|
Fig. 7
Membrane tubules connect the CV/developing cilium and the PM creating an extracellular membrane channel. a, b CLEM/FIB-SEM analysis of a 3 h starved GFP-EHD1 + SMO-tRFP cell showing a CV (a) and a short intracellular cilium (b) connected to GFP-EHD1-positive tubular membranes. Fluorescence images (upper left and middle panels, scale bar: 1 μm) and longitudinal FIB-SEM section of the BB/cilium (upper right panel, scale bar: 200 nm). Below are representative FIB-SEM xy image sections (183 sections in a and 157 sections in b) showing a continuous membrane tubule connecting the CV and short intracellular cilium to the PM, respectively (Scale bar: 1 μm). Blue circles highlight the BB region. Traces for the extracellular membrane channel (EMC) connected to the CV (a) or short intracellular cilium (b) are shown in green offset from FIB-SEM EMC structures (highlighted in yellow). The cilium outline is traced in red. Merged ciliary structure traces and 3D segmentation of the CV and short intracellular cilium and the EMC connected to the PM from the xy FIB-SEM images are shown in the last two lower panels. The raw FIB-SEM images for a can be found in Supplemental Movie 3 and b in Supplemental Movie 4. c CLEM/FIB-SEM of 3 h starved GFP-CENTRIN1 cell (fluorescence images in upper left panel, scale bar: 2 μm). Representative FIB-SEM images (213 xy images) (bottom left and middle left panels, scale bars: 500 nm) and 3D segmentation (middle right panel) showing an EMC connecting the CV and the PM. FIB-SEM images (right panels, scale bar: 100 nm) showing different cross-sections of the EMC connected to the plasma membrane (PM) (top right panels). A view of the cell surface showing the EMC opening at the PM (arrow) and the 3D segmentation of the EMC opening (green) at the PM (bottom right panels). A summary of CLEM FIB-SEM data sets is shown in Supplementary Table 2