Generation of S100A4 Knockout Mice
mice were generated through a research agreement with Ozgene Pty Ltd (Bentley DC, WA, Australia). A genomic clone of the S100A4 gene was isolated from a mouse C57BL/6 genomic DNA library. The targeting vector introduced a loxP site 305 base pairs upstream of the start methionine, which is present in the second exon of the S100A4 gene. A phosphoglycerine kinase (PGK) Neo cassette flanked by loxP sites was inserted 426 base pairs downstream of the stop codon in exon 3 (A). The entire targeting vector was sequenced because the 5′ and 3′ homology arms contain exonic and intronic sequences from upstream and downstream S100 family members. The targeting vector was electroporated into C57BL/6-derived Bruce4 embryonic stem (ES) cells (Kontgen et al., 1993
). ES cells that integrated the targeting vector were identified by Southern blot, and the PGK Neo cassette was removed by treatment with Cre recombinase. S100A4-targeted ES cells were microinjected into C57BL/6 blastocysts and chimeric mice were bred to generate heterozygous F1 mice on an inbred C57BL/6 genetic background. Floxed mice were crossed with Cre-deleter C57BL/6 mice (Schwenk et al., 1995
), which resulted in the complete removal of the S100A4 coding exons on one chromosome. Mice were bred to homozygosity and to remove Cre recombinase. Mice were maintained and inbred in a pathogen-free barrier facility. All experiments were performed according to protocols approved by the Animal Welfare Committee at the Albert Einstein College of Medicine (Bronx, NY).
Figure 1. Generation of S100A4 knockout mice. (A) Map of the S100A4 genomic locus with the S100A4 coding exons indicated in red. The targeting vector introduced loxP sites (black circles) that flank the S100A4 gene and a PGK Neo cassette for ES cell selection. (more ...)
Wild-type and S100A4-null animals were genotyped from tail DNA by polymerase chain reaction (PCR) analysis. The following primers were used for PCR of the S100A4 locus: forward primer (5′-AGCTGGGGTTTTTCCACTTT-3′) and reverse primer (5′-ATCCAACCCTTCATGGACAG-3′). Expected PCR products are 2.1- and 0.5-kb fragments for wild-type and S100A4−/− mice, respectively.
Isolation of Bone Marrow Macrophages
BMMs were isolated from 6- to 12-wk-old mice as described previously (Stanley, 1997
). In brief, bone marrow cells from femurs and tibias were seeded onto T-75 flasks, containing α-minimal essential medium (MEM) Plus (Invitrogen, Carlsbad, CA) supplemented with 15% fetal calf serum (FCS) and 10% WEHI conditioned medium. After 3 d, contaminating red blood cells, fibroblasts, and mature macrophages were removed by subtractive adherence to the plates, and nonadherent cells were transferred to fresh α-MEM Plus containing 15% FCS and 1000 U/ml human recombinant colony-stimulating factor-1 (CSF-1) (kindly provided by Dr. E. Richard Stanley, Albert Einstein College of Medicine). After an additional 2–3 d, adherent cells were cultured in α-MEM Plus containing 15% fetal bovine serum (FBS) and 10,000 U/ml human recombinant CSF-1 (standard growth medium). Day 5–14 BMMs were used for all experiments. For CSF-1 stimulation experiments, BMMs were CSF-1-starved for 16–20 h before the start of the experiment.
For BMM transduction, the human S100A4 and TurboRFP lentiviral expression plasmids were purchased from Open Biosystems (Huntsville, AL). To generate infectious lentiviral particles, human embryonic kidney (HEK)293T cells were cotransfected with the second generation packaging plasmids pCMV-dR8.2 dvpr and pCMV-VSVG (Addgene, Cambridge, MA) and the S100A4 or TurboRFP LentiORF plasmids. Virus was collected after 3 d and used to infect BMMs at day 8 of culture for 24 h in the presence of 5 μg/ml polybrene (Sigma-Aldrich, St. Louis, MO). Infected cells were cultured in BMM standard growth medium containing 1 μg/ml blasticidin S (MP Biomedicals, Solon, OH) for an additional 3–5 d and then used for experiments.
Antibodies and Reagents
The mouse monoclonal β-actin antibody was from Sigma-Aldrich. Antibodies to S100A4 were described previously (Li and Bresnick, 2006
). Antibodies to the C termini of human nonmuscle myosin-IIA and myosin-IIB, which react with the human, rat, and mouse myosins (Choi et al., 1996
; Lo et al., 2004
; Dulyaninova et al., 2007
), were used for blots against whole cell lysates. The antibody against human nonmuscle myosin-IIC, which reacts with monkey and mouse myosin-IIC (Buxton et al., 2003
; Golomb et al., 2004
), was a kind gift from Dr. Robert Adelstein (National Heart, Lung, and Blood Institute, Bethesda, MD). The BT561 human NMHC-IIA polyclonal antibody (Biomedical Technologies, Stoughton, MA) and antibodies to full-length platelet myosin-IIA (kind gift from Dr. Robert Adelstein) were used for Triton-insoluble fractions. Phospho-regulatory light chain (RLC) antibodies (T18/S19) were purchased from Cell Signaling Technology (Danvers, MA). Paxillin antibodies were from Millipore Bioscience Research Reagents (Temecula, CA). The paxillin pY118 and Pyk2 pY402 antibodies were from BioSource International (Camarillo, CA). Human fibronectin and the Pyk2, CD16/32, and phycoerythrin (PE)-Cy7–conjugated CD11b antibodies were from BD Biosciences (San Jose, CA). The fluorescein isothiocyanate (FITC)-conjugated GR-1, Alexa 488-conjugated F4/80, and allophycocyanin (APC)-conjugated CD11b antibodies were from Invitrogen. Recombinant mouse CSF-1 was purchased from R&D Systems (Minneapolis, MN). (−)-Blebbistatin was from Toronto Research Chemicals (North York, ON, Canada).
Eight- to 12-wk-old mice were injected intraperitoneally with 1 ml of sterile 3.8% of aged thioglycollate (Sigma-Aldrich). Seventy-two hours after injection mice were carbon dioxide asphyxiated, and cells were collected by intraperitoneal lavage using a total of 9 ml of ice-cold phosphate-buffered saline (PBS) supplemented with 0.5% bovine serum albumin (BSA) and 5 mM EDTA. Trace contaminating erythrocytes were selectively lysed by the addition of 10 ml of 1× RBC lysis buffer (150 mM NH4Cl, 0.1 mM EDTA, and 10 mM KHCO3) and incubated on ice for 15 min. Cells were counted with a hemocytometer and adjusted to 5 × 106/ml with cold PBS containing 0.5%BSA. In total, six to 11 mice were used for each time point in three independent experiments.
Cells derived from peritoneal exudates and BMMs were resuspended in PBS containing 0.5% BSA, and Fc receptors were blocked with CD16/32 antibodies. Peritoneal cells (5 × 105) were stained with FITC-Gr-1 or Alexa 488-F4/80 and PE-Cy7-CD11b antibodies. BMMs were reacted with Alexa 488-F4/80 and APC-CD11b antibodies. After staining, cells were washed twice with PBS and then resuspended and fixed in 1% paraformaldehyde/PBS. For flow cytometric analysis, forward and side scatter gates were set to exclude dead cells and aggregates. Data were collected using a FACScan cytometer (BD Biosciences) and analyzed with FlowJo version 8.7 software (TreeStar, (Ashland, OR).
The BAC1.2F5 macrophage cell line was cultured in α-MEM containing 10% FBS and 3000 U/ml human recombinant CSF-1. MDA-MB-231 and COS-7 cells were maintained in DMEM supplemented with 10% fetal bovine serum.
Intracellular S100A4 Concentration
To determine the total intracellular S100A4 protein concentration, wild-type BMMs or MDA-MB-231 cells were trypsinized and imaged with an UPlanFl 10×, 0.3 numerical aperture (NA) objective. The cell diameter was measured for 100 cells, and the total cell volume was calculated using the equation for the volume of a sphere, 4/3(πr3). Known cell numbers of BMMs or MDA-MB-231 cells were lysed directly in 2× Laemmli sample buffer and loaded onto a 12% Tricine SDS-polyacrylamide gel along with a standard curve of purified S100A4. S100A4 blots were processed as described below. The relative amount of S100A4 was estimated by densitometry using ImageQuant version 5.0 (GE Healthcare, Little Chalfont, Buckinghamshire, United Kingdom).
Triton Cytoskeleton Assay
Triton-insoluble fractions of CSF-1–stimulated (20 ng/ml) wild-type and S100A4−/− macrophages were prepared by lysis in ice-cold Triton X (Tx)-100 buffer (50 mM Tris-HCl, pH 7.4, 100 mM NaCl, 50 mM KCl, 5 mM MgCl2, 0.5% Triton X-100, 5 mM dithiothreitol (DTT), 1 mM phenylmethylsulfonyl fluoride (PMSF), and 5 μg/ml each of chymostatin, leupeptin, and pepstatin A). After 5 min of incubation on ice, the supernatant, which contains Triton X-100–soluble proteins, was removed. The Triton-insoluble fraction was washed twice with ice-cold PBS, resuspended in 2× Laemmli sample buffer, boiled, and separated on 8% SDS-polyacrylamide gel electrophoresis (PAGE) followed by immunoblotting with antibodies to myosin-IIA or β-actin. For whole cell lysates, cells were washed with cold PBS and lysed in ice-cold Tx-100 buffer containing 1% SDS, 1 mM EDTA, and 1 mM DTT. Protein concentrations were determined with the DC protein assay (Bio-Rad Laboratories, Hercules, CA) using BSA as a standard. The relative amounts of cytoskeletal myosin-IIA, actin, and paxillin were determined by densitometry using ImageQuant version 5.2.
For experiments using blebbistatin, CSF-1–starved cells were treated with 5 μM active (−)-blebbistatin or with vehicle alone (0.05% dimethyl sulfoxide [DMSO]) for 15 min. Cells were stimulated with 20 ng/ml CSF-1 for 15 min, lysed with ice-cold Tx-100 buffer, and processed as described above.
To prepare whole cell extracts, BMMs were lysed in a buffer (50 mM Tris-HCl, pH 7.4, 100 mM NaCl, 50 mM KCl, 0.5 mM DTT, 1 mM PMSF, and 5 μg/ml each of chymostatin, leupeptin, and pepstatin) supplemented with 2% SDS. For S100A4 or β-actin immunoblots, lysates were separated on a 12% Tricine SDS-polyacrylamide gel and transferred to a polyvinylidene difluoride membrane. For myosin-II immunoblots, lysates were separated on a 6% glycine SDS-polyacrylamide gel and transferred to a nitrocellulose membrane.
For phospho-paxillin and phospho-Pyk2 immunoblots, BMMs were lysed in the buffer described above supplemented with 20 mM NaF and a 1:100 dilution of phosphatase inhibitor cocktails I and II (Sigma-Aldrich). Lysates were separated on an 8% Tricine SDS-polyacrylamide gel. Immunoreactive proteins were detected using the SuperSignal West Pico chemiluminescent detection system (Pierce Chemical, Rockford, IL). The relative amounts of pY118 paxillin/total paxillin and pY402 Pyk2/total Pyk2 were estimated by densitometry. β-Actin immunoblots were used as a loading control. Phosphorylation was expressed as the fold change relative to unstimulated cells.
For phospho-RLC antibody (T18/S19) immunoblots, total cell lysates from CSF-1–stimulated cells were prepared by direct addition of ice-cold 10% trichloroacetic acid supplemented with 10 mM DTT to the cell culture dish. Cell samples were collected by scraping followed by microcentrifugation. Cell pellets were washed twice with ice-cold acetone containing 10 mM DTT and resuspended in 2× Laemmli sample buffer and separated on 15% SDS-PAGE. Relative phosphorylation of the RLC was estimated by densitometry as described above.
Rac Activation Assay
At specific times after CSF-1 stimulation, dishes were placed on ice and BMMs were washed with ice-cold PBS containing 0.5 mM sodium vanadate. Cells were lysed in a buffer containing 25 mM Tris-HCl, pH 7.5, 150 mM NaCl, 5 mM MgCl2, 1% NP-40, 1 mM DTT, 5% glycerol, 0.5 mM sodium vanadate, 1 mM PMSF, and 5 μg/ml each of chymostatin, leupeptin, and pepstatin. Total protein concentrations were determined using the DC protein assay. Active guanosine triphosphate (GTP)-bound Rac1 pull-down assays were performed with a Rac1 activation kit (Pierce Chemical). One-twelfth of the total lysate was used for the detection of total Rac1. The remaining lysate (0.8 mg of total protein) was used to pull down GTP-Rac1 as per the manufacturer's instruction. Total cell lysates and Rac1 pull-downs were resolved by 12% Tricine SDS-PAGE. Proteins were detected with the Rac1 antibody supplied in the kit. The relative amounts of active and total Rac1 were estimated by densitometry. Relative Rac1 activation was determined by dividing the values obtained by densitometry of Rac1-GTP pull downs by the values obtained for total Rac1. Activation was expressed as the fold change relative to unstimulated cells.
Boyden Chamber Assays
CSF-1–starved BMMs were seeded into the top chamber of an 8-μm transwell in CSF-1–free medium or in medium containing 20 ng/ml recombinant mouse CSF-1 for chemotaxis and chemokinesis experiments, respectively. The transwells were placed into wells containing medium supplemented with 20 ng/ml CSF-1, and the cells were allowed to migrate at 37°C for 3 h or for 5 h (rescue experiments). For controls, CSF-1–free medium was placed into the lower well. Membranes were fixed with 3.7% formaldehyde and stained with 4,6-diamidino-2-phenylindole (DAPI). From nine to 20 random fields of cells on the underside of the membrane were counted using an UPlanFl 10×, 0.3 NA objective. Migration toward CSF-1 was expressed as the fold change relative to migration in the absence of CSF-1 in the lower well.
BMMs (8 × 104) were plated on 35-mm bacterial plastic Petri dishes in standard macrophage growth medium. For imaging, the cells were maintained at 37°C using a PDMI-2 stage microincubator (Harvard Apparatus, Holliston, MA), and the pH was maintained by perfusing the medium with 5% CO2. Evaporation was prevented by overlaying the medium with mineral oil. Phase images were acquired with an UPlanFl 10×, 0.3 NA objective every 5 min for 5–6 h.
For kymography of randomly migrating cells, 8 × 104 BMMs were plated on 35-mm glass-bottomed dishes (MatTek, Ashland, MA) coated with 3 μg/cm2 (13.2 μg/ml) fibronectin. Before imaging, the medium was replaced with prewarmed growth medium containing 20 mM HEPES. The temperature was maintained at 37°C using a heated stage (WPI, Sarasota, FL). Images were recorded every 20 s over a 20-min period using a UPlanFl 20×, 0.5 NA objective.
BMMs (8 × 104
) were plated on 35-mm glass-bottomed dishes coated with fibronectin as described above. A Femtojet micromanipulator and pump (Eppendorf-Brinkmann Instruments, Westbury, NY) were used to control the position and the flow from the micropipette. A micropipette was filled with 120 ng/ml recombinant mouse CSF-1 and 0.5 mg/ml 10-kDa rhodamine-dextran and placed ~60 μm from the edge of a quiescent cell. Images were recorded every 20 s over a 30-min period using a UPlanFl 20×, 0.5 NA objective with the magnification selection knob set to 1.5. Cell perimeters were traced and the coordinates of the cell centroid for each frame of the film were determined using ImageJ (National Institutes of Health, Bethesda, MD; Rasband, 1997–2007
). Cell motility parameters and difference pictures were calculated using macros developed by the Analytical Imaging Facility and the Gruss-Lipper Biophotonics Center (Albert Einstein College of Medicine). Directionality is defined as the net path divided by the total path length. The angle θ describes the motility path of a cell and is defined by two reference lines; the line from the cell centroid to the tip of the micropipette at t = 0, and a line from the cell centroid at t = 0 to the cell centroid at t = final.
For assays performed in the presence of blebbistatin, CSF-1–starved macrophages were treated with 5 μM (−)-blebbistatin or DMSO (0.05%) for 15 min before directional stimulation with 120 ng/ml CSF-1.
Cells directionally stimulated with CSF-1 for 15 min were fixed by the addition of an equal volume of 8% paraformaldehyde to the cell culture medium (final concentration, 4%). Fixed cells were permeabilized with 0.2% Triton X-100 for 5 min, blocked with 10% horse serum and 1% BSA in Tris-buffered saline, and stained with the pY118 paxillin antibody. For the analysis of paxillin pY118 immunofluorescence in directionally stimulated cells, cell perimeters were traced and the coordinates of the cell centroid were determined. A 90° angle was drawn from the cell centroid to the cell edge along the side of the cell facing the micropipette. The mean pixel intensity was determined for the cellular region delineated by the 90° angle (cell front) and for the entire cell. The data are expressed as the ratio of the mean pixel intensity of the cell front (region facing the micropipette) to the entire cell.
All images were acquired using IPLab Spectrum software and a CoolSNAP HQ interline 12-bit, cooled charge-coupled device camera (Roper Scientific, Trenton, NJ) mounted on an IX70 microscope (Olympus, Melville, NY).
For kymographs of randomly migrating cells, cells were overlaid with a template of eight equally spaced lines radiating from the cell nucleus to the cell periphery (Miller et al., 2004
). Kymographs were generated and analyzed with ImageJ and Excel software (Microsoft, Redmond, WA) at all eight positions for each cell. For directionally stimulated cells, kymographs were generated by drawing a line from the tip of the micropipette to the cell centroid before CSF-1 stimulation. The frequency, persistence, and velocity of lamellipodial protrusions and retractions were quantified as described previously (Hinz et al., 1999
Unpaired Student's t tests were performed to assess the statistical significance of all assays.