Figure 1.
Cholesterol depletion collapses caveolae and causes loss of cavin-2 in a switch-like fashion (A).
Freeze drying electron micrograph depicting the inner surface of the adipocyte plasma membrane before (panels A and B) and after (panels C and D) cholesterol depletion by 20 mM MβCD for 60 min. (left and right panels respectively, scale bar represents 200 nm). (B). 3T3-L1 adipocytes were treated with 20 mM MβCD for the indicated time points, lysates were prepared in RIPA buffer and analyzed by SDS-PAGE followed by Western blotting as described in Methods. (C & D). Cholesterol was determined as described in the Methods section for whole cell extracts and/or membranes (C, 90 min.) or at the times indicated (D) after MβCD exposure. The amount of cavin-2 was determined using a Fujifilm LAS-4000 Image Analyzer. The experiments of 1A, 1B and 1D are representative of >4 such experiments and C shows the mean ± S.E. for triplicate cell preparations. The value for cavin-2 at time zero was set to 1.0 and subsequent time points are expressed as ratios of this.
Figure 2.
Cavin-2 is degraded by the proteosome and Cavin-1 redistributes to the cytosol following cholesterol depletion.
3T3-L1 adipocytes were treated with or without MβCD (20 mM, 90 minutes) and subjected to either (A) immunostaining with the indicated antibodies (red) and for nuclei with DAPI (4′,6-diamidino-2-phenylindole, blue) and analyzed by confocal microscopy as described in Methods or (B), subjected to subcellular separation into membrane and cytosolic fractions. Following centrifugation, an equal proportion of each fraction (ca. 6X more cytosol protein than membrane) were analyzed by SDS-PAGE and Western blotting. Glyceraldehyde phosphate dehydrogenase (GAPDH) is a loading control for the cytosolic fraction. (C) Cultured at cells were treated with or without methylβ-cyclodextrin in combination with the proteasome inhibitor MG-132 (10 µM), the lysosomal inhibitor chloroquine (40 µM), or the inhibitors alone for 90 minutes and cell extracts were prepared in lysis buffer and analyzed by SDS-PAGE and Western blotting for the proteins indicated. Shown are representative experiments.
Figure 3.
Cholesterol repletion restores cavin-2 levels, which allows return of cavin-1 to the plasma membrane.
3T3-L1 adipocytes were treated without or without methyl-β-cyclodextrin (20 mM, 90 minutes), and the medium was changed to that containing either 10% FBS, 10% FBS depleted of lipoproteins (LPDS), or, 10% FBS lipoprotein depleted serum containing cholesterol loaded cyclodextrin (25 µM cholesterol). Following incubation for the times indicated (time 0 being after MβCD removal), cell lysates were analyzed by SDS-PAGE and Western blotting (A) or were processed for immunofluorescence with antibodies for cavin-1 and -2 (B) and for nuclei (DAPI) as in prior figures. The percent cavin-2 rim staining was determined by scoring 104+/−3 cells that were also positive for DAPI staining.
Figure 4.
ShRNA mediated knockdown of cavin-2 causes redistribution of cavin-1 to the cytosol.
3T3-L1 adipocytes stably expressing shRNA directed against enhanced green fluorescent protein (eGFP) or cavin-2 were processed for freeze drying electron microscopy (A) or subjected to immunostaining (B) with the indicated antibodies (red) and DAPI (blue), and analyzed by confocal microscopy and quantitatively analyzed as in Figure 3 for 102 cells or (C) subcellular fractionation into membrane and cytosolic fractions as in Figure 2B. Following fractionation, proteins were analyzed by SDS-PAGE and Western blotting, with glyceraldehyde phosphate dehydrogenase (GAPDH) being used as a loading control for the cytosolic fraction. In Figure 4A, panel B, black arrows point to torus shaped caveolae and white arrows to flattened caveolae and the scale bar is 200 nm. In 4B, the amount of cavin-2 in whole cell lysates is shown. These are representative of 3 such experiments.
Figure 5.
Cholesterol depletion by simvistatin and cholesterol-binding antibiotics causes loss of cavin-2 in fat cells (A) and fibroblasts (B).
Adipocytes or NIH-3T3 fibroblasts were treated with or without Simvastatin (10 µM, 18 hrs) methyl-β-cyclodextrin (20 mM, 90 minutes), nystatin (76 µM, 4 hours) or filipin (7.6 µM, 4 hours). Whole cell extracts were then prepared in RIPA buffer and analyzed by SDS-PAGE and Western blotting as in prior figures.
Figure 6.
Cav1 null adipocytes lack caveolae but cavin-2 is membrane associated and redistributes to the cytosol upon cholesterol depletion.
Cav1 null adipocytes were treated with or without MβCD as in previous figures and lysates (A) were prepared and analyzed by Western blot or (B) labeled with anti-cavin-2 and secondary antibody prior to analysis by confocal microscopy.