The I1 dynein-associated tether and tether head complex is a conserved regulator of ciliary motility

Fu, Gang; Wang, Qian; Phan, Nhan; Urbańska, Paulina; Joachimiak, Ewa; Lin, Jianfeng; Włoga, Dorota; Nicastro, Daniela

doi:10.1091/mbc.e18-02-0142

Cited by 56 publications

(90 citation statements)

References 55 publications

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“…We rescued the pf16 mutant with wild-type PF16 C-terminally tagged with BCCP. Axonemes of the rescued strain were isolated, and the biotinylated tag was enhanced with streptavidin-nanogold, which is visible as additional cryo-EM density in comparison to the wild-type structure Fu et al, 2018). Tomograms of the pf16;PF16::BCCP rescue showed only "9+2" axonemes and all CA projections were restored to the wild-type architecture with and without (control) addition of streptavidin-gold ( We were unable to rescue the pf16 mutant with N-terminally tagged PF16, possibly because the PF16 N-terminus is required for protein-protein interactions and assembly of the C1a-e-c supercomplex, or the N-terminal BCCP tag (9 kDa) disrupts PF16 folding or its transport into the axoneme.…”

Section: Pf16 a C1a Projection Subunit Is Required For C1a-e-c Compmentioning

confidence: 99%

“…The pf28 and pf16 (Zhao et al, 2019), which served as the control. As previously described (Fu et al, 2018),…”

Section: Strains and Culture Conditionsmentioning

confidence: 99%

“…Frame alignment for data collected on the K2 camera, alignment of tilt serial images and tomogram reconstruction were performed as previously described (Fu et al, 2018). In brief, motion correction of the frames was done with a script extracted from the IMOD software (Kremer et al, 1996).…”

Section: Image Processingmentioning

confidence: 99%

“…The "9+2" axonemal core structure of motile cilia consists of nine outer doublet microtubules (DMTs) surrounding two singlet microtubules (C1 and C2) that form the central apparatus (CA), or central pair complex. Attached to this axonemal microtubule scaffold are hundreds of proteins (Pazour et al, 2005), including the inner and outer arm dynein motors, and regulatory complexes forming part of the signal transduction pathway(s) that coordinate dynein activity to generate ciliary motility (Summers and Gibbons, 1971;Sale and Satir, 1977;Lin and Nicastro, 2018;Witman et al, 1978;Smith and Sale, 1992;Piperno et al, 1994;Smith and Lefebvre, 1997a;Porter and Sale, 2000;Smith, 2002;Mitchell, 2004;Nicastro et al, 2006;Dymek and Smith, 2007;Wirschell et al, 2007;Bower et al, 2009;Heuser et al, 2009;a;Yamamoto et al, 2013;Loreng and Smith, 2017;Fu et al, 2018;Kubo et al, 2018).…”

Section: Main Text Introductionmentioning

confidence: 99%

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Structural organization of the C1a-e-c supercomplex within the ciliary central apparatus

Zhao

Dymek

et al. 2019

Preprint

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Running title: C1a-e-c supercomplex of motile cilia Summary Fu et al. use a wild-type vs. mutant comparison and cryo-electron tomography of Chlamydomonas flagella to identify central apparatus (CA) subunits and visualize their location in the native 3D CA structure. The study provides a better understanding of the CA and how it regulates ciliary motility. Abbreviations:BCCP, biotin-carboxyl-carrier-protein; CA, central apparatus; CCD, charge-coupled device; cryo-ET, cryo-electron tomography; DMT, doublet microtubule; FAP, flagellar associated protein; IDA, inner dynein arm; MS, mass spectrometry; N-DRC, nexin-dynein regulatory complex; ODA, outer dynein arm; PCD, primary ciliary dyskinesia; psu, peripheral subunit. AbstractNearly all motile cilia contain a central apparatus (CA) composed of two connected singletmicrotubules with attached projections that play crucial roles in regulating ciliary motility.Defects in CA assembly usually result in motility-impaired or paralyzed cilia, which in humans causes disease. Despite their importance, the protein composition and functions of the CA projections are largely unknown. Here, we integrated biochemical and genetic approaches with cryo-electron tomography to compare the CA of wild type Chlamydomonas with CA mutants.We identified a large (>2 MDa) complex, the C1a-e-c supercomplex, that requires the PF16 protein for assembly and contains the CA components FAP76, FAP81, FAP92, and FAP216. We localized these subunits within the supercomplex using nanogold-labeling and show that loss of any one of them results in impaired ciliary motility. These data provide insight into the subunit organization and three-dimensional (3D) structure of the CA, which is a prerequisite for understanding the molecular mechanisms by which the CA regulates ciliary beating. Despite many biochemical and structural studies of the CA (Witman et al., 1978; protein composition, 3D organization, and functional mechanism(s) of the CA in ciliary motility are not fully understood. Our recent mass spectrometry (MS) study compared the proteomes of Chlamydomonas wild-type and mutant axonemes, and identified 44 new candidate CA proteins assigned to the C1 or the C2 microtubule (Zhao et al., 2019).However, questions about the organization, assembly, and function of the CA and its projections remain, making the CA the structurally and functionally least understood axonemal complex to date.Here we combined biochemical, genetic, and structural analyses to investigate the protein composition and molecular organization of a group of interconnected CA projections, here termed the C1a-e-c supercomplex, in wild-type and CA mutants of Chlamydomonas. Sucrose gradient sedimentation and MS revealed that several CA proteins identified in this study, FAP76, FAP81, FAP92 and FAP216, are associated with the protein PF16, previously assigned to the C1 microtubule but not to a specific projection. Chlamydomonas mutants that lacked any of these proteins showed impaired motility. Structural comparisons of flagella from wild-ty...

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Section: Pf16 a C1a Projection Subunit Is Required For C1a-e-c Compmentioning

confidence: 99%

“…The pf28 and pf16 (Zhao et al, 2019), which served as the control. As previously described (Fu et al, 2018),…”

Section: Strains and Culture Conditionsmentioning

confidence: 99%

Section: Image Processingmentioning

confidence: 99%

Section: Main Text Introductionmentioning

confidence: 99%

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Structural organization of the C1a-e-c supercomplex within the ciliary central apparatus

Zhao

Dymek

et al. 2019

Preprint

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“…In Tetrahymena, CFAP57 is placed adjacent to the FAP43/44 complex based on proximity mapping (6). Comparative proteomics analysis of I1/f and FAP43/44 mutants showed that the I1 dynein and the T/TH complex assemble independently of each other (56). The fap57 mutants assemble the Il/f twoheaded dynein complex properly as well (Table 3).…”

Section: Discussionmentioning

confidence: 99%

Mutation ofCFAP57causes primary ciliary dyskinesia by disrupting the asymmetric targeting of a subset of ciliary inner dynein arms

Bustamante-Marin

Horani

Stoyanova

et al. 2019

Preprint

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word count: 247 Text word count, without methods: 3856 Abstract (247/ 250 words)Primary ciliary dyskinesia (PCD) is characterized by chronic airway disease, male infertility, and randomization of the left/right body axis, and is caused by defects of motile cilia and sperm flagella. We screened a cohort of affected individuals that lack an obvious TEM structural phenotype for pathogenic variants using whole exome capture and next generation sequencing. The population sampling probability (PSAP) algorithm identified one subject with a homozygous nonsense variant [(c.1762C>T) p.(Arg588*) exon 11] in the uncharacterized CFAP57 gene. In normal human nasal epithelial cells, CFAP57 localizes throughout the ciliary axoneme. Analysis of cells from the PCD patient shows a loss of CFAP57, reduced beat frequency, and an alteration in the ciliary waveform. Knockdown of CFAP57 in human tracheobronchial epithelial cells (hTECs)recapitulates these findings. Phylogenetic analysis showed that CFAP57 is conserved in organisms that assemble motile cilia, and CFAP57 is allelic with the BOP2 gene identified previously in Chlamydomonas. Two independent, insertional fap57Chlamydomonas mutant strains show reduced swimming velocity and altered waveforms. Tandem mass spectroscopy showed that CFAP57 is missing, and the "g" inner dyneins (DHC7 and DHC3) and the "d" inner dynein (DHC2) are reduced. Our data demonstrate that the FAP57 protein is required for the asymmetric assembly of inner dyneins on only a subset of the microtubule doublets, and this asymmetry is essential for the generation of an effective axonemal waveform. Together, our data identifies mutations in CFAP57 as a cause of PCD with a specific defect in the inner dynein arm assembly process. Significance (119/120 words)Motile cilia are found throughout eukaryotic organisms and performs essential functions.Primary ciliary dyskinesia (PCD) is a rare disease that affects the function of motile cilia.By applying a novel population sampling probability algorithm (PSAP) that uses large population sequencing databases and pathogenicity prediction algorithms, we identified a variant in an uncharacterized gene, CFAP57. This is the first reported example of PCD caused by a mutation that affects only a subset of the inner dynein arms, which are needed to generate the waveform. CFAP57 identifies an address for specific dynein arms. These findings demonstrate the effectiveness of the PSAP algorithm, expand our understanding of the positioning of dynein arms, and identify mutations in CFAP57 as a cause of PCD.

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Algal Ciliary Motility

Hunter

Penny

Dutcher

2021

Encyclopedia of Life Sciences

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The microtubule‐based cilium (also known as a flagellum) extends from the surface of most eukaryotic cells. This long, slender organelle is biochemically complex and is conserved in many eukaryotic organisms. The unicellular alga, Chlamydomonas reinhardtii, serves as a tractable organism for studying the assembly and function of cilia. Cilia provide locomotion and environmental sensing. To produce motility, specialised molecular motors called outer and inner dynein arms generate force to produce waveform. These motors are regulated by other macromolecular complexes in the cilium. The ease of genetic and biochemical approaches combined with electron microscopy has provided the ability to perform in‐depth studies of the regulation of ciliary assembly and function. Results from Chlamydomonas researchers have broadened our knowledge of motile cilia diseases in humans. There are still many outstanding questions to be addressed. Key Concepts Chlamydomonas reinhardtii is a haploid, motile, single‐celled alga that is useful for the study of cilia. The Chlamydomonas cilium is a complex structure that generates asymmetric and symmetric waveforms to allow forward and backward swimming, respectively, and orientation to environmental signals. During ciliary motility, dynein arms pull microtubule doublets past each other to generate sliding, which is converted into axonemal bending, although the mechanism for how this occurs is still controversial. Outer dynein arms control the ciliary beat frequency by changing the velocity of doublet sliding. Inner dynein arms in conjunction with the radial spokes and the central pair apparatus, control the bend amplitude and the shape of the waveform. The axoneme is divided into 96 nm repeating segments that contain regulatory, enzymatic and structural components essential for motility; these include the inner and outer dynein arms, N‐DRC, MIA complex and radial spokes as well as two central microtubules. IFT (intraflagellar transport) is essential for building the cilium by transporting cargo into (anterograde) and out of (retrograde) the cilium. Cell biological research about ciliary components, including the central pair apparatus, radial spokes and N‐DRC, provides key information about how ciliary motility is regulated. Genetic, biochemical and microscopic methods used in Chlamydomonas have allowed molecular resolution of interactions between regulatory complexes. Studies of Chlamydomonas cilia have provided additional insights to the structure, function and regulation of cilia, and have helped with the understanding of human cilia‐related diseases called ciliopathies.

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The I1 dynein-associated tether and tether head complex is a conserved regulator of ciliary motility

Cited by 56 publications

References 55 publications

Structural organization of the C1a-e-c supercomplex within the ciliary central apparatus

Structural organization of the C1a-e-c supercomplex within the ciliary central apparatus

Mutation ofCFAP57causes primary ciliary dyskinesia by disrupting the asymmetric targeting of a subset of ciliary inner dynein arms

Algal Ciliary Motility

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