Generation of WASP and Scar1 Derivatives
DNA encoding WASP tagged at its N terminus with both Met Arg Gly Ser (MRGS) 6xHis and FLAG epitopes was amplified by polymerase chain reaction (PCR) from a human WASP cDNA (a generous gift of Arie Abo, PPD Discovery, Menlo Park, CA). The PCR product was subcloned into pFastBac1 (Invitrogen, Carlsbad, CA) and pGEX4T-1 (Amersham Biosciences, Piscataway, NJ) to generate the plasmids pDY10-1 and pDY7, respectively. DNAs encoding WASP truncation derivatives BGPWCA, GPWCA, PWCA, and WCA were also amplified by PCR and were subcloned into pDY10-1 to generate plasmids pDY10-2 (BPGWCA), pDY10-3 (GPWCA), pDY10-4 (PWCA), and pDY10-5 (WCA). DNA encoding WASP-CA was generated by PCR and subcloned into pDY7 to make plasmid pDY33. WASP ΔP was amplified by PCR from WASPΔP (a generous gift of Arie Abo) and subcloned into pDY10-1.
The DNA encoding Scar1 tagged at its N terminus with MRGS 6xHis and FLAG epitopes was amplified by PCR and subcloned into pDY10-1 to generate pTD9 and into pDY7 to generate pTD1. The Scar1 truncation derivative PWCA was generated by PCR and subcloned into pTD9 to make pTD10. The Scar1 WCA and CA derivatives were generated by PCR and subcloned into pTD1 to make pTD3 and pTD4.
Expression and Purification of Recombinant Proteins
Recombinant WASP and its truncation derivatives BGPWCA, GPWCA, PWCA, WCA, and WASP ΔP, and recombinant Scar1, its derivative PWCA, and VASP were expressed in Sf9 cells by using the baculovirus expression system. Baculovirus strains were generated and used for infections according to the Bac-to-Bac baculovirus expression system (Invitrogen). After 60 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 and frozen in liquid N2. To prepare the lysate, cells were thawed and centrifuged at 200,000 × g.
To purify the recombinant proteins, Sf9 lysates were supplemented with 20 mM imidazole, incubated wtih Ni2+-NTA-agarose (QIAGEN, Valencia, CA) resin for 1 h at 4°C, washed with wash buffer (50 mM NaH2PO4, pH 8.0, 300 mM KCl, 20 mM imidazole), and eluted with elution buffer (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 control buffer [20 mM 3-(N-morpholino)propanesulfonic acid (MOPS), pH 7.0, 100 mM KCl, 2 mM MgCl2, 5 mM EGTA, 1 mM EDTA, 0.5 mM dithiothreitol (DTT), 10% [vol/vol] glycerol].
Recombinant GST-WASP WCA, WASP-CA, SCAR-WCA, and SCAR-CA proteins were expressed as glutathione S-transferase (GST) fusions in Escherichia coli BL21-CodonPlus-RP cells (Stratagene, La Jolla, CA). Cells were grown to an OD600 of 0.5 and induced with 0.4 mM isopropyl β-d-thiogalactoside at 37°C for 3 h. Proteins were bound to glutathione-Sepharose (Amersham Biosciences), washed with phosphate-buffered saline, and eluted with 10 mM glutathione. Eluted proteins were further purified by gel filtration chromatography as described above. The GST tag was cleaved by incubation with thrombin (~ 0.5 U/mg protein) for 5 min at 25°C, and the cleavage reaction was stopped by incubation with benzamidine Sepharose (Sigma-Aldrich, St. Louis, MO) for 20 min at 4°C. WASP and Scar1 derivatives were isolated from GST by Ni2+-NTA-agarose (QIAGEN) affinity chromatography as described above and transferred into control buffer by using Nap5 spin columns (Amersham Biosciences). All protein concentrations were determined by the Bio-Rad protein assay (Bio-Rad, Hercules, CA) with bovine serum albumin as a standard.
Recombinant human GST-WT profilin and GST-H133S profilin (plasmids kindly provided by Changsong Yang and Sally Zigmond, University of Pennsylvania, Philadelphia, PA) were expressed in E. coli
strain BL21 (DE), cleaved with thrombin, and purified as described previously (Yang et al., 2000
). Recombinant GST-Cdc42 V12 (plasmid kindly provided by Arie Abo) was expressed in E. coli
strain BL21 (DE). Protein was purified by glutathione affinity and gel filtration chromatography as described above and charged with guanosine-5′-O
-(3-thio)triphosphate (GTPγS) as described previously (Ma et al., 1998
). VASP was prepared as described previously (Skoble et al., 2001
Pyrene-Actin Polymerization Assays
Human platelet Arp2/3 complex (Welch and Mitchison, 1998
), rabbit skeletal muscle actin (Spudich and Watt, 1971
), and pyrene-labeled actin (Kouyama and Mihashi, 1981
) were prepared as described previously. Pyrene-actin polymerization assays were performed as described previously (Cooper et al., 1983
) with the following modifications. Pyrene-actin and unlabeled actin were thawed and transferred into fresh G-buffer (2 mM Tris, pH 7.4, 0.2 mM CaCl2
, 0.2 mM ATP, 0.2 mM DTT) by passing them over a 1-ml Sepharose G25 (Amersham Biosciences) spin column and were then mixed to generate 3 μM monomer solution with 10% pyrene actin. Arp2/3 complex (10 nM) or an equal volume of control buffer was mixed with initiation buffer (20 mM MgCl2
, 10 mM EGTA, 5 mM ATP) and 20 nM WASP/Scar1 derivatives in control buffer. This solution and the G actin solution (final concentration 2 μM) were incubated at room temperature for 1 min and then mixed to initiate actin polymerization.
To measure the effects of GST Cdc42-V12 and PIP2 [PI(4,5)P2] on the activities of the WASP truncation derivatives, 10 nM Arp2/3 complex or control buffer was mixed with initiation buffer and 30 nM of each WASP derivative. To test the effect of PIP2 alone, the premix was added to 2 mM PIP2 (Calbiochem, San Diego, CA) and Cdc42 buffer (14 mM MOPS, pH 7.0, 68 mM KCl, 1.4 mM MgCl2, 22.4 mM EGTA, 0.14 mM ATP, 0.34 mM DTT, 7% [vol/vol] glycerol, 1 mM GTPγS, 20.7 mM MgCl2). To test the effect of Cdc42 alone, 10 μl of 2 μM GST Cdc42-V12 and 1 μl of control buffer were added to the premix. To test the effect of both PIP2 and Cdc42, 30 μM PIP2 and 300 nM GST Cdc42-V12 in Cdc42 buffer were added to the premix. The premix ± Cdc42-V12, ± PIP2 and 2 μM G actin were incubated at room temperature for 1 min and then mixed to initiate actin polymerization. Assembly kinetics was monitored using a Fluorolog 3 fluorometer (Instruments S.A.; excitation wavelength 365 nm, emission wavelength 407 nm) maintained at a temperature of 25°C.
Bead Motility Assays
For coating beads with protein, 1 μl of carboxylated polystyrene beads (0.5 μm in diameter, 2.68% solids; Polysciences, Warrington, PA) was incubated with various concentrations of purified proteins in 20 μl of final volume control buffer (20 mM MOPS, pH 7.0, 100 mM KCl, 2 mM MgCl2
, 5 mM EGTA, 1 mM EDTA, 0.5 mM DTT, 10% [vol/vol] glycerol) for 1 h at room temperature, pelleted, and washed with XB (100 mM KCl, 0.1 mM CaCl2
, 2 mM MgCl2
, 5 mM EGTA, 10 mM K-HEPES, pH 7.7), and then resuspended in XB. The final amount of protein on the beads was determined by Western blotting with an anti-FLAG antibody by using known amounts of the FLAG-tagged proteins as a standard. For motility assays, beads were coated with a final concentration of 10–20 pmol of protein bound to 1 μl of bead slurry. For motility assays, beads were added to X. laevis
egg extract (Theriot et al., 1994
) supplemented with N
-hydroxysuccinimidyl 5-carboxytetramethyl rhodamine-labeled actin (Kellogg et al., 1988
) and 20× ATP-regenerating mix (150 mM creatine phosphate, 20 mM ATP, 2 mM EGTA, pH 7.7, 20 mM MgCl2
). The extract was squashed between a slide and a coverslip and viewed after 5-min incubation at room temperature for WASP/Scar1 derivative-coated beads or 1-h incubation at room temperature for ActA-coated beads. To determine rates of movement, differential interference contrast or fluorescence images were recorded every 10 s, and rates of movement were determined by averaging the distance moved in a 60-s time interval by using MetaMorph software (Universal Imaging, Downington, PA).
Statistical analysis was performed using MINITAB software. Motility rates measured for the derivative-coated beads were compared using an analysis of variance test followed by a Tukey's multiple comparison test (set at 5%).
Control resin (glycine-Sepharose) or poly-l-proline (Sigma-Aldrich)-Sepharose resin was blocked for at least 2 h in 30 mg/ml bovine serum albumin and washed 3× with XB (100 mM KCl, 0.1 mM CaCl2, 2 mM MgCl2, 5 mM EGTA, 10 mM K-HEPES, pH 7.7). Resin was then added to X. laevis egg extract and incubated for 30 min at 4°C. The degree of depletion was determined by immunoblotting with an anti-Xenopus profilin antibody (see below) and comparing to known dilutions of control extract. Addback was done using 2 μM recombinant human profilin (WT or H133S) and/or 0.2 μM recombinant human VASP or an equivalent volume of buffer (20 mM MOPS, pH 7.0, 100 mM KCl, 2 mM MgCl2, 5 mM EGTA, 1 mM EDTA, 0.5 mM DTT, 10% [vol/vol] glycerol). After addition of profilin/VASP/buffer, the extract was incubated on ice for 20 min, and motility assays were performed as described above, except that slides were incubated for 20 min at room temperature before viewing. For poly-l-proline (PLP) addition experiments, soluble 10 μM PLP dissolved in XB, or XB alone was added to extract and incubated for 20 min. The extract was incubated on ice for 20 min and motility assays were performed as described above.
To determine rates of movement, differential interference contrast or fluorescence images were recorded every 5 s and rates of movement were determined by averaging the distance moved in 20-s time intervals by using MetaMorph software. For PLP depletion experiments, statistical analysis was performed as described above. For PLP addition, statistical significance was examined using a Student's t test.
Carboxylated polystyrene beads (3 μl, 0.5 μm in diameter, 2.68% solids; Polysciences) were coated with 20 pmol of WASP or WASP GPWCA, and washed with control buffer (20 mM MOPS, pH 7.0, 100 mM KCl, 2 mM MgCl2, 5 mM EGTA, 1 mM EDTA, 0.5 mM DTT, 10% [vol/vol] glycerol). Coated beads were incubated with platelet extract, washed with control buffer, and proteins bound were determined by immunoblotting by using an anti-WIP antibody (kindly provided by Ines Anton and Narayanaswamy Ramesh, Harvard Medical School, Boston, MA).
Profilin was purified from Xenopus
egg extract by poly-l
-proline affinity chromatography by using the procedures described in Janmey (1991)
. Purified profilin was used to immunize rabbits, and anti-Xenopus
profilin antibodies were purified from serum as described previously (Harlow and Lane, 1999