Extended Data Fig. 1: Structural comparison of the 96-nm axonemal repeat between WT and mutant axonemes from Tetrahymena (a-f) and mouse respiratory (g-x) cilia.
From: Mouse radial spoke 3 is a metabolic and regulatory hub in cilia
a-c) Tomographic slices (a-c) and an isosurface rendering (d) of the averaged 96-nm axonemal repeat show the full-length RS3 in Tetrahymena WT (a, d), whereas class-averaging of a CSC-mutant (CFAP61-KO) (b, c) reveals that large parts of RS3 are missing from about one third of the repeats (c) (based on data previously published7). The CSC subunits CFAP61 (green) and CFAP251 (red) are colored at the base of RS3 (d). e, f) Negative staining EM of Tetrahymena WT (e) and CFAP61-KO (f) axonemes after mild salt-extraction (0.6 M NaCl) shows in WT three RS per 96-nm repeat (from distal to proximal: RS3/orange, RS2/blue and RS1/green), whereas in CFAP61-KO RS3 is missing. g-r) Tomographic slices (g-i, k-m, o-q) and isosurface renderings (j, n, r) of the averaged 96-nm repeats from WT (g-j), AK1−/− (k-n), and AK7−/− (o-r) mouse respiratory cilia show RS3 in cross-sectional view (g, k, o) and in longitudinal view (h, l, p), and the RS bases in longitudinal bottom view, similar to the isosurface renderings. White arrowheads indicate missing structures in the mutants, that is the missing distal part of RS3 in AK7−/− (o, p), and a protrusion from the DMT wall between RS1 and RS2 (red arrowheads and colored red in j and r) that is missing in AK1−/− (m, n). s-x) Tomographic slices of class averages of RS3 from AK7−/− axonemes show RS3 in the same orientations as in (o-p). The classification analysis revealed that all AK7−/− repeats have truncated RS3, but with slightly different levels of truncation (indicated by white arrowheads), from the shortest (class 1) to the longer one (class 3). The percentage of subtomograms included in each class average is indicated. Note that, the distal side of the cilium on the right in (a-d), otherwise, cross-sections are viewed from distal to proximal orientation, and in longitudinal views with distal side of the cilium on the left. Scale bars: (a-c) 20 nm, (e-f) 100 nm, (g-i, k-m, o-q) 20 nm, (s-x) 20 nm.
