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. 2020 May 4;9:e55038. doi: 10.7554/eLife.55038

Figure 5. Chol accumulation reduces the caveolae diameter in 3T3 adipocytes.

(A) Representative overlays of light microscopy images with corresponding electron micrographs showing localization of caveolae (Cav1-mCh in red) and nuclei (DAPI in cyan) for untreated Cav1-mCh HeLa cells (control) or cells treated with Bodipy-labeled Chol or LacCer. Dotted boxes show regions of higher magnification in corresponding panels below. N, nucleus; PM, plasma membrane. White arrows denote surface connected caveolae and black arrows indicate surface adjacent caveolae. Scale bars, 1 μm; inset scale bars, 100 nm. (B, C) Electron micrographs of control 3T3-L1 adipocytes (B) and 3T3-L1 adipocytes treated with Bodipy-Chol (C). Top and bottom panels show two representative images per sample: one with clear surface connected necks and one without. Cells were chemically fixed, embedded in resin, and processed for electron microscopy. Scale bars, 100 nm. (D, D’) Scatter plots showing the quantification of neck diameter (D) and bulb width (D’) of surface connected caveolae in 3T3-L1 adipocytes. Bulb width and neck diameter are highlighted in (B), upper panel. n ≥ 30, mean ± SEM. (E, E’) Scatter plots showing the quantification of surface area (E) and bulb width (E’) of surface adjacent caveolae in 3T3-L1 adipocytes. n ≥ 120, mean ± SEM. ***, p≤0.001.

Figure 5—source data 1. Excel file containing source data pertaining to Figure 5D and E.

Figure 5.

Figure 5—figure supplement 1. Correlative electron microscopy of caveolae.

Figure 5—figure supplement 1.

(A) Cav1-mCh HeLa cells were induced with Dox and transiently expressed EHD2-GFP. Light microscopy image showing localization of caveolae (Cav1-mCh) and nuclei (DAPI) within cells (LM, left panel). Middle panel depicts corresponding EM images. Overlay of LM and EM images shows correlation of fluorescently labeled structures to ultrastructure in same cells (right panel). Scale bar, 2 μm. (B) Representative overlays of light microscopy images with corresponding electron micrographs showing localization of caveolae (Cav1-mCh in red) and nuclei (DAPI in cyan) for Cav1-mCh HeLa cells treated with Bodipy-labeled SM C12. Dotted boxes show regions of higher magnification in corresponding panels below. N, nucleus; PM, plasma membrane. White arrows denote surface connected caveolae and black arrows indicate surface adjacent caveolae. Scale bars, 1 μm; inset scale bars, 100 nm.
Figure 5—figure supplement 2. Expression of EHD2 and Cav1 is upregulated in 3T3-L1 adipocytes.

Figure 5—figure supplement 2.

(A) Representative immunoblots showing protein expression of EHD2 and Cav1 during 3T3-L1 differentiation. Clathrin HC served as loading control. (B) Representative electron micrographs of 3T3-L1 adipocytes. Caveolae (indicated by arrow) can be clearly distinguished from clathrin-coated pits (indicated by asterisk). Scale bar, 100 nm. (C) Incorporation rate of Bodipy-Chol into the PM of live 3T3-L1 adipocytes. Cells were treated with fusogenic liposomes (final total lipid concentration 7 nmol/ml). Total fluorescence intensity (FI) of the Bodipy signal was measured within circular ROIs in a confocal section using spinning disk microscopy. Ten ROIs were analyzed using the Zeiss Zen system software. n = 2, mean ± SEM.
Figure 5—figure supplement 2—source data 1. Excel file containing source data pertaining to Figure 5—figure supplement 2C.