Figure 6.

2D STED microscopy reveals structural changes in the axoneme for IFT25 knockout cells. SNAP-SMO (SMO) and α-tubulin (αTub) are stained with Atto647N and Star520SXP-labeled antibodies, respectively, in chemically fixed MEF cells and are subsequently imaged using a two-color confocal and two-color STED microscope. Yellow double arrows indicate both magnitude shift and direction between the two red and green images. (a) In control WT MEF cells, (i–v) the ciliary membrane exhibits a cylindrical shape, whereas the axoneme is frequently observed to not extend to the tip of the cilia. (ii) The dotted line indicates where the axoneme ends. (b) In IFT25 cells, results similar to the 3D SR data are observed, where (i) kinks and (ii) narrowing of both the ciliary membrane and axoneme are pointed out in white arrows. (iii) Bulging and budding often occurs at the tip, and the axoneme appears to extend all the way to the tip and (iv) sometimes has an uneven diameter throughout the cilium. (v) Severe swelling of the ciliary membrane and the axoneme is also observed, although this is relatively rare. Scale bars, 1 μm. Image contrast within each cell line condition is identical for both channels. To see this figure in color, go online.