Generation and analysis of ARPC3+/− gene-trap mice
ES cell clone (XG476) carrying the ARPC3 gene disruption was purchased from the Bay genomics (version CC183108.1) and were injected into the blastocyst of the C57BL6 blastocysts (3.5 dpc) with Nikon micromanipulators. The blastocysts were cultured in KSOM+AA for 2–3 h and were transplanted into the uterus of 2.5(dpc) pseudo-pregnant recipient F1 mothers (CBAXC57Bl/10). The offspring born with agouti coat color reflect the contribution of the ES cells to the developing embryo. Successful transmission of the mutation was initially determined by mating the chimeras to wild-type C57BL/6 females. Offspring with agouti coat color represent the transmission of the 129 ES cell background and were genotyped to identify the presence of the mutated allele. The mouse line was further established by intercrossing the heterozygous male and females. The ARPC3+/− line was established by backcrosses with mice of the C57BL/6 genetic background.
ARPC3+/+, ARPC3+/−, and ARPC3−/− ES cell derivation and maintenance
females (4–8 wk old) were mated with (ARPC3+/−
) males. The uterine horns and uterus were dissected from the 3.5-d pregnant female mice and the blastocysts were collected in M2 medium and washed and cultured in a drop of KSOM+AA media overlaid with mineral oil at 37°C in a humidified atmosphere with 5% CO2
. ES cell derivation was performed with slight modification of the protocols described previously (Egli et al., 2007
). In brief, blastocyst zonae pellucidae was removed by treating with acidic Tyrode’s solution (Millipore) and washed with KSOM+AA medium. The blastocysts were transferred individually into individual wells of 4-well IVF plates (Thermo Fisher Scientific) coated with 0.1% (wt/vol) gelatin and filled with a layer of iMEF (Global Stem) in 500 µl of ES cell derivation medium. This medium contains Knockout DMEM (Invitrogen), 20% Knockout FBS (Invitrogen), 2 mM l
-glutamine (Invitrogen), 50 U/ml penicillin/50 µg/ml streptomycin (Invitrogen), 0.1 mM β-mercaptoethanol (BD), 0.1 mM MEM nonessential amino acids (Invitrogen), 1,000 U/ml recombinant mouse LIF (Millipore), and mitogen-activated protein kinase inhibitor (PD98059; Cell Signaling Technology) at 37°C in an incubator with 5% CO2
. The blastocysts were allowed to attach to the iMEF feeder layer without disturbance for 6 d and cultured up to 12 d by changing the ES derivation medium every second day. The inner cell mass was mechanically detached using a glass pipette tip and transferred into a 12-well plate containing iMEFs. The ES cell colonies were cultured further, washed with calcium- and magnesium-free PBS, and trypsinized with 0.25% trypsin/EDTA for 2–5 min at 37°C. The cells were centrifuged and transferred into a 6-well plate and cultured in Knockout DMEM-FBS medium until they were confluent. At this stage, each ES clone was plated in duplicates in 6-well plates with one set being used for genotyping and the other for further colony expansion. To scale up the ES cell culture, cells were cultured in T75 flasks filled with iMEF and Knockout DMEM-FBS medium until they reached 70–80% confluence. The ES cells were frozen in 10% DMSO and 90% FBS and stored in liquid nitrogen.
ES cell differentiation into fibroblast using retinoic acid
ES cells were separated from the iMEFs by transferring into a new dish every 45 min, taking advantage of the fact that iMEFs are more adherent. The ES cells in supernatant were spun down at 1,000–1,200 rpm for 5 min and used either for genotyping or differentiation. The ES cells were seeded into pregelatin-coated T75 flask(s) or 150-mm dishes with ES cell medium containing LIF. Cells were allowed to settle down on plates overnight and replaced with fibroblast medium (DMEM high-glucose [Invitrogen], 10% FBS, 1% nonessential amino acids [Invitrogen], 0.1% 2-mercaptoethanol, 50 U/ml penicillin, and 50 µg/ml streptomycin) containing 3.33 × 107
M (0.33 µM) retinoic acid (Smith, 1991
). The media was changed twice at 24-h intervals. Cells were cultured for up to 7 d, with daily changes of medium after the initial 72 h. Undifferentiated ES cells were separated from the fibroblasts by consecutive transfer to a new dish every 30 min. The fibroblast cells re-adhere to the substrate faster than the undifferentiated ES cells. The differentiated fibroblast cells were able to survive for ~10 d after 7 d of differentiation and 2–3 passages.
Genotyping of ARPC3+/+ and ARPC3−/− ES cells and fibroblast cells and RT-PCR
Genomic DNA from ARPC3+/+, ARPC3+/−, and ARPC3−/− ES cells and fibroblast cells was isolated by homogenizing the cells in 20–40 µl extraction buffer and 5–10 µl tissue preparation buffer (Sigma-Aldrich). Homogenized samples were incubated for 20 min at room temperature, 5 min at 95°C, and then neutralized with 20–40 µl of neutralization buffer. The supernatant was used for the PCR genotyping using Red extract PCR mixture (Sigma-Aldrich) with the following primers: P1: ARPC3-Wt-F 5′-TGCAGGCATACCACTCTTCTCTCA-3′; P2: ARPC3-Wt-R 5′-AGCACCACGAATTGAGGCTAGAGT-3′; and P3: ARPC3-GT-R 5′-AAAGGGTCTTTGAGCACCAGAGGA-3′.
RT-PCR analysis in fibroblast cells was performed by isolating the total RNA from ARPC3+/+, ARPC3+/−, and ARPC3−/− fibroblasts using an RNA extraction kit (QIAGEN). cDNA was synthesized from total RNA using Superscript III (Invitrogen) reverse transcription with random hexamer primers (Invitrogen). To examine the expression of ARPC3 wild-type or gene-trap truncated mRNA, cDNA was amplified using the following primers: P4: Xg476-wt-E2F 5′-GGACACCAAGCTCATCGGTAACAT-3′; P5: Xg476-wt-E4R 5′-GATGTAGATCAATGTCCTGTCCGC-3′; and P6: Xg476-βgeo-R 5′-ATTCAGGCTGCGCAACTGTTGGG-3′.
ARPC3+/−, ARPC3+/−, and ARPC3−/− ES cells were cultured on a 0.1% (wt/vol) gelatin-coated glass-bottom dish for 3 d. Cells were washed and fixed with 4% paraformaldehyde in PBS for 25 min. Fixed cells were permeabilized by treatment with PBS/0.1% Tween 20 for 5 min and blocked by incubation with 3% BSA in PBS for 1 h. Cells were then incubated in PBS with 3% BSA containing Oct4 antibodies (mouse; Abcam) at a dilution of 1:100 overnight at 4°C. Cells were washed three times with PBS/0.1% Tween 20 and then incubated for 1 h with Alexa 488–conjugated mouse anti–rabbit secondary antibody at a dilution of 1:300 for 45 min at 37°C. Cells were washed with PBS/ 0.1% Tween 20 and then counterstained with fluorescent phalloidin (Invitrogen) for actin filaments and DAPI for DNA. The cells were mounted and imaged under a confocal microscope (LSM-510-LIVE; Carl Zeiss).
fibroblast cells were grown on glass coverslips (or) glass-bottom dishes for 24 h. Two different fixation protocols were used. For staining IB10, Arp2, and mDia1, the cells were fixed with 4% paraformaldehyde in PBS at 37°C for 30 min. After fixation and permeabilization as described above, cells were blocked with fetal bovine serum or 3% BSA for 1 h and incubated with the primary antibodies against: IB10 (1:75; Abcam), Arp2 (1:200 and 1:75 for two different batches; Santa Cruz Biotechnology, Inc.), ARPC3 (1:50; BD), and mDia1 (1:200; Santa Cruz Biotechnology, Inc.) overnight at 4°C. After three washes with PBS/ 0.2% Tween 20, the cells were incubated with a corresponding secondary antibody coupled to Alexa Fluor 488 (Invitrogen) at a dilution of 1:300 at 37°C for 1 h. Cells were washed with PBS/0.2% Tween 20 and then counterstained with Alexa Fluor 546 phalloidin (Invitrogen) and DAPI. For straining with anti-mDia2 (a gift from Dr. S. Narumiya, Kyoto University, Kyoto, Japan), cells were fixed with 10% TCA on ice for 15 min as described in Watanabe et al. (2008)
. The fixed cells were washed with PBS containing 30 mM glycine (G-PBS) and permeabilized with 0.2% Tween 20 in G-PBS for 5 min on ice, followed by three washes with G-PBS and processed as described above. For fascin antibody (1:50; Millipore) staining, the cells were fixed with methanol at −20°C for 15 min.
Alkaline phosphatase assay
ARPC3+/−, ARPC3+/−, and ARPC3−/− ES cells were cultured and fixed with 4% paraformaldehyde in PBS for 30 min at 37°C. Cells were washed in PBS and incubated with recommended substrate solutions (Vector Laboratories) for 20–30 min at room temperature until sufficient staining developed. Cells were imaged under a microscope (Axiovert; Carl Zeiss) using Plan Neofluor 10x/0.30 Ph1 (DIC I) 10x objectives.
Cell spreading and wound-healing assays
For observing cell spreading, ARPC3+/+ and ARPC3−/− fibroblast cells were trypsinized with 0.05% trypsin/EDTA for 5 min and centrifuged. The cells were resuspended in fibroblast medium and placed on a glass-bottom dish (MatTek Corp.) coated with 5 µg/ml of bovine fibronectin (Sigma-Aldrich) for 20 min in a 37°C incubator. The cell spreading was recorded on a microscope equipped with a 37°C incubator and 5% CO2 for a period of 2 h with frames taken every 2 min. Phase-contrast imaging was performed either with a microscope (LSM-510; Carl Zeiss) equipped with a Plan-Apochromat 20x/0.6 Ph2 air objective (Carl Zeiss) or with a microscope (Eclipse TE2000-E; Nikon) equipped with a Plan-Fluar 10x Ph1 DLL objective and a CCD camera (CoolSNAP; Photometrics). For the LSM-510 microscope, images were collected using the maximum field of view and 512 × 512 image size. The overall spreading area was measured by outlining the cell boundary every 10 frames using custom ImageJ segmentation software (described below).
For the wound-healing assay, ARPC3+/+ and ARPC3−/− fibroblast cells were cultured in fibroblast medium as described previously. Glass-bottom dishes were coated with 5 µg/ml fibronectin at 37°C for 1–2 h, washed with PBS, and attached to a culture insert (Ibidi). Fibroblast cells were trypsinized with 0.05% trypsin for 5 min, centrifuged, and resuspended in fibroblast medium. The cells suspensions (85 µl) were seeded at a density of 1.5–2.0 × 105 cells/ml and cultured overnight at 37°C with 5% CO2. The next day, cells were rinsed with PBS at least twice and switched to DMEM with 0.5% serum for 12 h. The culture insert was removed to create the “wound”, and cells were rinsed with PBS and fed with fibroblast culture medium supplemented with 10% FBS. The wounds were imaged with phase contrast on the LSM-510 microscope using a Plan-Neofluar 10x/0.30 Ph1 objective at different time points, and healing was quantified by manually measuring the wound areas that remained using Axiovision software (Carl Zeiss). For the cell-tracking analysis movies were made for 10 h with frames taken every 10 min; leading edge dynamics movies were 1.5 h long with an interval of 3 s/frame.
Chemotaxis was assayed following Ibidi protocol with several modifications. In brief, ARPC3+/+ and ARPC3−/− fibroblast cells were trypsinized and counted. The cell suspension was diluted to ~3 × 106 cells/ml. The μ-Slide Chemotaxis slides (Ibidi) were coated with 5 µg/ml of fibronectin at 37°C for 1–2 h and washed with PBS. The C, D, E, and F ports were closed with plugs and 6 µl of cell suspension was applied onto filling port (A) of the μ-Slide using a 20-µl pipettor and 6 µl of air was aspirated from the opposite filling port (B). The slide was placed in a sterile 10-cm Petri dish with a wet tissue around the slide and incubated in the tissue culture hood for 15 min and transferred to a 37°C incubator until the cells were attached. All plugs were gently removed from the filling ports and both reservoirs were filled with 70 µl chemoattractant-free medium. One of the filling ports was filled with 18 µl chemoattractant (500 ng/ml EGF) solution by removing 18 µl of medium from the other port on the same side of the device. Once again, all the ports were closed with plugs. Cell migration was recorded by mounting the μ-Slide on the stage of an inverted microscope with a 37°C incubator and 5% CO2. For the trajectory analysis, movies were made using the LSM-510 microscope with an A-Plan10x/0.25 Ph1 air objective or the Eclipse TE2000-E microscope as described above for a period of 12 h with frames taken every 10 min.
Cell trajectory analysis
Movies were acquired on an inverted microscope as described above in the wound-healing and chemotaxis assay sections. To generate the trajectories for the wound-healing assay, single cells at the wound edge were tracked by manually selecting the nucleus throughout the movie using a custom plug-in written for ImageJ (National Institutes of Health [NIH], Bethesda, MD). For the chemotaxis assay, single cells were tracked by selecting the center of mass throughout the length of the movie using the Chemotaxis and Migration tool for ImageJ from Ibidi. For both assays, speed, velocity, straightness, and α value were obtained by using Mathematica software (Wolfram Research Inc.). The trajectory path length and the displacement from the initial to the end point of each trajectory was computed. The speed and mean velocity are given by the ratio of the path length and the displacement to the total trajectory time, respectively. The trajectory straightness is computed as the ratio of displacement to path length. The mean squared displacement was found for each trajectory according to the following formula:
is the two-dimensional position at time t
, and < > denotes time averaging. This quantity was fit by nonlinear least squares to the following general formula: MSD
(τ) = 4D
, where D
is the apparent diffusion coefficient and α is a factor indicating nonrandom diffusion. For purely random motion (e.g., Brownian diffusion), α is 1 and MSD follows a straight line. For directed motion, α is greater than 1, and for confined motion α is smaller than 1.
For analysis methods involving leading edge tracking (cell spreading or leading edge dynamics), phase-contrast images were segmented using a custom plug-in written for ImageJ, which links to the OpenCV library (Willow Garage) following the example of the CellTrack program (Sacan et al., 2008
). In brief, each phase-contrast image frame was processed by first performing Canny edge detection followed by a single binary dilation. Subsequently, the binary image was outlined using the cvFindContours subroutine of OpenCV. Finally, the outline containing the largest area was selected and filled to create a cell mask.
For the kymograph analysis of leading edge dynamics, phase-contrast time-lapse movies were made with the LSM-510 microscope using a Plan-Apochromat 20x/0.6 Ph2 air objective for a period of 1.5 h with frames taken every 3 s. The cells chosen for analysis were single cells migrating in the wound. These cells were cropped from the full-size images and the leading edge boundary was masked frame by frame (see above) to reduce noise without altering the position of the cell edge. Masked images were further processed by automatic generation of several equally spaced (5° in angular measure) 1-pixel-wide lines from a manually chosen central point to the leading edge of the cell and subsequent calculation of the distance to the leading edge along each line in the direction of individual protrusions using a custom-written ImageJ plug-in. We note that the protrusion and retraction rate determined using this plug-in was confirmed by those determined with manual kymographs of the FLPs in the mutant cells. The distances generated above were subsequently analyzed using Mathematica software. Each kymograph was smoothed using a moving averaging window of 30 time points and regions of leading edge protrusion and retraction were determined as follows. First, the smoothed kymograph was segmented into 10 time point segments. The slopes of these segments were calculated from the starting and ending points of the segments. Slopes between 10% and −10% of the maximal and minimal slope for each trajectory were eliminated. Segments with contiguous regions with slopes of the same sign were flagged as protruding (positive slope) or retracting (negative slope). The durations and average slopes of these contiguous regions were averaged to give the final duration and rate measurements for each trajectory. In addition, for each time step the angular correlation C
) of the leading edge dynamics was computed. This quantity is defined as:
) is the velocity as a function of angular position ϕ
along the leading edge, ψ
is the angular shift, and < > denotes averaging over ϕ
. If the adjacent regions of the leading edge are concerted in velocity the width of the angular correlation function (at half of its maximal value) is large, whereas for uncorrelated velocity distributions this width tends to zero.
Online supplemental material
Fig. S1 shows generation of ARPC3+/−
gene-trap mouse and ARPC3+/+
ES cells. Fig. S2 shows characterization of ARPC3+/+
ES cells. Fig.S3 shows characterization of ARPC3+/+
fibroblast cells by immunofluorescence staining. Video 1 shows a spreading ARPC3+/+
fibroblast cell. Video 2 shows a spreading ARPC3−/−
fibroblast cell. Video 3 shows ARPC3+/+
fibroblast cells migrating toward the wound. Video 4 shows ARPC3−/−
fibroblast cells migrating toward the wound. Video 5 shows trajectories of individual ARPC3+/+
fibroblast cells during wound closure. Video 6 shows trajectories of individual ARPC3−/−
fibroblast cells. Video 7 shows ARPC3+/+
fibroblast cells responding to stimulation with 25 ng/ml EGF. Video 8 shows ARPC3−/−
fibroblast cells responding to stimulation with 25 ng/ml EGF. Video 9 shows trajectories of individual ARPC3+/+
fibroblast cells migrating in the presence of an EGF gradient. Video 10 shows trajectories of individual ARPC3−/−
cells migrating in the presence of an EGF gradient. Table S1 shows leading edge protrusion and retraction rates and durations. Table S2 shows quantification of leading edge dynamics. Online supplemental material is available at http://www.jcb.org/cgi/content/full/jcb.201112113/DC1