Scaffold subunits support associated subunit assembly in the Chlamydomonas ciliary nexin–dynein regulatory complex

Abstract

The nexin–dynein regulatory complex (N-DRC) in motile cilia and flagella functions as a linker between neighboring doublet microtubules, acts to stabilize the axonemal core structure, and serves as a central hub for the regulation of ciliary motility. Although the N-DRC has been studied extensively using genetic, biochemical, and structural approaches, the precise arrangement of the 11 (or more) N-DRC subunits remains unknown. Here, using cryo-electron tomography, we have compared the structure of Chlamydomonas wild-type flagella to that of strains with specific DRC subunit deletions or rescued strains with tagged DRC subunits. Our results show that DRC7 is a central linker subunit that helps connect the N-DRC to the outer dynein arms. DRC11 is required for the assembly of DRC8, and DRC8/11 form a subcomplex in the proximal lobe of the linker domain that is required to form stable contacts to the neighboring B-tubule. Gold labeling of tagged subunits determines the precise locations of the previously ambiguous N terminus of DRC4 and C terminus of DRC5. DRC4 is now shown to contribute to the core scaffold of the N-DRC. Our results reveal the overall architecture of N-DRC, with the 3 subunits DRC1/2/4 forming a core complex that serves as the scaffold for the assembly of the “functional subunits,” namely DRC3/5–8/11. These findings shed light on N-DRC assembly and its role in regulating flagellar beating.

Nexin-Dynein regulatory complex component DRC7 but not FBXL13 is required for sperm flagellum formation and male fertility in mice

“…We investigated the interaction of N-DRC components. We focused on DRC3, GAS8, and TCTE1 because these DRCs were shown to be localized adjacent to DRC7 in Chlamydomonas flagella ( Fig 1A) [13][14][15]. We co-expressed FLAG-tagged mouse DRC7 and PA-tagged mouse DRC3, GAS8, or TCTE1 in HEK293T cells and confirmed that mouse DRC7 interacts with mouse DRC3 and TCTE1 but not RSPH4A, a radial spoke protein, using immunoprecipitation analysis ( Fig 3A).…”

“…Despite showing testisenriched expression, Fbxl13 is not necessary for male fertility, sperm formation, or sperm motility. According to the previous report in Chlamydomonas, DRC6 (FBXL13 ortholog) is located in the most distal region adjacent to the B-tubule [10,13]; therefore, FBXL13 may not play critical roles compared to proteins localized at the base of the N-DRC. It is also possible that there is either a paralogue or an analog that replaces FBXL13 in mouse flagella.…”

“…Fbxl13 and Drc7 are evolutionarily conserved genes in many eukaryotes, including Chlamydomonas, Trypanosoma, mouse, and human (S1 and S2 Figs, S1 Table). Localization of each N-DRC component has been analyzed with biochemical and cryo-electron tomography analyses of Chlamydomonas wild-type and mutant axonemes ( Fig 1A) [13,14]. To investigate spatial expression of Fbxl13 and Drc7 in mice, we conducted multi-tissue RT-PCR using cDNA obtained from adult mice.…”

“…Drc7 is also expressed abundantly in testis with weak expression in brain, thymus, lung and ovary ( Fig 1B), suggesting that Fbxl13 and Drc7 may have some roles in spermatogenesis. In mice, the first wave of spermatogenesis occurs within the first 35 days [13,14]. The numbers shown in the N-DRC correspond to each DRC component.…”

“…The N-DRC is a large complex that links the A-tubule of one doublet to the B-tubule of the adjacent doublet [10][11][12]. In the unicellular flagellate Chlamydomonas reinhardtii, 11 distinct subunits that compose the N-DRC have been identified [10,13,14]. Further studies using Chlamydomonas mutants revealed that the N-DRC plays critical roles in regulating flagellar motility [15][16][17].…”