Fig. 2: Cryo-ET reveals that the slit diaphragm (SD) resembles a bilayered fishnet.

a Orthogonal planes through the SD average provide an overview of the 3D geometry. b–d 1 nm thick slices through the tomographic reconstructions show the nephrocyte SD from three different perspectives: b top view revealing the fishnet pattern of the SD between the two plasma membranes (PM); c membrane view revealing two parallel lines of “pearls on a string”; and d classical view displaying two parallel electron-dense lines reflecting the bilayered architecture of the SD. (e–g) 1 nm thick slices through the 3D volume of the SD obtained after subtomogram averaging, shown from the same perspectives as in (b–d) (pixel size: 2.7 Å). The slice showing the membrane view (f) illustrates the cross-section of the fishnet pattern at the center of the fishnet, parallel to the membrane. The slice showing the classical view (g) illustrates one of the cross-sections at the center of the fishnet perpendicular to the direction of the SD line, and thus appears as two dots. The result was obtained by averaging 595 particles selected from 16 tomograms, collected on 3 lamellae from three different Drosophila larvae and three different electron microscopy grids. h–j Isosurface representation of the cryo-ET map illustrating the 3D bilayered fishnet architecture, shown from the same perspectives as in (e–g) (pixel size: 2.7 Å). Individual strands criss-cross the space between the two plasma membranes, creating the fishnet pattern. k Stereo-pair of the SD at an oblique angle showing the bilayered architecture, with each of the layers resembling a fishnet.