In vivo imaging of radial spoke proteins reveals independent assembly and turnover of the spoke head and stalk

Abstract

Radial spokes (RSs) are multiprotein complexes regulating dynein activity. In the cell body and ciliary matrix, RS proteins are present in a 12S precursor, which is converted into axonemal 20S spokes consisting of a head and stalk. To study RS assembly 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 pfl, lacking spoke heads, and pf14, lacking RSs entirely. RSP3 and RSP4 mostly co-migrated by intraflagellar transport (IFT). Transport was elevated during ciliary assembly. IFT of RSP4-FP depended on RSP3. To study RS assembly independently of ciliogenesis, strains expressing FP-tagged RS proteins were mated to untagged cells with, without, or with partial RSs. RSP4-FP is added a tip-to-base fashion to preexisting pf1 spoke stalks while de novo RS assembly occurred lengthwise. In wild-type cilia, the exchange rate of head protein RSP4 exceeded that of the stalk protein RSP3 suggesting increased turnover of spoke heads. The data indicate that RSP3 and RSP4 while transported together separate inside cilia during RS repair and maintenance. The 12S RS precursor encompassing both proteins could represent transport form of the RS ensuring stoichiometric delivery by IFT. (196 of 200)