Journal: Current Biology
Article Title: EHD Proteins Cooperate to Generate Caveolar Clusters and to Maintain Caveolae during Repeated Mechanical Stress
Figure Lengend Snippet: Caveolin1-GFP Clusters Are Generated by EHD Proteins (A) TIR microscopy to show the distribution of caveolin1-GFP in gene-edited WT and ΔEHD1,2,4 NIH 3T3 cells. Scale bar, 10 μm. (B) Quantification of puncta size in TIR images as shown in (A), shown as frequency distribution of all sizes detected in 10 cells of each genotype shown. (C) Confocal and stimulated emission depletion (STED) microscopy of WT cells, ΔEHD1,2,4 cells, and ΔEHD1,2,4 cells expressing mCherry-EHD1, mCherry-EHD2, and mCherry-EHD4 by transient transfection. All cells are gene edited to express caveolin1-GFP. The STED images are of the boxed region in the confocal images. Scale bars, 5 μm (in confocal images) and 1 μm (in STED images). (D) Electron micrographs of ΔEHD1,2,4 cells expressing mitochondrially targeted APEX, mCherry-EHD1, mCherry-EHD2, and mCherry-EHD4 by transient transfection. Cells were stained with diaminobenzidine, producing electron-dense deposits in the mitochondria of transfected cells. Two cells are shown; arrows highlight mitochondria, and the boxed regions are shown at higher magnification in the additional panels. Scale bars, 500 nm.
Article Snippet: For 3′3’-diaminobenzidine (DAB) staining of EHD1,2,4/mitochondrial APEX transfected cells, transiently transfected and non transfected cells grown on MatTek dishes were fixed in pre-chilled 2% glutaraldehyde in 0.1M cacodylate buffer plus 2 mM calcium chloride for 1 hr on ice.
Techniques: Generated, Microscopy, Expressing, Transfection, Staining