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Physiological consequences of caveolin/cavin loss.

<p>(A) Confluent monolayer of CAV1−/− MEFs co-expressing CAV1-YFP and Cavin-1-mCherry was subjected to a scratch-wound assay. Internalization assay was performed with anti-CD44 mAb for 2 min at 37°C. Cells were acid washed before fixation and internalized anti-CD44 mAb was labeled with AF-647 secondary antibody. Arrow indicates direction of wound. (B) CAV1−/− MEFs expressing CAV1-YFP were labeled for Cdc42 with specific primary antibody followed by AF-555 secondary antibody labeling. For quantitative analysis of Cdc42 expression, line scans were performed in 30–40 CAV1-YFP-expressing cells at random positions on the PM. The plot profile represents average fluorescence (± SEM) of Cdc42 labeling in mentioned areas of PM from all cells and the bar graph represents amplitude together with error obtained from nonlinear Gaussian analysis. (C) Pearson coefficient analysis of the image data set from (B). (D) CAV1−/− MEFs were co-transfected with CRIB-Ypet and CAV1-mCherry and quantitative colocalization analysis was performed at the random regions of PM. For 20–30 co-transfected cells, line scans and nonlinear Gaussian analysis were performed as mentioned in (B) at random positions on the PM. (E) WT, CAV1−/−, and Cavin-1−/− MEF monolayers were subjected to a scratch-wound assay and the extent of migration of respective cells into the wound area was measured at 0, 4, 8, and 12 h time points. Percentage of wound closure is plotted. (F) At time points 0, 4, 8, and 12 h a scratch-wound assay, followed by internalization assay, was applied to WT, CAV1−/−, and Cavin-1−/− MEFs as mentioned in (A). For labeling endogenous CAV1 and Cavin-1 respective primary antibodies followed by AF-488 secondary antibody were used. The plot profile represents average pixel intensity across the leading to trailing edge for CD44, CAV1, and Tfn-647. Arrows indicate CD44 labeling. In (C) data represents mean ± SEM of data pooled from three independent experiments; n = 40 cells. In (B, D, E) data represent mean ± SEM of three independent experiments. *p<0.05, **p<0.01, ***p = 0.001, ****p<0.0001 (two-tailed t-test). Scale bar: 10 µm.</p

Caveolae generation down-regulates CLIC endocytosis.

<p>(A) Cell lysates from untransfected Cavin-1−/− MEFs or Cavin-1−/− MEFs transiently transfected with CAV1-YFP or Cavin-1-mCherry or co-transfected with CAV1-YFP and Cavin-1-mCherry were immunoblotted for CAV1 and Cavin-1. Actin was used as loading control. For quantification, the integrated intensity of protein bands was calculated. (B) Representative image of cell from (A) showing colocalization between CAV1 and Cavin-1 at different subcellular locations. Insets depict CAV1, Cavin-1 and merged labeling at the plasma membrane and cytosolic regions, marked by the square. 40 cells from (A) were subjected to Pearson correlation analysis, and a representative graph is shown. (C) Cells from (A), growing on coverslips, were subjected to internalization assay with anti-CD44 mAb and Tfn-647 for 2 min at 37°C. Cells were acid washed prior to fixation and internalized anti-CD44 mAb was labeled with AF-555 secondary antibody. 40–50 cells were quantified for normalized fluorescent intensity of endocytic markers. (D) FRAP analysis for CAV1 diffusion at the plasma membrane of Cavin-1−/− MEFs expressing CAV1-YFP alone or co-expressing CAV1-YFP and Cavin-1-mCherry. Fluorescence recovery curve and lateral diffusion analysis (inset) from 15 cells (mean ± SD) are shown. In (B) data represent mean ± SEM of data pooled from three independent experiments and in (C) data represent mean ± SEM of three independent experiments. ***p<0.0005,****p<0.0001 (two-tailed t-test). Scale bar: 10 µm.</p

CD44 as a marker of CLIC/GEEC endocytosis.

<p>(A) WT MEFs were incubated with anti-CD44 mAb and Tfn-647 for 2 min and 10 min at 37°C. Cells were placed on ice and acid washed before fixation. Internalized anti-CD44 mAb was labeled with anti-mouse Alexa Fluor-555 (AF-555) secondary antibody. For labeling endogenous CAV1 and Cavin-1, respective primary antibodies were used, followed by respective secondary AF antibodies. (B) WT MEFs were treated with 60 µM dynasore and 30 µM 7-KC respectively or left untreated (control) for 30 min at 37°C prior to uptake of endocytic markers as mentioned in (A). The bar graph represents the quantitation of internalized endocytic markers. (C) Internalization assay with anti-CD44 mAb and Tfn-647 was performed in WT, CAV1−/−, and Cavin-1−/− MEFs for 2 min at 37°C as mentioned in (A). Arrows indicate CD44-labeled puncta. (D) 40–50 cells from (C) were quantified for normalized fluorescent intensity of endocytic markers. (E) Whole cell lysates from WT, CAV1−/−, and Cavin-1−/−MEFs were immunoblotted for CD44. A representative immunoblot is shown in which actin was used as a loading control. The bar graph represents quantitation of CD44 protein levels calculated by measuring band intensities, by densitometry, in WT, CAV1−/−, and Cavin-1−/− MEFs from three independent experiments. (F) Both nonpermeabilized and permeabilized WT, CAV1−/−, and Cavin-1−/− MEFs were labeled with primary anti-CD44 mAb followed by secondary AF-488 labeling and Phalloidin-AF-596 (F-actin, not shown) as an internal control. 50–60 cells from each condition and cell type were quantified for average fluorescent intensity ratio of CD44 and F-actin (CD44:F-Actin). The graph represents ratio of CD44:F-actin values obtained in nonpermeabilized (surface) cells normalized by the corresponding value obtained in permeabilized (total) cells. (G) Internalization assay with Dex-488 was performed in WT, CAV1−/−, and Cavin-1−/− MEFs for 5 min at 37°C. 40–50 cells from each cell type were quantified for normalized fluorescent intensity of internalized Dex-488. In (F) data represent mean ± SEM of data pooled from three independent experiments, and statistical significance was calculated by one-way ANOVA analysis. In (B, D, E, G) data represent mean ± SEM of three independent experiments. *p<0.02,**p<0.01,***p<0.001,****p<0.0001 (two-tailed t-test). Scale bar: 10 µm.</p

Cavin-1 and Cavin-3, but not Cavin-2 and Cavin-4 negatively regulate CLIC endocytosis.

<p>(A) CAV1−/− MEFs were transiently transfected with pIRES-GFP, pIRES-Cavin-1, pIRES-Cavin-2, pIRES-Cavin-3, and pIRES-Cavin-4 respectively and internalization assay was performed with anti-CD44 mAb and Tfn-647 for 2 min at 37°C. Cells were acid washed prior to fixation and internalized anti-CD44 mAb was labeled with AF-555 secondary antibody. (B) 40–50 cells from each transfection were quantified for normalized fluorescent intensity of endocytic markers. (C,D) 3T3-L1 cells were transiently transfected with siRNA directed specifically to Cavin-1 and Cavin-3 respectively. Uptake and quantification of internalized marker were performed as mentioned in (A,B). In (B–D) data represent mean ± SEM of three independent experiments. *p<0.05,**p<0.005,****p<0.0001 (two-tailed t-test). Scale bar: 10 µm.</p

Caveolar proteins specifically down-regulate CLIC endocytosis.

<p>(A) In CAV1−/− MEFs transiently expressing CAV1-YFP and Cavin-1-GFP respectively, internalization assay was performed with anti-CD44 mAb and Tfn-647 for 2 min at 37°C. Cells were acid washed prior to fixation, and internalized anti-CD44 mAb was labeled with AF-555 secondary antibody. Arrows indicate CD44-labeled puncta. (B,C) 40–50 cells of each transfection from (A) were quantified for normalized fluorescent intensity of endocytic markers. (D) Cavin-1−/− MEFs were transiently transfected with Cavin-1-GFP, and uptake and quantification of internalized anti-CD44 mAb and Tfn-647 were performed as mentioned in (A,B). (E,F) CAV1−/− MEFs were transiently transfected with Myc-CAV2 and RFP-CAV3 respectively. Uptake and quantification of internalized anti-CD44 mAb and Tfn-647 were performed as mentioned in (B). (G) In CAV1−/− MEFs transiently expressing GFP vector, CAV1-YFP, and Cavin-1-GFP respectively, constitutive internalization of HRP (10 µg/ml) was performed for 2 min at 37°C followed by DAB reaction on live cells. Cells were fixed, embedded, and sectioned. Quantification of HRP-labeled CLICs per cell, using 16-20 cells from each transfection, is shown. For more images representing experimental variation obtained for uptake analysis, performed in untransfected and CAV1-YFP/Cavin-1-GFP-expressing CAV1−/− MEFs, see supporting information <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.1001832#pbio.1001832.s013" target="_blank">Figure S12</a>, <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.1001832#pbio.1001832.s014" target="_blank">Figure S13</a>, and <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.1001832#pbio.1001832.s015" target="_blank">Figure S14</a>. In (B–G) data represent mean ± SEM of three independent experiments. *p<0.05,***p<0.0005,****p<0.0001 (two-tailed t-test). Scale bar: 10 µm.</p

CAV1 alters general membrane characteristics.

<p>(A) WT and CAV1−/− MEFs were transiently transfected with GP1-YFP; CAV1−/− MEFs were also co-transfected with GPI-YFP plus CAV1-mCherry or CAV1G83S-mCherry. FRAP analysis for GPI-YFP diffusion was performed, and FRAP curves and diffusion coefficients (D) calculated from FRAP measurements in at least 30 cells per condition are shown. (B) CAV1−/− MEFs were transiently transfected with PA-CD44 and co-transfected with PA-CD44 and CAV1 respectively. Fluorescence decay curves and diffusion coefficients (D) for PA-CD44, calculated from photo-activation measurements in at least 20 cells per condition, are shown. (C) CAV1−/− MEFs were either treated with 0.05% Tween20 at 37°C prior to performing internalization assay with anti-CD44 mAb and Tfn-647 or incubated with endocytic markers at 41°C. Cells were acid washed prior to fixation and internalized anti-CD44 mAb was labeled with AF-555 secondary antibody. 40–50 cells from each treatment were quantified for normalized fluorescence intensity of endocytic markers. (D,E) CAV1−/− MEFs transiently expressing CAV1-YFP and Cavin-1-GFP respectively, were either treated with Tween20 or incubated with endocytic markers at 41°C. Uptake and quantification of internalized markers was performed as mentioned in (C). (F) In CAV1−/− MEFs transiently expressing CAV1-YFP, internalization assay were performed with Dex-488 for 5 min either at 41°C or at 37°C. 40–50 cells from each condition were quantified for fluorescence intensity of the internalized Dex-488. (G) Representative images for filipin labeling performed for CAV1-YFP-expressing treated and untreated CAV1−/− MEFs and a bar graph representing quantification for normalized fluorescence intensity of filipin are shown. In (A) and (B) data represent average mean ± SEM of pooled data from three independent experiments. Diffusion coefficients were obtained by non-linear regression of recovery and fluorescence decay curves, as described in the <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.1001832#s4" target="_blank">Materials and Methods</a> section. In (C–F) data represent mean ± SEM of three independent experiments. *p<0.01, **p = 0.007, ***p<0.005, ****p<0.0001 (two-tailed t-test). Scale bar: 10 µm.</p

Loss of Cavin-1 up-regulates endocytic activity in muscle fibers.

<p>(A) Muscle fibers, isolated from Cavin-1−/− and WT mice, were incubated with anti-CD44 mAb for 3 min at 37°C. Fibers were acid washed on ice and internalized anti-CD44 mAb was labeled with AF-555 secondary antibody. 8–10 muscle fibers from three independent experiments were quantified for total fluorescence intensity of internalized CD44 mAb. (B,C) HRP uptake was performed in WT (B) and Cavin-1−/− (C) muscle fibers followed by DAB reaction. Arrows indicate DAB-positive intracellular structures. (D) Quantitation of DAB-labeled structures, as described in the <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.1001832#s4" target="_blank">Materials and Methods</a> section, is shown. In (A, D) data represent mean ± SEM of three independent experiments. ***p<0.001, ****p<0.0001 (two-tailed t-test). Scale bars: (A) 10 µm, (B) 1 µm, (C) 500 nm.</p

Scaffolding domain of CAV1 is crucial for inhibition of CLIC endocytosis.

<p>CAV1−/− MEFs were transiently transfected with (A) CAV1G83S-mCherry, (B) RFP-CAV3 and Cav3G55S-HA (C) CAV1Δ80-100-HA and (D) CAV1-SD-GFP (GFP-tagged minimal caveolin scaffolding domain) respectively. (A–D) Internalization assay was performed with anti-CD44 mAb and Tfn-647 for 2 min at 37°C. Cells were acid washed prior to fixation and internalized anti-CD44 mAb was labeled with AF-555 secondary antibody. 40–50 cells from each transfection were quantified for normalized fluorescence intensity of endocytic markers. In (A–D) data represent mean ± SEM of three independent experiments. **p = 0.002,***p = 0.0007, ****p<0.0001 (two-tailed t-test).</p

Cavin-1 regulates cholesterol distribution and Cdc42 activity.

<p>(A) Filipin labeling was performed for Cavin-1-GFP-expressing CAV1−/− MEFs, and for quantification average fluorescence intensity measured from 40–50 transfected cells was normalized to untransfected cells. (B) Whole cell lysates from CAV1−/− MEFs expressing CAV1-YFP and Cavin-1-GFP respectively were immunoblotted for Cdc42 and glyceraldehyde 3-phosphate dehydrogenase (GAPDH) was used as a loading control. The bar graph represents quantitation of protein levels, calculated by measuring band intensities by densitometry. (C) Pearson coefficient analysis determined at the plasma membrane for CAV1/Cavin-1 (positive control), Cavin-1/Cdc42, and randomized Cavin-1/Cdc42. (D) CAV1−/− MEFs were co-transfected with CRIB-YPet and Cavin-1-mCherry and quantitative colocalization analysis was performed at the PM ruffles. For 30 cells, line scans (10 pixels' length) were performed at random positions on PM ruffles and cytosol. The resultant fluorescent intensities profiles were plotted against each other and subjected to linear regression analysis. (E) CAV1−/− MEFs were transiently transfected with Cdc42-CyPet alone, with Cdc42-CyPet and CRIB-YPet simultaneously, or with Cdc42-CyPet, CRIB-YPet, and Cavin-1-Flag. Lifetime of CyPet was analyzed by FLIM-FRET and a representative graph showing lifetime of CyPet in 25 cells, mean ± SEM, is shown. (F) In Cavin-1−/− and CAV1−/− MEFs internalization assay was performed with anti-CD44 mAb and Tfn-647 for 2 and 10 min at 37°C. Endogenous CAV1 and Cavin-1 was labeled with respective primary antibodies followed by AF-488 secondary antibody labeling and for internalized anti-CD44 mAb labeling AF-555 secondary antibody was used. In (A,B,D) data represent mean ± SEM of three independent experiments. In (C) data represent average mean ± SEM of pooled data from three independent experiments; n = 40 cells. **p<0.01,***p<0.001 ****p<0.0001 (two-tailed t-test). Scale bar: 10 µm.</p