Figure 6. Subtomogram averaging of the intramembranous beads/ciliary necklace along the ciliary membrane.

(A) Sections from raw tomograms from mouse rod photoreceptors displaying intramembranous beads periodically arranged on the ciliary plasma membrane (left displaying a slice from the outside view of the CC; right showing a slice through the center of the CC). The beads were categorized into two groups based on spacing, ciliary pocket region (orange arrows, ∼22-nm spacing), and CC region (purple arrows, ∼40 nm). (B) Spacing between beads for each of the two groups along the ciliary membrane (∼22 nm, n = 6 cells for ciliary pocket; and ∼40 nm, n = 7 cells for CC). The box-and-whisker plot shows the median (horizontal line), upper and lower quartile (box edges), and range of measurements (error bars), as well as individual points. (C) Conventional electron micrographs of mouse rod CC displaying the same intramembranous beads (average spacing 38.5 nm apart in this image, magenta arrows) along the CC. The dashed-line box in the image on the far left indicates the region shown at a higher magnification in the (middle) image immediately to its right. Dashed-line boxes on the image to the far right are the regions shown at a higher magnification below. (D) Map of the ciliary necklace beads obtained by subtomogram averaging was fit (in multiple copies) into a raw tomogram of a CC and the resulting map shown in a surface view from outside the CC (top, far left), in a projection view sliced through the CC membrane (immediately to the right of the surface view), and a magnified surface view (lower left). The series of images on the right show different high-magnification views of the average bead (cyan) and the portion of the CC membrane into which it was fit (gray). The longest dimension of the averaged structure is about 40 nm (magenta bars with inverted arrowheads), and the narrower dimension is about 10 nm (pink double-headed arrows). (E, F) Conventional transmission electron microscopy (TEM) images of CC showing the beads. (E, F) Dashed-line box in the upper image of (E) shows the region shown at higher magnifications below and in panel (F), to the right. The magenta bar with two inverted arrowheads represents 40 nm, and the cyan-outlined arrowheads indicate the ridge-like subdomains of the structure that yield the “bead” appearance. (D, G) is a similar view of a portion of a map from cryo-ET obtained as described in panel (D) above. (H) Plot of spacings measured for bead ridges in our TEM images and subtomogram averages, or in a previously published report of freeze-fracture/SEM studies. Whisker plots show medians, lower and upper quartiles, and range of the data, with n = 5 cells for both TEM and subtomogram average results.