Fig. 3

Ultrastructural analysis of N-cadherin and Myo10 in iTNTs. a Low-magnification electron micrograph displaying N-Cadherin immunogold-labeled CAD cells connected by iTNTs. (b-top) White dashed rectangle in a correspond to two high-magnification non-consecutive cryo-tomogram slices (25 nm in thickness) shown in b-top and b-bottom (Supplementary Movie 4). Green arrowheads in b and spheres in c indicate 10 nm gold particles attached to a polyclonal secondary antibody that binds to an N-Cadherin primary antibody. Red arrows indicate a long thin thread filament surrounding iTNTs anchored to the membrane of iTNTs by N-Cadherin molecules (green arrows). c Segmentation rendering of the tomogram described in b; N-Cadherin (green beads), thin thread surrounding iTNTs (green arrowheads). d Epifluorescence micrographs of CAD cells overexpressing GFP-Myo10 connected by TNTs stained with WGA (red). Yellow arrowheads indicate GFP-Myo10 signal in TNTs. e Low-magnification electron micrograph corresponding to TNTs shown in d. f, g High-magnification cryo-tomography slices corresponding to the yellow dashed squares in e. Yellow arrowheads in d mark GFP-Myo10 vesicles (Supplementary Movies 6 and 7). Orange arrows indicate vesicle-iTNT connections. h Rendering of tomogram shown in g (Supplementary Movie 7). Scale bars: a, e, 2 μm, b, f, 100 nm; d, 5 μm; g, h, 50 nm