Douglas Tritschler

The nexin-dynein regulatory complex subunit DRC1 is essential for motile cilia function in algae and humans

Primary ciliary dyskinesia (PCD) is characterized by dysfunction of respiratory cilia and sperm flagella and random determination of visceral asymmetry. Here, we identify the DRC1 subunit of the nexin-dynein regulatory complex (N-DRC), an axonemal structure critical for the regulation of dynein motors, and show that mutations in the gene encoding DRC1, CCDC164, are involved in PCD pathogenesis. Loss-of-function mutations disrupting DRC1 result in severe defects in assembly of the N-DRC structure and defective ciliary movement in Chlamydomonas reinhardtii and humans. Our results highlight a role for N-DRC integrity in regulating ciliary beating and provide the first direct evidence that mutations in DRC genes cause human disease.

The N-DRC forms a conserved biochemical complex that maintains outer doublet alignment and limits microtubule sliding in motile axonemes

The nexin–dynein regulatory complex (N-DRC) is implicated in the control of dynein activity as a structural component of the nexin link. This study identifies several new subunits of the N-DRC and demonstrates for the first time that it forms a discrete biochemical complex that maintains outer doublet integrity and regulates microtubule sliding.

Building blocks of the nexin-dynein regulatory complex in Chlamydomonas flagella

The directional flow generated by motile cilia and flagella is critical for many processes, including human development and organ function. Normal beating requires the control and coordination of thousands of dynein motors, and the nexin-dynein regulatory complex (N-DRC) has been identified as an important regulatory node for orchestrating dynein activity. The nexin link appears to be critical for the transformation of dynein-driven, linear microtubule sliding to flagellar bending, yet the molecular composition and mechanism of the N-DRC remain largely unknown. Here, we used proteomics with special attention to protein phosphorylation to analyze the composition of the N-DRC and to determine which subunits may be important for signal transduction. Two-dimensional electrophoresis and MALDI-TOF mass spectrometry of WT and mutant flagellar axonemes from Chlamydomonas identified 12 N-DRC-associated proteins, including all seven previously observed N-DRC components. Sequence and PCR analyses identified the mutation responsible for the phenotype of the sup-pf-4 strain, and biochemical comparison with a radial spoke mutant revealed two components that may link the N-DRC and the radial spokes. Phosphoproteomics revealed eight proteins with phosphorylated isoforms for which the isoform patterns changed with the genotype as well as two components that may play pivotal roles in N-DRC function through their phosphorylation status. These data were assembled into a model of the N-DRC that explains aspects of its regulatory function.

In Situ Localization of N and C Termini of Subunits of the Flagellar Nexin-Dynein Regulatory Complex (N-DRC) Using SNAP Tag and Cryo-electron Tomography

Background: Techniques to localize proteins in situ at high resolution are important but limited. Results: Combining SNAP tag technology with cryo-electron tomography, we precisely localized proteins within the N-DRC that are important for ciliary motility. Conclusion: The developed method was applied to localize proteins with ∼3 nm resolution without interfering with the complex function. Significance: The method is a powerful tool for studies of proteins in situ. Cryo-electron tomography (cryo-ET) has reached nanoscale resolution for in situ three-dimensional imaging of macromolecular complexes and organelles. Yet its current resolution is not sufficient to precisely localize or identify most proteins in situ; for example, the location and arrangement of components of the nexin-dynein regulatory complex (N-DRC), a key regulator of ciliary/flagellar motility that is conserved from algae to humans, have remained elusive despite many cryo-ET studies of cilia and flagella. Here, we developed an in situ localization method that combines cryo-ET/subtomogram averaging with the clonable SNAP tag, a widely used cell biological probe to visualize fusion proteins by fluorescence microscopy. Using this hybrid approach, we precisely determined the locations of the N and C termini of DRC3 and the C terminus of DRC4 within the three-dimensional structure of the N-DRC in Chlamydomonas flagella. Our data demonstrate that fusion of SNAP with target proteins allowed for protein localization with high efficiency and fidelity using SNAP-linked gold nanoparticles, without disrupting the native assembly, structure, or function of the flagella. After cryo-ET and subtomogram averaging, we localized DRC3 to the L1 projection of the nexin linker, which interacts directly with a dynein motor, whereas DRC4 was observed to stretch along the N-DRC base plate to the nexin linker. Application of the technique developed here to the N-DRC revealed new insights into the organization and regulatory mechanism of this complex, and provides a valuable tool for the structural dissection of macromolecular complexes in situ.

The role of the dynein light intermediate chain in retrograde IFT and flagellar function in Chlamydomonas

D1bLIC is a subunit of the retrograde IFT motor. Knockdown or knockout of D1bLIC has dose-dependent effects on flagellar assembly, length, motility, and signaling. iTRAQ-based proteomics identifies novel proteins altered in d1blic mutant flagella. TIRF microscopy reveals the kinetics and remodeling of the retrograde motor at the flagellar tip.

Mutation of Growth Arrest Specific 8 Reveals a Role in Motile Cilia Function and Human Disease.

Ciliopathies are genetic disorders arising from dysfunction of microtubule-based cellular appendages called cilia. Different cilia types possess distinct stereotypic microtubule doublet arrangements with non-motile or ‘primary’ cilia having a 9+0 and motile cilia have a 9+2 array of microtubule doublets. Primary cilia are critical sensory and signaling centers needed for normal mammalian development. Defects in their structure/function result in a spectrum of clinical and developmental pathologies including abnormal neural tube and limb patterning. Altered patterning phenotypes in the limb and neural tube are due to perturbations in the hedgehog (Hh) signaling pathway. Motile cilia are important in fluid movement and defects in motility result in chronic respiratory infections, altered left-right asymmetry, and infertility. These features are the hallmarks of Primary Ciliary Dyskinesia (PCD, OMIM 244400). While mutations in several genes are associated with PCD in patients and animal models, the genetic lesion in many cases is unknown. We assessed the in vivo functions of Growth Arrest Specific 8 (GAS8). GAS8 shares strong sequence similarity with the Chlamydomonas Nexin-Dynein Regulatory Complex (NDRC) protein 4 (DRC4) where it is needed for proper flagella motility. In mammalian cells, the GAS8 protein localizes not only to the microtubule axoneme of motile cilia, but also to the base of non-motile cilia. Gas8 was recently implicated in the Hh signaling pathway as a regulator of Smoothened trafficking into the cilium. Here, we generate the first mouse with a Gas8 mutation and show that it causes severe PCD phenotypes; however, there were no overt Hh pathway phenotypes. In addition, we identified two human patients with missense variants in Gas8. Rescue experiments in Chlamydomonas revealed a subtle defect in swim velocity compared to controls. Further experiments using CRISPR/Cas9 homology driven repair (HDR) to generate one of these human missense variants in mice demonstrated that this allele is likely pathogenic.

The nexin link and B-tubule glutamylation maintain the alignment of outer doublets in the ciliary axoneme

We developed quantitative assays to test the hypothesis that the N‐DRC is required for integrity of the ciliary axoneme. We examined reactivated motility of demembranated drc cells, commonly termed “reactivated cell models.” ATP‐induced reactivation of wild‐type cells resulted in the forward swimming of ∼90% of cell models. ATP‐induced reactivation failed in a subset of drc cell models, despite forward motility in live drc cells. Dark‐field light microscopic observations of drc cell models revealed various degrees of axonemal splaying. In contrast, >98% of axonemes from wild‐type reactivated cell models remained intact. The sup‐pf4 and drc3 mutants, unlike other drc mutants, retain most of the N‐DRC linker that interconnects outer doublet microtubules. Reactivated sup‐pf4 and drc3 cell models displayed nearly wild‐type levels of forward motility. Thus, the N‐DRC linker is required for axonemal integrity. We also examined reactivated motility and axoneme integrity in mutants defective in tubulin polyglutamylation. ATP‐induced reactivation resulted in forward swimming of >75% of tpg cell models. Analysis of double mutants defective in tubulin polyglutamylation and different regions of the N‐DRC indicate B‐tubule polyglutamylation and the distal lobe of the linker region are both important for axonemal integrity and normal N‐DRC function.

DRC2/CCDC65 is a central hub for assembly of the nexin–dynein regulatory complex and other regulators of ciliary and flagellar motility

DRC2 is a subunit of the nexin–dynein regulatory complex linked to primary ciliary dyskinesia. Little is known about the impact of drc2 mutations on axoneme composition and structure. We used proteomic and structural approaches to reveal that DRC2 coassembles with DRC1 to attach the N-DRC to the A-tubule and mediate interactions with other regulatory structures.