Scaffold subunits support associated subunit assembly in theChlamydomonasciliary 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 eleven (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 sub-complex 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 which is now shown to contribute to the core scaffold of the N-DRC and C-terminus of DRC5. Our results reveal the overall architecture of N-DRC, with the three subunits, DRC1/2/4 forming a core complex that serves as the scaffold for the assembly of the "functional subunits" associate, namely DRC3/5-8/11. These findings shed light on N-DRC assembly and its role in regulating flagellar beating. Significance StatementCilia and flagella are small hair-like appendages in eukaryotic cells that play essential roles in cell sensing, signaling, and motility. The highly conserved nexin-dynein regulatory complex (N-DRC) is one of the key regulators for ciliary motility. At least 11 proteins (DRC1-11) have been assigned to the N-DRC, but their precise arrangement within the large N-DRC structure is not yet known. Here, using cryo-electron tomography combined with genetic approaches, we have localized DRC7, the sub-complex DRC8/DRC11, the N-terminus of DRC4, and the C-terminus of DRC5. Our results provide insights into the N-DRC structure, its function in the regulation of dynein activity, and the mechanism by which n-drc mutations can lead to defects in ciliary motility that cause disease.Cilia and flagella are dynamic microtubule (MT)-based organelles that emanate from the surface of many eukaryotic cells and are involved in cell sensory functions, motility, and signaling. Defects in cilia assembly or function have been associated with multiple human disorders collectively known as ciliopathies, such as polycystic kidney disease, Bardet-Biedl syndrome, infertility, hydrocephalus, and primary ciliary dyskinesia (1, 2).The microtubule-based axoneme forms the core structure of motile cilia and is highly conserved, from the green algae Chlamydomonas reinhardtii to differentiated cells in the human body. The "9 + 2" axoneme is comprised of nine outer doublet microtubules (DMTs) and a central-pair complex (CPC) composed of two singlet microtubules and associated projections ( Fig. 1A). Each DMT is b...

Structural organization of the intermediate and light chain complex of Chlamydomonas ciliary I1 dynein

“…55 Recent studies have combined biochemical, genetic, and high resolution imaging approaches, such as cryo-ET and sub-tomogram averaging, to locate proteins, visualize their 3D structure and characterize their function(s) within cilia, including elucidating the conformational changes of axonemal structures in actively beating cilia. 5,13,18,37,[56][57][58] Using these approaches, we investigated the protein composition and structural F I G U R E 5 Model of the structural organization of the I1 ICLC. A-C, isosurface renderings (A, B) and schematic drawing (C) of Chlamydomonas wild-type I1 dynein viewed from longitudinal front (A, C) and back (B) orientations.…”

“…Cilia are composed of hundreds of different proteins, eg, more than 700 proteins in Chlamydomonas (http://labs.umassmed.edu/chlamyfp/index.php) 54 and often the mutation of a single protein results in impaired ciliary motility; yet the location of most ciliary proteins in the intact organelle remains unclear 55 . Recent studies have combined biochemical, genetic, and high resolution imaging approaches, such as cryo‐ET and sub‐tomogram averaging, to locate proteins, visualize their 3D structure and characterize their function(s) within cilia, including elucidating the conformational changes of axonemal structures in actively beating cilia 5,13,18,37,56–58 . Using these approaches, we investigated the protein composition and structural differences between Chlamydomonas wild type and I1‐dynein ICLC mutants, ida7‐1 and tctex2b (Table S1).…”

Proteomic analysis of microtubule inner proteins (MIPs) in Rib72 null Tetrahymena cells reveals functional MIPs

“…Cryo-ET and image processing of isolated axonemes from Chlamydomonas wild-type and fap115 mutant were performed as reported previously ((Stoddard et al, 2018) and (Gui et al, 2019), respectively). The fap115 mutant strain was obtained from the CLiP collection (CLiP ID: LMJ.RY0402.105592) (Li et al, 2016).…”

Functional unknomics: closing the knowledge gap to accelerate biomedical research

“…CFAP43 (orthologous to CG17687) is mutated in patients with multiple morphological abnormalities of the sperm flagella (MMAF), and CFAP52 (orthologous to CG10064) is mutated in laterality disorder, a condition caused by defects in ciliary beating during development (Coutton et al, 2018; Ta-Shma et al, 2015). A further semi-sterile hit, CG14183, is an ortholog of DRC11, a subunit of the nexin-dynein regulatory complex that regulates flagellar beating in Chlamydomonas (Gui et al, 2019). These findings prove the value of the Unknome database approach to identifying new genes of biological significance, and validate the RNAi-based screening approach.…”