Polarization of metastatic cells is dependent on caveolin-1.

<p>(<b>A</b>) Total protein extracts were prepared from both parental MDA-MB-231 and cells stably transduced with a control shRNA targeting luciferase (sh-ctrl) or a shRNA targeting caveolin-1 (shRNA-caveolin-1 #5, sh-cav1). Extracts were separated by SDS-PAGE (35 µg total protein per lane) and analyzed by Western Blotting with antibodies against caveolin-1 and β-actin. (<b>B</b>) Confluent monolayers of parental, shRNA-control (sh-ctrl) or shRNA-caveolin-1 (sh-cav1) treated MDA-MB-231 cells were wounded with a pipet tip and fixed at 0 (left panel) and 60 minutes (right panel) after monolayer injury. Samples were stained with anti-caveolin-1 (green) and anti-Golgin-97 (red) antibodies, whereas the nuclei were stained with DAPI (blue). The first layer of cells facing the wounded area is distinguished from the rest by a white line. Scale bar, 20 µm. (<b>C</b>) Caveolin-1 localization was analyzed as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0033085#pone-0033085-g001" target="_blank">Figure 1C</a>, by measuring the ratio of fluorescence intensity “rear/front”. Data are the mean ± SEM, n = 3. (<b>D</b>) Polarization of both parental (-) and shRNA treated MDA-MB-231 cells with shRNA-luciferase (sh-ctrl) or shRNA-caveolin-1 (sh-cav1) was measured from (C) as described in the <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0033085#s4" target="_blank">Materials and Methods</a>, by analyzing the outer layer of cells facing the wounded area (B). The percentage of polarized cells was evaluated at 0, 60 and 360 minutes after wounding. Data were averaged from three independent experiments (mean ± SEM). *Comparison with control shRNA P<0.05. (<b>E</b>) B16-F10 cells transfected with either pLacIOP (mock) or pLacIOP-caveolin-1 (cav1) were treated or not with 1mM IPTG for 24 hours, grown in monolayers and wounded with a pipet tip to allow migration for 1 hours. Samples were stained with anti-Gigantin-1 polyclonal antibody (blue), phalloidin-Alexa488® (green) and propidium iodide (red). The outer layer of cells facing the wounded area is outlined with a white line. (<b>F</b>) Cell polarization was measured as described in the <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0033085#s4" target="_blank">Materials and Methods</a>, by analyzing the outer layer of cells facing the wounded area. The percentage of polarized cells was evaluated 0-360 minutes after wounding the monolayer. Data were averaged from three independent experiments (mean ± SEM). Statistically significant differences compared with pLacIOP-transfected B16-F10 (mock) cells are indicated (**, P<0.01 at time 360 min; *, P<0.05 at time 60 min).</p

Caveolin-1 favors directionality and persistence of migration in MDA-MB-231 cells.

<p>(<b>A</b>) Migration of MDA-MB-231 cells treated with shRNA targeting either luciferase (sh-ctrl) or caveolin-1 (sh-cav1) was assessed in a Boyden chamber assay. Cells (5×10⁴) were seeded on fibronectin-coated (2 µg/ml) transwell plates and allowed to migrate for 2 hours. Cells that migrated to the lower side were detected with crystal violet staining (mean ± SEM, n = 3). **P<0.01. (<b>B</b>) Parental and shRNA treated MDA-MB-231 cells were grown as confluent monolayers, wounded with a pipet tip and migration was recorded by time-lapse video microscopy (total 15 hours, 12 minutes frame interval). Cell tracks were determined by using the <i>Image J</i> software (<i>“Manual Tracking”</i> plug-in). Tracks of single cells at the wounded edge are shown (upper panel). The edge of the wound is indicated with a white line. Single cell tracks are shown in a Cartesian coordinate system for each cell type (lower panel). (<b>C</b>) Instant velocity was analyzed for each cell type in (B) and plotted as a function of time (0-15 hours). (<b>D</b>) Mean velocity values were obtained from (C) and plotted for each cell type. (<b>E</b>) Persistency of migration was calculated as the ratio between the net distance and the total distance of migration in (B). Average values were obtained for parental, sh-ctrl and sh-cav1 cells (mean ± SEM). (<b>F</b>) Directionality of migration was obtained from the analysis shown in (B), lower panels. Tracks that lay within a 60° angle with respect to the direction of cell movement were considered as oriented (shaded region). The percentage of cells with orientated tracks was calculated and plotted (mean ± SEM, n = 3). *P<0.05.</p

Caveolin-1 aminoacid tyrosine-14 is required for directionality and persistence of migration in B16-F10 cells.

<p>(<b>A</b>) B16-F10 cells transfected with pLacIOP (mock), pLacIOP-caveolin-1 (WT) or the pLacIOP-caveolin-1/Y14F mutant (Y14F) were treated with 1mM IPTG for 24 hours. Then, cell migration was assessed in a Boyden chamber assay by seeding cells (5×10⁴) on fibronectin-coated (2 µg/ml) transwell plates and allowing migration for 2 hours. Cells that migrated to the lower side were detected by crystal violet staining (mean ± SEM, n = 3). *P<0.05. Bottom panels show total protein extracts, separated by SDS-PAGE (35 µg total protein per lane) and analyzed by Western Blotting. Numbers below panels indicate relative caveolin-1 levels (caveolin-1/WT = 22.0±1.8, caveolin-1/Y14F = 16.6±3.8; values compared with the mock condition and obtained as the mean from three independent measurements ± SEM). (<b>B</b>) Confluent monolayers of B16-F10 cells transfected with pLacIOP (mock), pLacIOP-caveolin-1 (WT) or pLacIOP-caveolin-1/Y14F (Y14F) were wounded with a pipet tip and recorded by time-lapse video microscopy for 10 hours (12-min frame interval). Cell tracks were determined as indicated in Figure legend 3B. Tracks of single cells at the wounded edge are shown (upper panel). The edge of the wound is outlined by a white line. Single cell tracks are shown in a Cartesian coordinate system for each cell type (lower panel). (<b>C</b>) The mean velocity ( µm/min) was derived from the instant velocity (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0033085#pone.0033085.s003" target="_blank">Figure S3</a>), as described in (D). The persistency and directionality of migration were obtained as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0033085#pone-0033085-g003" target="_blank">Figures 3E and 3F</a>, respectively. Data shown are the mean ± SEM (n = 3). *P<0.05.</p

Inhibition of Src family kinases decreases cell migration and phosphorylation of caveolin-1 on tyrosine-14. (A)

<p>MDA-MB-231 cells stably transduced with shRNA either targeting Luciferase (sh-ctrl) or caveolin-1 (sh-cav1) were pre-incubated with DMSO (D) or 10 µM PP2 for 30 minutes, wounded with a pipet tip, stimulated (+) or not (-) with 3% of serum and images were recorded at 0 and 16 hours post-wounding. The wounded area was measured with the <i>Image J</i> software and the percentage of wound closure was plotted (upper panel). Total protein extracts were prepared and analyzed by Western blotting with antibodies against caveolin-1, pY14-caveolin-1 and actin. (<b>B</b>) Confluent monolayers of B16-F10 cells transfected with either pLacIOP or pLacIOP-caveolin-1 were pre-incubated with 1 mM IPTG for 24 hours. Then, cells were treated with DMSO (D) or PP2, wounded with a pipet tip, stimulated with 3% of serum and analyzed at 0 and 7 hours post-wounding, as described in (A). Total protein extracts were prepared and analyzed by Western blotting. Values in (A) and (B) were averaged from 3 independent experiments (mean ± SEM). *P<0.05.</p

Caveolin-1 enhances focal adhesion turnover in metastatic cells.

<p>(<b>A</b>) B16-F10 cells transfected with pLacIOP (mock) or pLacIOP-caveolin-1 (cav1) were transfected with pEGFP-vinculin for 24 hours, seeded on fibronectin-coated coverslips (2 µg/ml) and grown in the presence of 1 mM IPTG for 24 hours (see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0033085#s4" target="_blank">Materials and Methods</a>). Then, cells were serum-starved for 4 hours, pulsed with 10% serum and recorded by time-lapse video microscopy for 60 minutes (1-min frame interval). Zoomed areas are shown at selected times for mock and cav1 cells. Focal adhesions (FA, arrows) and focal contacts (FC, arrowheads) were defined by size. Images are representative of 3 independent experiments. (<b>B</b>) Kinetics of FAs and FCs were measured from experiments shown in A. FA disassembly and FC lifetime were measured for at least 6 structures per experiment (mean ± SEM, n = 3). *P<0.05. (<b>C</b>) B16-F10 mock or cav1 cells were seeded onto fibronectin-coated coverslips (2 µg/ml), grown in the presence of 1 mM IPTG for 24 hours and treated with 10 µM nocodazole in serum-free medium for 4 hours. Then, nocodazole was removed by wash-out with serum-free medium and cells were incubated by 0–20 minutes at 37°C. Subsequently, cells were fixed and stained with anti-vinculin antibody (red) and DAPI (blue) to label FAs and nuclei, respectively. (<b>D</b>) FAs were quantified from experiments described in (C) by using the <i>Image J</i> software. At least 10 images per time point were analyzed. Data were averaged from three independent experiments (mean ± SEM, n = 3). (<b>E</b>) B16-F10 mock (-), cav1 (WT) or cav1/Y14F (Y14F) cells were treated with 10 µM nocodazole, as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0033085#pone-0033085-g006" target="_blank">Figure 6C</a>. Nocodazole was subsequently washed-out for 60 minutes and FAs were analyzed with an anti-vinculin antibody (red) and nuclei with DAPI (blue). FAs were quantified as in (D). Data are representative from three independent experiments (mean ± SEM). *P<0.05. (<b>F</b>) MDA-MB-231 cells stably transduced with shRNA either targeting Luciferase (sh-ctrl) or caveolin-1 (sh-cav1) were transfected with pEGFP-vinculin for 24 hours, serum-starved for 4 hours, pulsed with 10% serum and recorded by time-lapse video microscopy, as described in (A). FA disassembly and FC lifetime were measured for 5 structures per experiment. Data were averaged from three independent experiments (mean ± SEM, n = 3). (<b>G</b>) Focal adhesion disassembly was measured after nocodazole removal in MDA-MB-231 cells treated with shRNA targeting luciferase (sh-ctrl) or caveolin-1 (sh-cav1), as described in (C) and (D). Data were averaged from 12 measurements (mean ± SD). Data are representative of two independent experiments. (<b>H</b>) FA assembly was evaluated as described in the <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0033085#s4" target="_blank">Materials and Methods</a>. MDA-MB-231 cells were treated with 10 µM nocodazole for 4 hours and subsequently washed-out for 45 min. Then, cells were pulsed with 10% serum for the indicated periods of time and FAs were evaluated as described in (D). Data were averaged from 12 measurements (mean ± SD). Data are representative of two independent experiments.</p

Caveolin-1 fails to accumulate at the trailing edge of migrating metastatic cells.

<p>(<b>A</b>) Total protein extracts from DI-TNC1, MEF-3T3 and MDA-MB-231 cells were separated by SDS-PAGE (35 µg total protein per lane) and analyzed by Western Blotting with antibodies against caveolin-1 and β-actin. (<b>B</b>) Confluent monolayers of DI-TNC1, MEF-3T3 and MDA-MB-231 cells were wounded with a pipette tip and fixed at 0 (left panel) and 60 minutes (right panel) after monolayer injury. Cells were stained with a rabbit polyclonal anti-caveolin-1 antibody followed by an anti-rabbit IgG antibody (green) and nuclei were visualized with DAPI (blue). The first layer of cells facing the wounded area is distinguished from the rest by a white line. Scale bar, 20 µm. (<b>C</b>) Caveolin-1 distribution was evaluated by measuring the fluorescence intensity in four randomly chosen regions of equal dimensions at the front and the rear of the cell with the <i>Image J</i> software, as detailed in the <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0033085#s4" target="_blank">Materials and Methods</a>. Data represent the ratio of fluorescence intensity “rear/front” (mean ± SEM; n =  3). Statistically significant differences compared with time 0 minutes for each cell type are indicated (**, P<0.01; *, P<0.05). (<b>D</b>) B16-F10 cells transfected with either pLacIOP (mock) or pLacIOP-caveolin-1 (cav1) were treated or not with 1mM IPTG for 24 hours and total protein extracts were prepared, separated by SDS-PAGE (35 µg total protein per lane) and analyzed by Western Blotting. (<b>E</b>) Confluent monolayers of pLacIOP-caveolin-1 transfected B16-F10 cells in the absence or presence of 1mM IPTG were wounded with a pipet tip and fixed either at 0 or 60 minutes after wounding. Samples were stained with anti-caveolin-1 polyclonal antibody (green) and DAPI (blue). The edge of the wound is outlined with a white line. Scale bar, 20 µm. (<b>F</b>) Caveolin-1 localization was analyzed at different time points, as described in C. Data are the mean ± SEM from three independent experiments.</p