Arp2/3 complex and actin from A. castellani
were purified as described previously (Zalevsky et al., 2001
). Bovine Arp2/3 complex was a gift from T. Pollard (Yale University, New Haven, CT), and recombinant human Arp2/3 complex was a gift from M. Welch (University of California, Berkeley, Berkeley, CA). N-WASP VCA and ScarVCA were expressed in E. coli
and purified as described previously (Zalevsky et al., 2001
The Arp2/3 complex was precipitated by chloroform/methanol extraction and resuspended in first-dimension rehydration buffer (8 M urea, 2% CHAPS, 0.5% pharmalyte, pH 3–10, 0.002% bromophenol blue, and 18 mM DTT). Protein samples were separated in the first dimension with an Ettan IGphor 3 IEF system (GE Healthcare) on Immobiline Dry Strips (pH 3–10NL, 7 cm; GE Healthcare) at 1,500 volt hours for 10 h. The focused proteins were then separated in the second dimension on Nu-PAGE 12% Bis-Tris precast gels (Invitrogen).
SDS-PAGE and immunoblotting
Arp2/3 complex was separated on 12.5% SDS-PAGE gels and transferred to Immobilon-P (Millipore) by standard methods. Immunoblots were incubated in 3% BSA in TBS-T (25 mM Tris, pH 7.5, 150 mM NaCl, and 0.1% Tween-20) for 2 h, and monoclonal anti-pTyr (Cell Signaling Technology) or polyclonal anti-pThr (Invitrogen) were added at 200 ng/ml. Primary antibodies were detected with horseradish peroxidase–conjugated anti–mouse and anti–rabbit antibodies (BioRad Laboratories), respectively, and visualized with an ECL chemiluminescence kit (Pierce).
Arp2/3 complex dephosphorylation
Arp2/3 complex was dephosphorylated with AP (New England Biolabs, Inc.). Arp2/3 complex was diluted 1:1 with 2 mM Tris, pH 8, and combined with HipH Buffer (50 mM Tris, pH 8, 1 mM Mg2Cl2, and 0.1 mM ZnCl2) containing 1 U AP and incubated at 30°C for 1.5 h. For mock-treated controls, Arp2/3 complex was incubated with heat-inactivated phosphatase (65°C for 30 min) in HipH reaction buffer. For YOP dephosphorylation, Arp2/3 complex was incubated with 1 U YOP (New England Biolabs, Inc.) in 50 mM Tris-HCl, pH 7, 100 mM NaCl, 5 mM DTT, and 0.01% Brij 35 at 30°C for 1 h. For PP2Cα dephosphorylation, the Arp2/3 complex was incubated with 1 U PP2Cα (EMD) in 25 mM MES, 50 mM NaCl, 2 mM MnCl2, and 1 mM DTT at 30°C for 1 h. The Arp2/3 complex was affinity purified after phosphatase treatment by using N-WASP VCA coupled to activated CH–sepharose 4B (GE Healthcare).
Pyrene actin polymerization assays were performed with 4 μM monomeric actin containing 5% pyrene-labeled actin in KMEI (50 mM KCl, 1 mM MgCl2
, 1 mM EGTA, and 10 mM imidazole, pH 7), 5 nM Arp2/3 complex, and 500 nM ScarVCA/N-WASP VCA domain. Ca-ATP actin was converted to Mg-ATP actin by incubation in ME (50 mM MgCl2
and 0.2 mM EGTA) before adding other assay components. Pyrene actin was excited in an RF-5301PC spectrophotometer (Shimadzu) at 365 nm, and fluorescence was measured at 407 nm at 1-s intervals. Actin filament barbed ends were calculated as described previously (Higgs, et al., 1999
). Pointed elongation from gelsolin-capped actin filaments was measured as described previously (Mullins et al., 1998
). Gelsolin-capped actin filaments (100 nM) were used for pointed end–binding assays with untreated and dephosphorylated Arp2/3 complex.
Quantification of Arp2/3 complex binding constants
Binding constants of Arp2/3 complex for NPFs were determined by using GST-ScarVCA and GST-N-WASP VCA covalently coupled to activated CH–sepharose 4B (GE Healthcare). GST-NPF–coupled beads were added to mock-treated or AP-treated Arp2/3 complex and incubated at room temperature for 30 min. NPF-coupled beads were spun at 700 g
for 5 min, the supernatant was removed, and beads were resuspended in SDS-PAGE sample buffer. Coomassie-stained gels were scanned and quantified using a LabWorks imaging system and LabWorks Software (UVI). The data were plotted and fitted using GraphPad Prism software (GraphPad Software, Inc.). Binding constants for Arp2/3 complex for actin filaments were determined by actin cosedimentation as described previously (Mullins et al., 1997
Proteins were separated on 12.5% SDS-PAGE gels, individual bands were excised, and gel slices were destained with 25 mM NH4HCO3/50% acetonitrile. After reduction with 10 mM DTT and alkylation with 55 mM iodoacetamide, proteins were digested with 12.5 ng/μl of recombinant porcine trypsin (Roche) in 25 mM NH4HCO3 at 37°C for 16 h. Peptides were extracted from the gel slices and cleaned using a ZipTipC18 (Millipore) pipette tip. Mass spectrometry was performed with a matrix-assisted laser desorption/ionization time-of-flight mass spectrometer (Voyager-DE STR; Applied Biosystems).
MTLn3 cell culture and metabolic labeling
MTLn3 rat adenocarcinoma cells (provided by J. Condeelis, Albert Einstein College of Medicine, New York, NY) were maintained as described previously (Segall et al., 1996
). For metabolic labeling, 3 × 106
cells were plated on 150-mm dishes and grown to ~85% confluence in MEMα without nucleosides supplemented with 10% FBS at 37°C, 5% CO2
. Cells were transferred to phosphate-free MEMα without nucleosides supplemented with 0.2 mCi/ml [32
P]orthophosphate for 4 h and then stimulated with 10 nM EGF. Cells were lysed at the indicated times with modified RIPA buffer (50 mM Tris, pH 7.5, 100 mM NaCl, 10% glycerol, 1% NP-40, 1 mg/ml aprotinin, 1 mM pefabloc, and 1 mg/ml leupeptin) containing phosphatase inhibitors (1 mM EGTA, 50 mM NaF, 10 mM sodium pyrophosphate, 1 mM β-glycerophosphate, and 1 mM sodium orthovanadate), and the Arp2/3 complex was immunoprecipitated with anti-ARPC1 antibody (a gift from M. Welch, University of California, Berkeley) as described previously (Machesky and Insall, 1998
S2 cell culture, Arp2 RNAi, and Arp2 mutants
Schneider S2 cells were cultured as described previously (Rogers et al., 2002
). Arp2 siRNA was performed with dsRNA to the 3′ untranslated region (UTR) coding sequence of D. melanogaster
Arp2 (5′-GUGUGUGUGCGGACCGCAAGAAAUAGGAUAAAAAAGUGAUAGAUUUCUUUUCUCUAUUUUCUATAGGUUUAAACCUUUCAGAUUUACGUGAUAUAUCCGUCUAUAUAUGUUUUUUUUUU-3′). The Arp2 3′ UTR was cloned into a TOPO plasmid (Invitrogen) containing a T7 promoter. RNA was amplified using a T7 Megascript RNA amplification kit (Ambion). Plasmids expressing D. melanogaster
Arp2 were constructed using Gateway cloning technology (Invitrogen). D. melanogaster
Arp2 was cloned from an S2 cDNA library, sequenced, and inserted into a pENTR-D-TOPO plasmid (Invitrogen). Arp2 was then cloned into an expression vector containing a single C-terminal GFP. Mutant Arp2 sequences were constructed using a QuikChange Mutagenesis kit (Stratagene). RNAi was performed over 7 d as described by Rogers et al. (2003)
. In brief, 5 μg Arp2 double-stranded RNA was added to 24-well plates containing 3 × 105
cells on day one and again on day three. Cells were transformed with plasmids using Cellfectin LTX (Invitrogen) on day five. Plasmid transfection in S2 cells was ~30% efficient. On day seven, S2 cells were prepared for microscopy as described previously (Rogers et al., 2003
) and mounted in fluorescent mounting medium (Dako). Images were acquired with a Roper SPOT charge-coupled device camera (Roper Scientific) on an Axiophot microscope (Carl Zeiss, Inc.) at 40× magnification at room temperature with Roper SPOT acquisition software. Images were assembled into figures using Photoshop (Adobe).
Arp2/3 structural model
Arp2/3 complex structure was analyzed using PyMol molecular viewing software and PDB 1K8K (Robinson et al., 2001
). Images were imported and notated using Illustrator software (Adobe).
Online supplemental material
Fig. S1 shows data related to AP treatment of Arp2/3 complex with mock-treated samples, N-WASP VCA, and phosphatase inhibitors. Fig. S2 shows the effect of AP treatment on Arp2/3 complex subunit association, NPF-binding, and F-actin binding. Fig. S3 shows mass spectrometry data for phospho-Arp2 and immunoblots of Arp2-GFP from S2 cells. Online supplemental material is available at http://www.jcb.org/cgi/content/full/jcb.200802145/DC1