Subunit interactions within the Chlamydomonas flagellar spokehead

Abstract

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 mechanochemical 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.

The DPY-30 Domain and Its Flanking Sequence Mediate the Assembly and Modulation of Flagellar Radial Spoke Complexes

“…The rest of the stalk proteins, including two RIIa domain-containing RSPs, are present in normal amounts. The phenotypes (69), further corroborated by recent cryoelectron tomography of pf24 and the other RS mutants (46) and chemical cross-linking (32), suggest that the two DPY-30 domain-containing RSPs are located in the neck region adjacent to the spokehead, whereas the two RIIa domain-containing proteins are positioned toward the radial spoke stalk base ( Fig. 1D and Table 1).…”

“…1) (3, 24, 46) and multiple short helices in the splayed spokehead (50). Additionally, it explains chemical cross-linking of RSP2 and spokehead proteins (32) and at least two proteins involved in the head-stalk connection (70). This subcomplex could further associate with other spoke proteins, such as the dimeric spoke HSP40, which depends on RSP2 for assembly (27).…”

Radial Spokes—A Snapshot of the Motility Regulation, Assembly, and Evolution of Cilia and Flagella

“…For example, in the spoke head, RSP4 and 6 are paralogs expressed from duplicated genes (Curry et al 1992), whereas RSP1 and 10 are homologs with membrane occupation and recognition nexus (MORN) repeats. Analysis of recombinant proteins and tagging indicate that each spoke head contains two copies of each protein that is inherently dimeric (Kohno et al 2011;Oda et al 2014b). Furthermore, the RIIa domains in RSP11 and RSP7, and the Dpy-30 domains in RSP2 and RSP23 (NDK5) in the stalk are dimerization and docking (DD) domains of striking similar tertiary structures .…”

Immunofluorescence analysis and diagnosis of primary ciliary dyskinesia with radial spoke defects

“…It is reported that the CP-RS interactions determine the waveform and the bend direction, whereas RS-IDA interactions influence the velocity of ciliary beating (48). In the alga, C. reinhardtii, the …”