Journal: Nature Communications

Article Title: Autophagy initiation by ULK complex assembly on ER tubulovesicular regions marked by ATG9 vesicles

doi: 10.1038/ncomms12420

Figure Lengend Snippet: HEK293 cells stably expressing GFP-ATG13 and transiently expressing mCherry-dgk1 (ER marker) were starved, subjected to live-cell imaging by wide-field microscopy and fixed on stage. ( a ) Fluorescent images of the frame capture just before the fixation, × 100 and × 10 DIC images of the fixed cells are shown. Red box in × 10 DIC image indicates the cell of interest. ( b ) Image of the resin-embedded sample. Cell of interest located in red box. ( c ) Resin blocks were trimmed down to a block face of 1 mm 2 and mounted on stub for imaging in an Auriga focused ion beam scanning electron microscopy (FIB-SEM, Carl Zeiss). Overview images before (left) and after milling (right) indicating the cell of interest with a red box. ( d ) Montage of an ATG13 particle formation from the live-cell imaging step and z stacks after fixation (particle ii in f ). ( e ) Overlays of light and electron microscopy images. Light and electron microscopy images were correlated using landmarks identified in both (shown in white and green lines, circles and triangles). ( f ) Three-dimensional (3D) opacity rendering of the FIB-SEM image stack. The areas outlined in red within the green boxes indicate ATG13 particles. Particle ii is the one that could be traced throughout the experiment and was identified in both live-cell and FIB-SEM imaging. ATG13 Particles in boxes i and iii could be identified from the wide-field and fluorescence image, but their provenance by live imaging could not because they were on a different focal plane from particle ii. ( g ) Magnification of the area within the green boxes in f (i–iii). Shown are the XY view from the middle of the ATG13 signal, and orthogonal XZ and YZ views along the thin white lines. ( h ) 3D Opacity rendering of the cropped FIB-SEM stacks in g with overlay of the ATG13 signal (red). Rendered in green are the membranes detected in the FIB-SEM stack that are in proximity of the ATG13 particle. Stars indicate mitochondrial membranes. Bars: 10 μm ( a ), 50 μm ( b ), 5 μm ( d – e ), 1 μm ( f ) and 0.25 μm ( g ).

Article Snippet: Durcupan-embedded cells were trimmed to a block face of <1 mm 2 , mounted on a stub and imaged in an Auriga Focused Ion Beam SEM (Carl Zeiss).

Techniques: Stable Transfection, Expressing, Marker, Live Cell Imaging, Microscopy, Blocking Assay, Imaging, Electron Microscopy, Fluorescence