Expression and characterization of BCCP-tagged RSPs in C. reinhardtii. (A) Organization of the C. reinhardtii axoneme. A base to tip view of the 9 + 2 structure is shown. Positions of outer and inner dynein arms (ODA and IDA), outer doublet microtubules (DMT), radial spokes (RS), central pair microtubules (C1 and C2), and C1a and C1b projections are indicated. The image was generated by superimposing the averaged tomograms of the DMT and CP onto an unaveraged tomogram of the wild-type 9 + 2 structure. Bar, 50 nm. (B) Predicted positions of RSPs in RSs based on previous mutant analyses (Yang et al., 2006; Pigino et al., 2011) superimposed on a side view of the averaged tomogram of DMT. The distal end of the flagellum is to the right. We call the upper surface of the RS heads the CP side in this article (broken line). Bar, 10 nm. (C) Immunoblot of tagged RSPs. Axonemal proteins from wild-type (wt) and RSP mutant strains were separated by SDS-PAGE and probed with horseradish peroxidase–conjugated streptavidin, anti-HA, or anti-GFP antibodies. The asterisks indicate the detected bands of RSPs. 3C, rsp3C; 3N, rsp3N; 4C, rsp4C; 4N, rsp4N; 6C, rsp6C; 6N, rsp6N; 11C, rsp11C; 4C-HA, rsp4C-3HA; 4C-GFP, rsp4C-GFP. (D) Immunofluorescence of axonemes. Left, phase contrast; right, Alexa Fluor 546. The axonemes with BCCP-tagged RSPs were incubated with Alexa Fluor–conjugated streptavidin, and the signals were detected by immunofluorescence microscopy. All of the BCCP-RSPs inside the axonemes were confirmed to be accessible to streptavidin. For the fluorescence image from wild type, the contrast was enhanced to show the absence of signals. Bar, 5 µm.