HeLa, a human cervix carcinoma cell line, and ECV304, a vascular endothelial cell line, were cultured in DME with high glucose (4.5 g/liter), l-glutamine, and sodium pyruvate (PAA Laboratories GmbH) supplemented with 10% (vol/vol) FCS. HCC1937 is a primary breast ductal carcinoma cell line cultured in RPMI 1640 medium with l-glutamine and 1 mM sodium pyruvate supplemented with 10% (vol/vol) FCS. CHO is a CHO cell line cultured in Ham’s F12 medium with l-glutamine and supplemented with 10% (vol/vol) FCS. MCF-7 is a human breast adenocarcinoma cell line cultured in MEM with Earle’s salts, without l-glutamine, supplemented with 1% (vol/vol) nonessential amino acids and 10% (vol/vol) FCS. All cell lines were obtained from the American Type Culture Collection.
Stable cell lines
CHO and MCF-7 cell lines were transfected with 5 µg pEGFP-BRCA11634-–1863 (pEGFP-CTD) DNA using Lipofectamine 2000 (Invitrogen) according to the manufacturer’s instructions. G418 (Sigma-Aldrich) was used as a selection drug at 800 µg/ml. Established stable CHO–EGFP-CTD and MCF-7–EGFP-CTD cell lines were cell sorted according to expression levels and referred to as CHO-High and MCF-7–High for high and CHO-Low for low expression levels.
The MCF-7 cell line was transfected with 5 µg of full-length BRCA1-pIRES2-EGFP and full-length BRCA1 with point mutation I26A-pIRES2-EGFP plasmid DNA using Lipofectamine 2000 according to the manufacturer’s instructions. G418 was used as a selection drug at 600 µg/ml.
To generate stable BRCA1-reconstituted HCC1937 cells, HCC1937 cells were electroporated with full-length BRCA1-pIRES2-EGFP, full-length BRCA1 with point mutation I26A-pIRES2-EGFP (provided by X. Yu, Mayo Clinic, Rochester, MN), or pEGFP-CTD DNA using an electroporation system (Gene Pulser II; Bio-Rad Laboratories) at 250 V and 960 µF for 51.1 ms. A few days after electroporation, the cells were put under 600 µg/ml G418 selection.
Membrane fraction preparation
HeLa P2 fractions were prepared as previously described (Coene et al., 2005
) with minor modifications. Nearly confluent HeLa cells were washed with ice-cold PBS, scraped, and harvested. The pellet was resuspended in 1 ml of isolation buffer (10 mM Tris-HCl, 10 mM NaCl, 1.5 mM MgCl2
O, 1 mM EDTA, 70 mM sucrose, and 210 mM mannitol, pH 7.5) with protease inhibitors (Roche) and homogenized with 40 strokes using a Balch homogenizer (custom made by EMBL workshop) on ice. The membrane fraction was collected by differential centrifugation, dissolved in 25 mM Tris-HCl, pH 8.0, and 10 mM CaCl2
O, and treated with 50 ng/ml proteinase K (Fluka; Sigma-Aldrich) in 10 mM Tris-HCl, pH 8.0, and 1 mM CaCl2
O for 30 min at RT. The reaction was stopped with 10 mM PMSF for 10 min at RT. For 1D SDS-PAGE, samples were denatured by adding sample buffer with 100 mM DTT (Sigma-Aldrich) and heated for 5 min at 95°C before loading. For 2D electrophoresis, samples were acetone precipitated and redissolved in IEF solubilization buffer (7 M urea, 2 M thiourea, 2% [wt/vol] CHAPS, 65 mM DTT, 2.0% [wt/vol] ampholine [IPG buffer; GE Healthcare], and trace bromophenol blue [Sigma-Aldrich]).
1D and 2D ligand overlay blotting
For 1D SDS-PAGE, membrane fractions (equivalent to 106 cells per lane) were resolved by SDS-PAGE using standard techniques. The gel was either stained with Imperial Protein Stain (Perbio Science) and processed for LC-MS/MS or blotted. Nitrocellulose membrane was incubated overnight with HeLa lysate. Untransfected, pEGFP-CTD–transfected (3.2 µg DNA per well of a 6-well plate), and pEGFP-NTA–transfected (3 µg DNA per well of a 6-well plate) HeLa cell lysates were made using radioimmunoprecipitation assay (RIPA) buffer (Millipore) and protease inhibitor cocktail tablets (Roche) according to the manufacturer’s instructions. Goat polyclonal anti-GFP antibody (dilution 1:5,000; Abcam) and donkey anti–goat HRP-labeled secondary antibody (dilution 1:4,000; Jackson ImmunoResearch Laboratories, Inc.) were used for detection.
For 2D electrophoresis, immobilized gel strips (Immobiline Drystrip; GE Healthcare) of 13 cm and a linear pH gradient range, pH 3–10, were rehydrated in IEF solubilization buffer. Protein (150 µg/strip) was loaded from a cup on the cathodic end. Proteins were electrofocused as follows: 30 min at 250 V, 3-h linear gradient at 250–8,000 V, and 14 h at 8,000 V, all at 20°C. IEF gel strips were equilibrated in 50 mM Tris-HCl, pH 8.8, 6 M urea, 30% (vol/vol) glycerol, 2% (wt/vol) SDS, and 65 mM DTT for 15 min followed by incubation in SDS equilibration buffer supplemented with 135 mM iodoacetamide (Fluka) for 15 min. Gel strips were placed onto the top of 10% SDS-PAGE and run at 8 W/gel for 2.5 h. 2D gels were either fixed in 10% (vol/vol) methanol and 7% (vol/vol) acetic acid and stained with SYPRO ruby (Sigma-Aldrich) for further imaging and spot excision or semidry transferred to nitrocellulose membrane and processed as described in the previous paragraph. For BRCA1 detection in CHO lysate on a Western blot, we used Ser988 (1:100) and Ser1497 (1:200) anti-BRCA1 antibodies (Santa Cruz Biotechnology, Inc.) as primary antibodies and donkey anti–goat HRP-labeled secondary antibody (dilution 1:4,000) for detection.
HeLa cells were treated with 150 nM PMA (Sigma-Aldrich) for 90 min at 37°C and lysed on ice using RIPA buffer (Sigma-Aldrich) supplemented with 1 µg/ml aprotinin, 1 µg/ml leupeptin, 1 mM NaF, 1 mM PMSF, and 1 mM Na3VO4. The lysate was rotated for 15 min at 4°C on an orbital rocker and centrifuged for 15 min at 14,000 g at 4°C. The supernatant was precleared by adding a 50% slurry of Protein A/G PLUS beads (Tebu-Bio) and rotating for 30 min at 4°C. 5 mg/ml of cell lysate was incubated with 2 µg each of mouse anti-BRCA1 Ab1 (MS110; Oncogene Science–Merck Chemicals), mouse antitryptase (AbCys), and mouse IgG1 pure (BD) antibodies and no antibody for 90 min at 4°C on an orbital shaker. A 50% slurry of Protein A/G PLUS beads was added overnight. Beads were washed three times with ice-cold RIPA buffer. Immunoprecipitated proteins were denatured by the addition of sample buffer, boiled for 5 min, resolved by 6% Tris-glycine SDS-PAGE, and analyzed by immunoblotting using mouse anti-BRCA1 Ab1 (1:50), mouse antitryptase (1:500), and rabbit antiezrin (1:1,000; Cell Signaling Technology) antibodies. For detection, we used goat anti–mouse HRP (Thermo Fisher Scientific) and goat anti–rabbit HRP (Dako) antibodies in combination with the SuperSignal West Dura Extended Duration Substrate kit (Thermo Fisher Scientific). Images were taken on an imaging system (ULTima16si Pro; Isogen Life Science).
The tubulin sequence was excised from an EGFP-tubulin vector (Takara Bio Inc.) by XhoI and XbaI digest (Roche). BRCA1 primers, including XhoI and XbaI restriction side ends, used in this study were BRCA1 (1,634–1,863 aa), 5′-GGCTGTCTAGAGTCAGTAGTGGCTGTG-3′, and BRCA1 (1,634–1,863 aa), 5′-AGATCTCGAGAGAAGCCAGAATTGACAGC-3′. DNA template pFLAG-YFP–full-length BRCA1 plasmid (gift from B. Henderson, Westmead Hospital, Sydney, Australia) was used. Inserts were digested with XhoI and XbaI, ligated in the vector, and transformed in Top10F’, and minipreps of the obtained colonies were prepared according to routine procedures. Sequence analysis was performed using the following primers: GFP forward, 5′-CATGGTCCTGCTGGAGTTCGTG-3′, and SV40 reverse, 5′-GGACAAACCACAACTAGAATGC-3′. The control pEGFP-NTA plasmid was a gift from Q. Sattentau (Dunn School of Pathology, Oxford, England, UK) and J. Komano (AIDS Research Center, Tokyo, Japan). Plasmids were transformed in DH5-α, and maxipreps of obtained colonies were prepared according to routine procedures. The control EGFP-neo was obtained from B. Wickstead (Dunn School of Pathology, Oxford, England, UK) by cutting pVP22 (Invitrogen) with HindIII–KpnI and ligating in EGFP from pGad8–VSG-S8 (Wickstead et al., 2003
). The EGFP-centrin plasmid was made in house (D.J. Vaux).
LC-MS/MS sample preparation and analysis were performed as follows: bands/spots were excised and diced. Gel fragments were destained using 25 mM ammonium bicarbonate (Fluka) in 50:50 double-distilled water/acetonitrile (HPLC grade; Sigma-Aldrich) followed by dehydration in 100% acetonitrile and vacuum drying (Speedvac). Proteins were reduced with 10 mM DTT and alkylated with 55 mM iodoacetamide. 200 ng trypsin (Promega) in 20 µl of 25-mM ammonium bicarbonate was added to the gel pieces, and proteins were digested at 37°C overnight. Digestion was stopped with formic acid (Fluka) to a concentration of ~0.1%, and peptides were extracted. Supernatants were pooled and evaporated to dryness. Peptides were resuspended in 0.1% formic acid and stored at −20°C before analysis. LC-MS/MS was performed on a mass spectrometer (Q-TOF Micro; Micromass) coupled to a capillary HPLC system (Waters). Proteins were identified by searching data files against Mascot (Matrix Science).
HeLa, ECV304, and HCC1937 cells were either cultured and transfected with 5 µg pEGFP-CTD/pEGFP-centrin/pEGFP-neo DNA using Lipofectamine 2000 according to the manufacturer’s instructions or cultured and prepared for immunofluorescence as previously described (Coene et al., 2005
). In brief, cells were seeded onto glass coverslips, grown until 60% confluence, transfected or not transfected, and fixed in 4% (vol/vol) PFA and 250 mM Hepes, pH 7.4, for 20 min at RT. Coverslips were quenched in 50 mM NH4
Cl in PBS for 10 min, permeabilized in 0.5% (vol/vol) Triton X-100 in PBS for 5 min, and blocked in 0.4% (vol/vol) fish skin gelatin (FSG; Sigma-Aldrich) in PBS for 30 min at RT. The primary antibodies were diluted in 0.4% (vol/vol) FSG in PBS and incubated for 1 h at RT. The Ser988 and Ser1497 anti-BRCA1 antibodies were incubated at 37°C for 1 h. Secondary antibodies were diluted in 0.4% (vol/vol) FSG in PBS and incubated for 1 h at RT. Washes were performed in PBS. Coverslips were rinsed in distilled water and mounted in Mowiol (Mowiol 4–88; Hoechst AG) in 0.2-M Tris-HCl, pH 8.5, supplemented with 0.1 µg/ml DAPI. The images were taken on a laser-scanning microscope (LSM 510 META; Carl Zeiss, Inc.) using a 63× oil lens, NA 1.4. Adjustments of brightness and contrast were applied using Photoshop (Adobe). For the competition and displacement assay, z stacks were taken with a 0.3-µm-thick optical section and a typical pixel size of 0.1 or 0.2 µm.
For some experiments, coverslips were rinsed with a jet of PBS directed from a wash bottle to provide mechanical shear stress that disrupted a proportion of the cells, leaving adherent membrane sheets behind. For other experiments, saponin treatment was performed as previously described (Beranger et al., 1994
Primary antibody dilutions used in this study were anti-BRCA1 antibodies Ab5 (1:50), Ser988 (1:50), Ser1497 (1:50), anti-ERM antibody (1:100; Cell Signaling Technology), 0.1 µg/ml TRITC-phalloidin (Sigma-Aldrich), and anti-FAK (1:50; BD). Secondary antibodies used were donkey anti–mouse Alexa Fluor 488 (1:500), donkey anti–goat Alexa Fluor 488 (1:500; both are in-house–labeled Jackson ImmunoResearch Laboratories, Inc. secondary antibodies using an Alexa Fluor 488–labeling kit from Invitrogen), donkey anti–rabbit Cy5 (1:500), donkey anti–mouse Cy5 (1:300; Jackson ImmunoResearch Laboratories, Inc.), and donkey anti–goat Alexa Fluor 647 (1:300; Invitrogen). For FRET, we used anti-pSer988 BRCA1 (1:50; Santa Cruz Biotechnology, Inc.), anti-ERM (1:100), and anti–human transferrin receptor (1:100; Invitrogen) antibodies labeled with donkey anti–goat Alexa Fluor 488 (1:500), donkey anti–rabbit Cy3 (1:300; Jackson ImmunoResearch Laboratories, Inc.), and donkey anti–mouse Cy3 (1:300; Jackson ImmunoResearch Laboratories, Inc.), respectively. As a positive control for FLIM, we used goat polyclonal anti-GFP antibody (1:1,000; Abcam) and donkey anti–goat Cy3 (1:300; Jackson ImmunoResearch Laboratories, Inc.).
HeLa cells were seeded and incubated at 37°C for a few hours. When they started to spread, cells were treated with 150 nM PMA for 90 min at 37°C, fixed, and processed for cryoimmuno-EM as previously described (Coene et al., 2005
). In brief, cells were fixed in 4% PFA/250 mM Hepes, pH 7.4, for 1 h at RT, embedded in 10% gelatin, and processed for cryosectioning. Primary antibodies were rabbit anti-ERM (1:10; Cell Signaling Technology) and mouse anti-BRCA1 Ab4 (1:5; Oncogene Science–Merck Chemicals). Secondary antibodies were goat anti–rabbit 10-nm gold and goat anti–mouse 5-nm gold conjugates (BB International). Images were collected on an electron microscope (Tecnai 12; FEI).
A microscope (Eclipse TE300; Nikon) incorporated in an imaging system (Radiance 2000 MP; Bio-Rad Laboratories) was used. A pulsed laser beam of 950 nm was obtained from a mode-locked Ti:Sapphire laser (Mira 900; Coherent, Inc.) for multiphoton excitation. Fluorescence lifetimes of EGFP were recorded using the direct detection system (Bio-Rad Laboratories) with a photomultiplier tube (H5783P; Hamamatsu) optimized for photon counting and FLIM and a fast-time–correlated single-photon–counting acquisition board (SPC-830; Becker & Hickl). Lifetime images were collected at 512 × 512 pixels with a 100-s scanning time and analyzed with SPCImage software (Becker & Hickl). Within SPCImage, mask polygons were drawn around regions of interest (ROIs), and binning was set to give an initial photon count level of ≥1,000. EGFP lifetimes within the cells were analyzed by matrix calculation on a pixel-by-pixel basis. A multiexponential decay model was used, as this gave the lowest χ2 value and the best fit. Lifetime histograms (t1) were exported to and analyzed in Excel (Microsoft). The EGFP lifetime in the absence of an acceptor was used to determine FRET efficiencies using the formula 100 × [1 − (t1/tunquenched EGFP)]. The acceptor used was Cy3, which was used to detect EGFP (positive control) or ERM.
FRET using the acceptor photobleaching method (Kenworthy, 2001
) was performed as previously described (Malhas et al., 2009
). This was performed on PMA-treated fixed cells after immunolabeling of ERM using anti-ERM antibody (1:100) and donkey anti–rabbit Cy3 as the acceptor and endogenous BRCA1 using Ser988 and donkey anti–goat conjugated to Alexa Fluor 488 as the donor. As a negative control, we used the same BRCA1–Alexa Fluor 488 donor but a mouse anti–human transferrin receptor primary antibody labeled with a Cy3-conjugated secondary antibody as an acceptor. An ROI containing membrane ruffles was selected, and both the donor and acceptor were detected for five scans. The ROI was then bleached with the 543-nm laser at 100% laser power. This was followed by scanning to detect any changes in the acceptor and donor intensities. FRET efficiency was then calculated using the following formula: 100 × [1 − (Ib
)], in which Ib
are the intensities of the donor before and after bleaching, respectively. Difference maps were calculated by subtracting “before” pixel values from ”after” pixel values and rescaling to fill the 8-bit histogram range (which required a scaling factor of 2.0).
PMA and cytochalasin B treatment
HeLa cells were transfected with pEGFP-CTD as described (see Immunofluorescence) and treated with 150 nM PMA for 90 min at 37°C. 20 µM cytochalasin B (Sigma-Aldrich) was added for 2 h at 37°C. Coverslips were fixed and further processed for immunofluorescence as described (see Immunofluorescence).
Competition and displacement
CHO-WT and CHO-High cells were fixed and immunostained with TRITC-phalloidin and anti-Ser988 BRCA1 antibody as described in Immunofluorescence. Confocal z stacks were obtained using an LSM 510 META and a 63× oil lens, NA 1.4. Adjustments of brightness and contrast were applied using Photoshop. To quantify the displacement, a journal (macro) was written in which we measured the proportion of overlapping actin and anti-BRCA1 staining in dual-channel immunofluorescence images using simple overlap parameters derived by Manders et al. (1993)
. We used the standard implementation of this parameter in the colocalization function of an image-processing program (MetaMorph; MDS Analytical Technologies; see MetaMorph colocalization macro for colocalization journal details). The percentage of values for endogenous BRCA1 overlap with F-actin was obtained for each plane in a z stack. For each z stack, only the colocalization values above a threshold cutoff (10% of the maximum value of all IF-actin
values in a specific z stack) were taken into account. Colocalization values of seven individual z stacks per cell type were pooled and plotted with corresponding means. Unpaired two-tailed t
test with Welch’s correction and nonparametric two-tailed Mann–Whitney test were performed. For the images in , z stacks resulting from this analysis were displayed as mean intensity z projections.
CHO-WT, CHO-High, and CHO-Low cells were seeded in equal densities (1–3 × 105 cells per well), fixed 3 h after seeding, mounted in Mowiol supplemented with DAPI, and imaged on a laser-scanning microscope (LSM 5 Pascal; Carl Zeiss, Inc.) using a 10× objective with NA 0.45. Phase contrast and EGFP images of five random fields on the coverslip were taken. The percentage of spread out cells per field was calculated. The percentage values for five fields from each of three independent experiments were pooled, averaged, and plotted with corresponding SDs after confirming no significant difference in variance for a condition between experiments using an F test. t tests were calculated. The area of the spread out cells versus rounded-up cells was measured using the Trace Region tool and Show Region Statistics option in MetaMorph software.
All cell lines were seeded in low densities in dishes (MatTek Corporation). 24 h after seeding, the cells were imaged on an LSM 5 Pascal system using a 10× objective with NA 0.45. Phase contrast and EGFP images of four random fields in the dish were taken per cell type. Each field was imaged over a time period of 1 h at 2-min intervals. For each cell line, 15 cells per field were manually tracked over 30 time points using the Track Points option in MetaMorph software. The mean speed of all 60 cells over 30 time points was calculated per cell type with corresponding t tests. For , the mean speed of all cells from two independent setups (120 cells) over 30 time points was calculated per cell type. The individual speed values per field, all in micrometers per minute, were grouped in bins ranging from 1 to 5 at 0.5 intervals for CHO cell lines, ranging from 1 to 10 at single intervals for MCF-7, MCF-7–-High, MCF-7–WT, MCF-7–I26A, HCC1937, HCC1937 + WT BRCA1, and HCC1937-I26A cell lines, ranging from 1 to 5 at single intervals for HCC1937–EGFP-CTD, and ranging from 0 to 2 at 0.2 intervals for HeLa-EGFP-centrin, and the frequency per bin was determined.
The mean of all frequencies per bin per cell type and corresponding SDs were calculated and plotted. To display the individual cell tracks, the Track Objects option in MetaMorph software was used.
CHO-WT, unsorted CHO–EGFP-CTD, and CHO-High cells were seeded in MatTek dishes and grown to confluence. Each cell layer was scrape wounded and imaged on an LSM 510 META microscope using the Multi Time Series software option. Phase contrast and EGFP images of four random wounds in the dish were recorded over a time period of 12 h at 2-min intervals. The wound perimeter was analyzed over time in a standardized way using an in-house analysis subroutine (see MetaMorph wound-healing macro for wound-healing MetaMorph journal details). For each two consecutive time points, the change in wound perimeter (width and length corrected) was calculated, summed, and referred to as the PVI. For each cell type, seven wounds from different experiments were analyzed using this journal, and the obtained PVI values per cell type were plotted in a scatter dot plot with the corresponding means. Nonparametric two-tailed Mann–Whitney U tests were performed.
MetaMorph colocalization macro
This is a description of the algorithm used in the MetaMorph journal Coloc_003_02.jnl. The method gives two values showing the percentage of fluorescence (above a defined threshold) in each channel (of a two-channel z-stack image) that occupies the same volume (pixel by pixel) as fluorescence (above a defined threshold) in the other channel. (a) It is assumed that there is no significant bleed through between channels. (b) It is assumed images have been filtered to reduce any significant noise. (c) It is assumed that each pixel represents a sum of background + fluorescence. (d) The background distribution is obtained from a background region in each section: MeanB = background mean in each section averaged over all sections; STDB = background SD in each section averaged over all sections. (e) The fluorescence distribution is obtained over the whole image in each section: minimum = (MeanB + n × STDB); n = 3 (channel A) and 1 (channel B; selectable by user); maximum = highest intensity in image; MeanF = mean of fluorescence distribution averaged over all sections; STDF = SD of fluorescence distribution averaged over all sections. (f) A fluorescence threshold is used to include just the bright focal signal: ThresholdF = MeanF + m × STDF; m = 0.5 (all channels). (g) ThresholdF is subtracted from the image. (h) The two image channels are merged after subtracting each threshold. (i) A 2D region for colocalization measurement is drawn and applied to all sections. (j) The colocalization percentage is obtained within the defined region for each image: IA = total intensity of all pixels in channel A; IAB = total intensity of all pixels in channel A with nonzero values in channel B; IAB/IA × 100% = percentage of channel A overlying nonzero channel B intensity. (k) Colocalization of channel B on channel A is estimated as IBA, IB, and IBA/IB. (l) The colocalization percentage as a function of focus (z) is found for the entire stack: IAB(z)/IA(z) = colocalization percentage for section (z). (m) Threshold values and all the colocalization parameters are recorded in text files.
MetaMorph wound-healing macro
This is a description of the algorithm used in the MetaMorph journal IWA_1_02.jnl to estimate closure and wound edge roughness in phase contrast time-lapse images of a closing in vitro wound. The method gives two values that are estimates for (a) the mean width of the wound and (b) the combined length of lines traced around both wound edges (reduced perimeter). Values are derived for each time point. (a) The wound orientation was determined interactively (one angle for the whole stack). (b) The image stack was rotated to bring the axis horizontal (aligned in X). (c) A four-pass filter was applied to all time points to enhance cell edges using the following four kernels sequentially: kernel 1 (5, 5, 5; −3, 0, −3; −3, −3, −3), kernel 2 (5, 5, −3; 5, 0, −3; −3, −3, −3), kernel 3 (5, −3, −3; 5, −3, −3; 5, −3, −3), and kernel 4 (−3, −3, −3; 5, 0, −3; 5, 5, −3). (d) A (sequential) Lomo filter (2-pixel-diam circle) reduced noise and joined touching cells. (e) A background region was defined within the wound area. (f) A threshold was determined from intensities in the background of each time point: threshold = (1 + n) × (mean background); n = 1 was empirically determined to clearly define the wound edge and kept constant in all wounds analyzed. (g) The wound area in each time point was found by the MetaMorph immunofluorescence tool. (h) Immunofluorescence always found a single object >10,000 pixels in area above the threshold. (i) The mean wound width and a reduced perimeter of the wound area were determined: width = (wound area)/(wound length); (reduced perimeter) = (perimeter of wound area) − 2 × width. (j) The threshold value, width, and reduced perimeter were written into an ASCII (American Standard Code for Information Interchange) file.
Online supplemental material
Fig. S1 shows a schematic presentation of the BRCA1 BRCT domains, cryoimmuno-EM of PMA-stimulated HeLa cells, a Western blot of CHO cells, and spreading of CHO-WT versus CHO-High cells. Fig. S2 shows EGFP-CTD and endogenous BRCA1 colocalization with F-actin and ERM. Fig. S3 shows endogenous BRCA1 colocalization with F-actin and ERM. Fig. S4 shows that EGFP-CTD is present in focal adhesion plaques. Fig. S5 shows EGFP-protein distribution after saponin treatment. Fig. S6 shows colocalization of EGFP-CTD, F-actin, and FAK after saponin treatment. Video 1 shows PM dynamics. Video 2 shows loss of cell–cell contacts and enhanced invasion capacity in wound healing. Video 3 shows wound healing in CHO-WT versus CHO-High. Table S1 shows protein identification by mass spectrometry from SDS-PAGE. Table S2 shows protein identification by mass spectrometry from 2D gel spots. Online supplemental material is available at http://www.jcb.org/cgi/content/full/jcb.201004136/DC1