrogen and stored at −80°C. The concentration of VCA was determined spectrophotometrically using an extinction coefficient 0.1%
of 0.467 cm−1
at 278 nm calculated from the amino acid sequence, and a mol wt of 12,900 D. Due to the acidic character of VCA, the Bradford protein assay gave underestimated values of the concentration.
Human Cdc42 was also expressed as a GST fusion protein in E. coli. Human cDNA was cloned in pGEX2T expression plasmid and was a kind gift from Dr. Alan Hall (Medical Research Council, University College, London, UK). Protein was expressed in E. coli JM109, purified on glutathione Sepharose as described, and kept on beads at 4°C in 10 mM Tris-Cl−, pH 7.6, 150 mM NaCl, 2 mM MgCl2, 0.1 mM DTT, and 0.1 mM GTP. When required, GST-Cdc42 was loaded with GDP or GTPγS on beads by a 1-h incubation at room temperature on a wheel in 50 mM Tris-Cl−, pH 7.6, 150 mM NaCl, 2 mM MgCl2, 1 mM DTT, 10 mM EDTA, and 0.1 mM GDP or GTPγS. The exchange reaction was stopped by addition of 10 mM MgCl2.
protein was purified as a GST fusion protein as described in Suzuki et al. 1996
Purification of Arp2/3 Complex from Bovine Brain
Bovine Arp2/3 complex was purified from bovine brain extracts by a novel two step method as follows. 100 ml of S2 was dialyzed against 50 mM MES, pH 6.8, containing 1 mM DTT, 1 mM EDTA, and 1 mM MgCl2 (buffer A), and loaded on an SP-Trisacryl column (2.5 × 12 cm) equilibrated in buffer A. After a wash step with 30 mM KCl, the Arp2/3 complex was eluted with 80 mM KCl in buffer A. This fraction was equilibrated in 20 mM Tris-Cl−, pH 7.5, containing 25 mM KCl, 1 mM DTT, 1 mM MgCl2, 0.5 mM EDTA, and 0.1 mM ATP-Tris, pH 7.5 (buffer B), and loaded on a GST-VCA glutathione Sepharose column (0.5 × 1 cm) in buffer B. Most of the proteins were washed off the column by buffer B, the remaining non-Arp2/3 proteins were eluted by 0.2 M KCl in buffer B, pending a small loss of Arp2/3 activity, and the pure Arp2/3 complex was eluted with 0.2 M MgCl2 in buffer B (see b for the purity of the Arp2/3 complex). After dialysis against buffer B, the protein was concentrated using a Centriprep 30 cartridge (Amicon). The Arp2/3 complex was supplemented with 0.2 M sucrose and stored at −80°C. The activity of Arp2/3 along the purification steps was monitored spectrofluorimetrically, using the ability of Arp2/3 to activate the branched polymerization of actin filaments in the presence of VCA. The assay (80 μl) contained 2.5 μM Mg-G-actin (10% pyrenyl-labeled), 0.5 μM VCA, and 10 μl of the tested fraction in 5 mM Tris-Cl−, pH 7.8 buffer, 1 mM DTT, 0.2 mM ATP, 0.1 M KCl, and 1 mM MgCl2. The time course of increase in pyrenyl-actin fluorescence was followed. The activity of Arp2/3 was detected by the steep sigmoidal increase in fluorescence, indicative of branched polymerization. The activity of Arp2/3 was easily observed, even in the S2 fraction. Alternatively, the activity of Arp2/3 was detected by the video-microscopy bacterial motility assay, using the property of Arp2/3 to initiate the formation of actin clouds at the surface of Listeria incubated in 4 μM rhodamine-labeled actin. The results of the two tests were in good agreement with each other, but the spectrofluorometric assay was more convenient and easily amenable to a quantitative estimate of the amount of Arp2/3 in the tested fractions by comparison with a calibration series of time courses performed with known concentrations of pure Arp2/3. The concentration of Arp2/3 complex was determined spectrophotometrically using an extinction coefficient of 224,480 M−1 · cm−1 at 278 nm, derived from the amino acid sequence of the seven polypeptides composing the 223,949-D complex. A good agreement was obtained between the spectrophotometric measurement and the bicinchoninic acid assay, with BSA as a standard. Typically, 1.2 nmol (0.26 mg) of pure Arp2/3 complex was obtained from 100 ml brain S2 supernatant.
Figure 2 Arp2/3 complex is essential for actin nucleation at the surface of E. coli (IcsA) and Listeria. a, Arp2/3 complex depletion in platelet extracts. Increasing amounts (20, 40, and 80 μg) of glutathione Sepharose-coupled GST or GST-VCA beads were (more ...)
Depletion of Arp2/3 Complex, VASP, and Profilin from Platelet Extracts
Arp2/3 complex was depleted from platelet extracts using a variation of the coprecipitation method described by Machesky and Insall 1998
. Various amounts of glutathione Sepharose-coupled GST-VCA beads (20, 40, and 80 μg) were incubated with 25 μl of platelet extracts for 30 min at 4°C on a rotating wheel. Beads were pelleted at 5,000 g
for 15 s. Depletion of the Arp2/3 complex was monitored by Western blotting of aliquots of protein extracts and beads. Add-back was done using the Arp2/3 complex purified from bovine brain.
VASP immunodepletion was carried out as described previously (Laurent et al. 1999
). Add-back was done using the recombinant VASP purified from insect cells.
Profilin depletion was carried out by two consecutive poly-l
-proline chromatography steps, as described by Laurent et al. 1999
. Profilin depletion was monitored by Western blotting. Add-back was done using the purified wild-type profilin or the H133S mutant profilin, described as unable to bind poly-l
-proline (Bjorkegren-Sjogren et al. 1997
Actin and Regulatory Proteins
Actin was purified from rabbit muscle and isolated as Ca-ATP-G-actin by gel filtration on Sephadex G-200 in G buffer (5 mM Tris-Cl−
, pH 7.8, containing 0.1 mM CaCl2
, 0.2 mM ATP, and 1 mM DTT). Actin was pyrenyl- or rhodamine-labeled, and converted into Mg-G-actin before polymerization, as described by Carlier et al. 1997
. Mg-actin was polymerized by addition of 1 mM MgCl2
and 0.1 M KCl (physiological ionic conditions). When actin was polymerized in the presence of gelsolin, no EGTA was added to the solution, and Ca-ATP-G-actin supplemented with the desired amount of gelsolin was polymerized by addition of 2 mM MgCl2
and 0.1 M KCl.
Gelsolin purified from human plasma was a kind gift from Dr. Yukio Doi (University of Kyoto, Kyoto, Japan).
Spectrin-actin seeds were isolated from human erythrocytes as described by Casella et al. 1986
Actin Polymerization Assays
Critical concentration plots were performed by serial dilution of pyrenyl-labeled F-actin solutions in F buffer (5 mM Tris-Cl−, pH 7.8, containing 1 mM DTT, 0.2 mM ATP, 0.1 mM CaCl2, 1 mM MgCl2, and 0.1 M KCl) and incubated overnight at room temperature. Polymerization assays were performed using the change in pyrenyl-actin fluorescence. Measurements were carried out in a Spex Fluorolog 2 instrument thermostated at 20°C, with excitation and emission wavelengths of 366 and 407 nm, respectively, and appropriate filters placed on the excitation and emisssion beams to eliminate stray light and bleaching, which interfere in polymerization in the presence of Arp2/3 (Pantaloni, D., manuscript in preparation).
The G-actin sequestering activity was measured by the shift in critical concentration plots with gelsolin-capped filament barbed ends. The equilibrium dissociation constant, Kd, for the actin-sequestering protein complex was derived from the measurements of the concentration, [A0], of unassembled actin at steady state (abscissa intercept of the critical concentration plots) and of the critical concentration, [AC], determined in the absence of sequestering agent. The following equation was used: Kd = [AC] · ([S0] − [A0] + [AC]) / ([A0] − [AC]), in which [S0] represents the total concentration of sequestering protein.
Rates of filament growth from barbed and pointed ends were measured using spectrin–actin seeds or gelsolin-capped filaments, added at time zero to a solution of 1 μM MgATP–G-actin (10% pyrenyl-labeled) in polymerization buffer. The gelsolin-capped filament solution contained 5 μM F-actin polymerized in the presence of 16.7 nM gelsolin for 2 h, and diluted 20-fold into the G-actin solution.
Frozen bacteria were thawed and spun down at 4,000 g
for 5 min at 22°C, resuspended at 6 × 109
bacteria/ml in XB buffer (10 mM Hepes, pH 7.7, 0.1 M KCl, 1 mM MgCl2
, 0.1 mM CaCl2
, and 50 mM sucrose). Motility assays were performed by mixing 4 μl of thawed extracts supplemented with 12 μl of a mixture containing 5 μM F-actin, 0.38% methyl cellulose, 6 mM DTT, 2 mM ATP/MgCl2
, 1.5 × 10−3
% DABCO, 1 μM rhodamine-actin, and 2 × 108
bacteria/ml. An aliquot of 2.5 μl was squashed between a 22 × 22 mm coverslip and a slide, sealed with VALAP, and incubated for 5 min at room temperature before motility measurements. Fluorescence microscopy observations and data acquisition were carried out as previously described (Laurent et al. 1999
). N-WASP-coated E
(IcsA) were prepared as follows: 20 μl of a suspension of 6 × 109
bacteria/ml were incubated with 5 μl of 1 μM N-WASP for 5 min at room temperature. Bacteria were pelleted, washed, and resuspended in 20 μl of XB buffer and kept on ice. The N-WASP–IcsA complex remains stable for several hours on ice.
Immunofluorescence Analysis of Shigella-infected HeLa Cells
HeLa cells were infected as follows: overnight culture of Shigella M90T strain was diluted in tryptic soy broth, grown to midexponential phase, centrifuged at 5,000 g for 5 min, washed, and resuspended at 108 bacteria/ml (M90T) in MEM, 50 mM Hepes, pH 7.5. HeLa cells, seeded on glass coverslips, were washed three times with MEM, overlaid with the bacterial suspension, and centrifuged for 10 min at 800 g. Bacterial entry was allowed by 30 min incubation at 37°C. Infected cells were washed with MEM five times and incubated with MEM, 50 mM Hepes, pH 7.5, with 50 μg/ml of gentamicin for a further 1 h at 37°C, to allow intracellular spread.
For immunofluorescence staining, infected cells were permeabilized with 0.2% Triton X-100 in 0.1 M MES, pH 7.4, 1 mM MgCl2, 1 mM EGTA, 4% PEG 6000, and then fixed for 30 min in 3.7% paraformaldehyde in PBS. After quenching with NH4Cl 50 mM in PBS for 10 min and saturation for 30 min in PBS-BSA 2%, infected cells were incubated with specific mono- or polyclonal antibodies. Affinity-purified antihuman N-WASP polyclonal NW011 antibodies and polyclonal Arp3 antibodies were used at 10 μg/ml concentration. Texas red-conjugated secondary antibodies (Nycomed Amersham Inc.) were used. F-actin was visualized by FITC-phalloidin (0.1 mg/ml; Sigma Chemical Co.). Preparations were examined with a confocal laser scanning microscope (Zeiss Axiophot).
In Vitro Protein Binding Assays
Glutathione Sepharose coupled to GST–IcsA or to GST was equilibrated in buffer P (20 mM Tris-Cl−, pH 7.5, 100 mM KCl, 1 mM MgCl2, and 0.1% BSA). 100 pmol of protein-bound beads and 80 pmol of purified N-WASP or VCA were mixed in 100 μl (total vol) and incubated for 1 h at 4°C on a rotating wheel. Supernatants were collected. Beads were rinsed twice with buffer P. Free and bound N-WASP and VCA were detected by Western blotting after resolving the proteins by SDS-PAGE.