PKD2 binds to the mastigoneme protein MST1. (A) PKD2-GFP pull-down assay using a wild-type control strain (g1) and a strain expressing both endogenous and GFP-tagged PKD2. Shown are the detergent extracts of isolated cilia (Input), the flow-throughs, and the eluates from the anti-GFP nanobody trap. Top: Western blot (WB) showing pull-down of both the GFP-tagged and the endogenous C-terminal fragment of PKD2 indicating PKD2 oligomers. Antibodies to IFT81 were used as loading control. Bottom: Silver-stained SDS-PAGE. Mass spectrometry identified the bands marked by arrows as MST1 and a mix of the PKD2-C-GFP and PKD2-N. (Ba–Bc) Schematic representation of Chlamydomonas with mastigonemes in blue (a), MST1 with the predicted signal sequence in green (b; trapezoids indicate glycosylation), and a single mastigoneme (c). (Bd) 20-nm-thick tomographic slice of a mastigoneme showing the periodic substructure (arrowheads) of the main filament and the thinner extended tip (arrow). Bar = 50 nm. (Be and Bf) 1-nm-thick tomographic slice (e) and a full-volume 3D isosurface rendering representation (f) of the subtomogram average based on 1,977 repeat units from 73 mastigonemes after applying fourfold symmetry. Bar = 5 nm. (C) Whole-mount negative stain of an intact Chlamydomonas wild-type cilium. Mastigonemes (arrowheads), released vesicle-like structures (arrows), and cilium (colored in red) are marked in panel a. Enlarged image of the boxed area in panel a showing the vesicle-like structures and mastigonemes (b). Bar = 500 nm. (D) Anti-GFP whole-mount immunogold staining of mastigonemes released from pkd2 PKD2-GFP cells (a–c) and control cilia (d). 10-nm gold complexes decorate the base (arrowheads) of many mastigonemes (arrows) of the PKD2-GFP strain. Bars = 200 nm.