Subunit interactions within the Chlamydomonas flagellar spokehead

The radial spoke (RS)/central pair (CP) system in cilia and flagella plays an essential role in the regulation of force generation by dynein, the motor protein that drives cilia/flagella movements. Mechanical and mechanochemicl interactions between the CP and the distal part of the RS, the spokehead, should be crucial for this control; however, the details of interaction are totally unknown. As an initial step toward an understanding of the RS-CP interaction, we examined the protein-protein interactions between the five spokehead proteins (radial spoke protein (RSP)1, RSP4, RSP6, RSP9, and RSP10) and three spoke stalk proteins (RSP2, RSP5, and RSP23), all expressed as recombinant proteins. Three of them were shown to have physiological activities by electroporation-mediated protein delivery into mutants deficient in the respective proteins. Glutathione S-transferase pulldown assays in vitro detected interactions in 10 out of 64 pairs of recombinants. In addition, chemical crosslinking of axonemes using five reagents detected seven kinds of interactions between the RS subunits in situ. Finally, in the mixture of the recombinant spokehead subunits, RSP1, RSP4, RSP6, and RSP9 formed a 7-10S complex as detected by sucrose density gradient centrifugation. It may represent a partial assembly of the spokehead. From these results, we propose a model of interactions taking place between the spokehead subunits.