Fig. 3. Ultrastructural analysis of EHD1 tubules. (A) HeLa cells were transiently transfected with Myc-EHD1 and processed for electron microscopy immunogold labeling using an anti-Myc antibody. Bound antibodies were detected using gold-conjugated protein A. (B–D) Correlative fluorescence/electron microscopy. HeLa cells were transiently transfected with a GFP–EHD1 construct on CELLocate grids, and live confocal images of a typical GFP–EHD1-expressing cell were obtained (B). Cells were then fixed with 4% paraformaldehyde and 0.05% glutaraldehyde, and enhanced gold labeling was performed for anti-GFP antibodies as described in Materials and methods. (C) and (D) are serial sections depicting the boxed region of interest in (B), and arrows in (C) and (D) mark the EHD1 tubular structure within the box. Particles indicate the presence of GFP–EHD1 along the tubular structure and in the cytosol. (E) Dynamics of EHD1 association with membranes. GFP–EHD1 was subjected to fluorescence recovery after photobleaching (FRAP) analysis. HeLa cells were transiently transfected with a GFP–EHD1 construct, and examined by live confocal image analysis 24 h later. An entire EHD1 tubular structure was photobleached (rectangular region of interest). GFP–EHD1 recovery to the bleached area was monitored every 3.3 s (see Supplementary time-lapse video). Images of live cells (B and E) are visualized as inverted images to facilitate analysis. M, mitochondria; PM, plasma membrane. Bars: (A), 200 nm; (B), 10 µm; (C and D), 200 nm; (E), 10 µm.