In vivo analyses of radial spoke transport, assembly, repair and maintenance

Abstract Radial spokes (RSs) are multiprotein complexes that regulate dynein activity. In the cell body, RS proteins (RSPs) are present in a 12S precursor, which enters the flagella and converts into the axoneme‐bound 20S spokes consisting of a head and stalk. To study RS dynamics in vivo, we expressed fluorescent protein (FP)‐tagged versions of the head protein RSP4 and the stalk protein RSP3 to rescue the corresponding Chlamydomonas mutants pf1, lacking spoke heads, and pf14, lacking RSs entirely. RSP3 and RSP4 mostly co‐migrated by intraflagellar transport (IFT). The transport was elevated during flagellar assembly and IFT of RSP4‐FP depended on RSP3. To study RS assembly independently of ciliogenesis, strains expressing FP‐tagged RSPs were mated to untagged cells with, without, or with partial RSs. Tagged RSPs were incorporated in a spotted fashion along wild‐type‐derived flagella indicating an exchange of RSs. During the repair of pf1 ‐derived axonemes, RSP4‐FP is added onto the preexisting spoke stalks with little exchange of RSP3. Thus, RSP3 and RSP4 are transported together but appear to separate inside flagella during the repair of RSs. The 12S RS precursor encompassing both proteins could represent a transport form to ensure stoichiometric delivery of RSPs into flagella by IFT.