Figure 2. Cryo-electron tomography (cryo-ET) analysis of rootlet striations.

(A) Schematic depiction of rootlet purification by membrane removal and gradient centrifugation (not shown). (B) Low-magnification cryoEM micrograph of a purified rootlet and associated ciliary cytoskeleton. (C) Negative stain EM of a purified rootlet, highlighting features visible on the rootlet surface. (D) CryoET projection image of a purified rootlet. The Fourier-filtered and thresholded striations are colored according to their appearance: D-bands in yellow and A-bands in green. Mean values of their spacing and the location of the centriole are indicated below, based on Figure 2—figure supplement 1H–J. (E) Central slice in a denoised and isotropically reconstructed electron tomogram showing two rootlet sub-fibers. (F) Example of fine features of D-bands in a cryo-ET slice and its segmentation (G) Example where D1 aligns with D2 of a neighboring sub-fiber. Larger view in Figure 2—figure supplement 1A. (H) Segmentation of the striations in the tomogram from panel D. D1–D1 contact of two sub-fibers is indicated by a white arrow. (I) Segmentation that shows amorphous features occur as two bands and connect to the rootlet surface densities. (J) Segmentation of amorphous material on the rootlet surface. The side view is shown in Figure 2—figure supplement 2H (H, J) The position of the A- and D-bands is shown by lines in the background. (E, H, J) Black arrows indicate the space between sub-fibers. (F, G, I) Fainter features not picked up by the automated segmentation were drawn with dotted lines.

Figure 2—figure supplement 1. Cross-striation analysis of purified rootlets.

(A) Low-magnification negative stain micrographs of purified rootlet sample with thicker and darker areas containing sample impurities and clustered rootlets. (B–D) Negative stain micrographs that were local contrast normalized for visualization of both thick and thin regions. Amorphous (A) and Discrete (DC, DA) striations are indicated with green and yellow, respectively. (B, C) Examples of the rootlet connected to centrioles. (D) Example of the rootlet tip. (E) Cryo-electron tomography (cryo-ET) projection image of a purified rootlet tomogram. (F) Fourier transform of panel E. (G) Inverse Fourier transform of dominant frequencies from panel F. The mean distance was obtained from analysis in panels H and I. (H) Example of sinusoid fitting to Fourier-filtered and thresholded striations of rootlet tomograms such as in panel G. (I) Distribution of sinusoid-fit values of 10 rootlets for each type of striation. (J) Phase offset in nanometer of the fitted sinusoid waves from each striation.

Figure 2—figure supplement 2. Segmentation of purified rootlets.

(A) Tomogram slice and segmentation where D1 aligns with D2 of a neighboring sub-fiber. The dotted square marks the location of Figure 2G. (B) Central slice in a denoised and isotropically reconstructed electron tomogram showing four rootlet sub-fibers. (C) Cartoon of sub-fiber arrangement in panel A. (D) Semi-automated segmentation of the striations in the tomogram from panel A by Eman2 TomoSeg. (E) Quantification of D-band alignment in 48 tomograms. (F) Full segmentation of the tomogram from panel A. (G) Segmentation of amorphous material on the rootlet surface. The side view is highlighted in blue. (H) Full segmentation of the tomogram from Figure 2. (I) Side view of segmented amorphous densities on surface of the rootlet from Figure 2. (J) Tomogram slice and segmentation highlighting connections to amorphous surface material.