The axoneme of motile cilia is the largest macromolecular machine of eukaryotic cells. In humans, impaired axoneme function causes a range of ciliopathies. Axoneme assembly, structure, and motility ...require a radially arranged set of doublet microtubules, each decorated in repeating patterns with non-tubulin components. We use single-particle cryo-electron microscopy to visualize and build an atomic model of the repeating structure of a native axonemal doublet microtubule, which reveals the identities, positions, repeat lengths, and interactions of 38 associated proteins, including 33 microtubule inner proteins (MIPs). The structure demonstrates how these proteins establish the unique architecture of doublet microtubules, maintain coherent periodicities along the axoneme, and stabilize the microtubules against the repeated mechanical stress induced by ciliary motility. Our work elucidates the architectural principles that underpin the assembly of this large, repetitive eukaryotic structure and provides a molecular basis for understanding the etiology of human ciliopathies.
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•High-resolution cryo-EM structure of a native axonemal doublet microtubule•Atomic model for the 48-nm repeat structure includes 38 proteins•Coherent register between different periodicities via interconnected networks•Insights into diverse MIP functions and roles in ciliopathies
Visualizing axonemal microtubules and the proteins that decorate them, on the outside and inside, points to how the underlying periodic architecture supports cilia function.
Asymmetries in the cilia of Chlamydomonas Dutcher, Susan K
Philosophical transactions of the Royal Society of London. Series B. Biological sciences,
02/2020, Letnik:
375, Številka:
1792
Journal Article
Recenzirano
Odprti dostop
The generation of ciliary waveforms requires the spatial and temporal regulation of dyneins. This review catalogues many of the asymmetric structures and proteins in the cilia of
, a unicellular alga ...with two cilia that are used for motility in liquid medium. These asymmetries, which have been identified through mutant analysis, cryo-EM tomography and proteomics, provide a wealth of information to use for modelling how waveforms are generated and propagated. This article is part of the Theo Murphy meeting issue 'Unity and diversity of cilia in locomotion and transport'.
In order to provide a comprehensive resource for human structural variants (SVs), we generated long-read sequence data and analyzed SVs for fifteen human genomes. We sequence resolved 99,604 ...insertions, deletions, and inversions including 2,238 (1.6 Mbp) that are shared among all discovery genomes with an additional 13,053 (6.9 Mbp) present in the majority, indicating minor alleles or errors in the reference. Genotyping in 440 additional genomes confirms the most common SVs in unique euchromatin are now sequence resolved. We report a ninefold SV bias toward the last 5 Mbp of human chromosomes with nearly 55% of all VNTRs (variable number of tandem repeats) mapping to this portion of the genome. We identify SVs affecting coding and noncoding regulatory loci improving annotation and interpretation of functional variation. These data provide the framework to construct a canonical human reference and a resource for developing advanced representations capable of capturing allelic diversity.
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•We sequence resolve and annotate 99,604 common human structural variants•55% of VNTRs map to the end of chromosomes and correlate with double-strand breaks•Alternate alleles facilitate accurate genotyping with short reads and new associations•We patch the reference and add diversity needed for developing a pan human genome
Long-read sequencing allows generation of a large catalog of human structural variants and the development of an algorithm for genotyping SVs from short-read data, clarifying the spectrum and importance of structural variation in the human genome.
Motile cilia assembly utilizes over 800 structural and cytoplasmic proteins. Variants in approximately 58 genes cause primary ciliary dyskinesia (PCD) in humans, including the dynein arm ...(pre)assembly factor (DNAAF) gene DNAAF4. In humans, outer dynein arms (ODAs) and inner dynein arms (IDAs) fail to assemble motile cilia when DNAAF4 function is disrupted. In Chlamydomonas reinhardtii, a ciliated unicellular alga, the DNAAF4 ortholog is called PF23. The pf23-1 mutant assembles short cilia and lacks IDAs, but partially retains ODAs. The cilia of a new null allele (pf23-4) completely lack ODAs and IDAs and are even shorter than cilia from pf23-1. In addition, PF23 plays a role in the cytoplasmic modification of IC138, a protein of the two-headed IDA (I1/f). As most PCD variants in humans are recessive, we sought to test if heterozygosity at two genes affects ciliary function using a second-site non-complementation (SSNC) screening approach. We asked if phenotypes were observed in diploids with pairwise heterozygous combinations of 21 well-characterized ciliary mutant Chlamydomonas strains. Vegetative cultures of single and double heterozygous diploid cells did not show SSNC for motility phenotypes. When protein synthesis is inhibited, wild-type Chlamydomonas cells utilize the pool of cytoplasmic proteins to assemble half-length cilia. In this sensitized assay, 8 double heterozygous diploids with pf23 and other DNAAF mutations show SSNC; they assemble shorter cilia than wild-type. In contrast, double heterozygosity of the other 203 strains showed no effect on ciliary assembly. Immunoblots of diploids heterozygous for pf23 and wdr92 or oda8 show that PF23 is reduced by half in these strains, and that PF23 dosage affects phenotype severity. Reductions in PF23 and another DNAAF in diploids affect the ability to assemble ODAs and IDAs and impedes ciliary assembly. Thus, dosage of multiple DNAAFs is an important factor in cilia assembly and regeneration.
Primary ciliary dyskinesia (PCD) is characterized by chronic airway disease, reduced fertility, and randomization of the left/right body axis. It is caused by defects of motile cilia and sperm ...flagella. We screened a cohort of affected individuals that lack an obvious axonemal defect for pathogenic variants using whole exome capture, next generation sequencing, and bioinformatic analysis assuming an autosomal recessive trait. We identified one subject with an apparently homozygous nonsense variant (c.1762C>T), p.(Arg588*) in the uncharacterized CFAP57 gene. Interestingly, the variant results in the skipping of exon 11 (58 amino acids), which may be due to disruption of an exonic splicing enhancer. In normal human nasal epithelial cells, CFAP57 localizes throughout the ciliary axoneme. Nasal cells from the PCD patient express a shorter, mutant version of CFAP57 and the protein is not incorporated into the axoneme. The missing 58 amino acids include portions of WD repeats that may be important for loading onto the intraflagellar transport (IFT) complexes for transport or docking onto the axoneme. A reduced beat frequency and an alteration in ciliary waveform was observed. Knockdown of CFAP57 in human tracheobronchial epithelial cells (hTECs) recapitulates these findings. Phylogenetic analysis showed that CFAP57 is highly conserved in organisms that assemble motile cilia. CFAP57 is allelic with the BOP2/IDA8/FAP57 gene identified previously in Chlamydomonas reinhardtii. Two independent, insertional fap57 Chlamydomonas mutant strains show reduced swimming velocity and altered waveforms. Tandem mass tag (TMT) mass spectroscopy shows that FAP57 is missing, and the "g" inner dyneins (DHC7 and DHC3) and the "d" inner dynein (DHC2) are reduced, but the FAP57 paralog FBB7 is increased. Together, our data identify a homozygous variant in CFAP57 that causes PCD that is likely due to a defect in the inner dynein arm assembly process.
A pair of extensively modified microtubules form the central apparatus (CA) of the axoneme of most motile cilia, where they regulate ciliary motility. The external surfaces of both CA microtubules ...are patterned asymmetrically with large protein complexes that repeat every 16 or 32 nm. The composition of these projections and the mechanisms that establish asymmetry and longitudinal periodicity are unknown. Here, by determining cryo-EM structures of the CA microtubules, we identify 48 different CA-associated proteins, which in turn reveal mechanisms for asymmetric and periodic protein binding to microtubules. We identify arc-MIPs, a novel class of microtubule inner protein, that bind laterally across protofilaments and remodel tubulin structure and lattice contacts. The binding mechanisms utilized by CA proteins may be generalizable to other microtubule-associated proteins. These structures establish a foundation to elucidate the contributions of individual CA proteins to ciliary motility and ciliopathies.
In motile cilia, a mechanoregulatory network is responsible for converting the action of thousands of dynein motors bound to doublet microtubules into a single propulsive waveform. Here, we use two ...complementary cryo-EM strategies to determine structures of the major mechanoregulators that bind ciliary doublet microtubules in Chlamydomonas reinhardtii. We determine structures of isolated radial spoke RS1 and the microtubule-bound RS1, RS2 and the nexin-dynein regulatory complex (N-DRC). From these structures, we identify and build atomic models for 30 proteins, including 23 radial-spoke subunits. We reveal how mechanoregulatory complexes dock to doublet microtubules with regular 96-nm periodicity and communicate with one another. Additionally, we observe a direct and dynamically coupled association between RS2 and the dynein motor inner dynein arm subform c (IDAc), providing a molecular basis for the control of motor activity by mechanical signals. These structures advance our understanding of the role of mechanoregulation in defining the ciliary waveform.
Mutations in transition zone genes change the composition of the ciliary proteome. We isolated new mutations in
(denotated as
in algae) that affect the localization of the transition zone protein ...NPHP4 in the model organism
NPHP4 localization is not affected in multiple new intraflagellar transport (IFT) mutants. We compared the proteome of cilia from wild-type and mutants that affect the transition zone (
) or IFT (
and
) by mass spectrometry. The
mutant cilia show the most dramatic increase in cytoplasmic proteins. These nonciliary proteins function in translation, membrane remodeling, ATP production and as chaperonins. These proteins are excluded in isolated cilia from
(IFT172) and
(DHC1b). Our data support the idea that RPGRIP1L, but not IFT proteins, acts as part of the gate for cytoplasmic proteins. The
cilia lack only a few proteins, which suggests that RPGRIP1L only has a minor role of in the retention of ciliary proteins. The
mutant shows the greatest loss/reduction of proteins, and one-third of these proteins have a transmembrane domain. Hence, IFT172 may play a role in the retention of proteins.
Summary Ciliopathies are a growing class of disorders caused by abnormal ciliary axonemal structure and function. Our understanding of the complex genetic and functional phenotypes of these ...conditions has rapidly progressed. Primary ciliary dyskinesia (PCD) remains the sole genetic disorder of motile cilia dysfunction. However, unlike many Mendelian genetic disorders, PCD is not caused by mutations in a single gene or locus, but rather, autosomal recessive mutation in one of many genes that lead to a similar phenotype. The first reported PCD mutations, more than a decade ago, identified genes encoding known structural components of the ciliary axoneme. In recent years, mutations in genes encoding novel cytoplasmic and regulatory proteins have been discovered. These findings have provided new insights into the functions of the motile cilia, and a better understanding of motile cilia disease. Advances in genetic tools will soon allow more precise genetic testing, mandating that clinicians must understand the genetic basis of PCD. Here, we review genetic mutations, their biological impact on cilia structure and function, and the implication of emerging genetic diagnostic tools.
Cilia/flagella and basal bodies/centrioles play key roles in human health and homeostasis. Among the organisms used to study these microtubule-based organelles, the green alga Chlamydomonas ...reinhardtii has several advantages. One is the existence of a temporary phase of the life cycle, termed the dikaryon. These cells are formed during mating when the cells fuse and the behavior of flagella from two genetically distinguishable parents can be observed. During this stage, the cytoplasms mix allowing for a defect in the flagella of one parent to be rescued by proteins from the other parent. This offers the unique advantage of adding back wild-type gene product or labeled protein at endogenous levels that can used to monitor various flagellar and basal body phenotypes. Mutants that show rescue and ones that fail to show rescue are both informative about the nature of the flagella and basal body defects. When rescue occurs, it can be used to determine the mutant gene product and to follow the temporal and spatial patterns of flagellar assembly. This review describes many examples of insights into basal body and flagellar proteins' function and assembly that have been discovered using dikaryons and discusses the potential for further analyses.
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