Structural organization of the C1a-e-c supercomplex within the ciliary central apparatus.
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ABSTRACT:
SUBMITTER: Fu G
PROVIDER: S-EPMC6891083 | biostudies-literature |
REPOSITORIES: biostudies-literature
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Project description:Nearly all motile cilia have a "9+2" axoneme containing a central apparatus (CA), consisting of two central microtubules with projections, that is essential for motility. To date, only 22 proteins are known to be CA components. To identify new candidate CA proteins, we used mass spectrometry to compare axonemes of wild-type Chlamydomonas and a CA-less mutant. We identified 44 novel candidate CA proteins, of which 13 are conserved in humans. Five of the latter were studied more closely, and all five localized to the CA; therefore, most of the other candidates are likely to also be CA components. Our results reveal that the CA is far more compositionally complex than previously recognized and provide a greatly expanded knowledge base for studies to understand the architecture of the CA and how it functions. The discovery of the new conserved CA proteins will facilitate genetic screening to identify patients with a form of primary ciliary dyskinesia that has been difficult to diagnose.
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Project description:Nearly all motile cilia and flagella (terms here used interchangeably) have a '9+2' axoneme containing nine outer doublet microtubules and two central microtubules. The central pair of microtubules plus associated projections, termed the central apparatus (CA), is involved in the control of flagellar motility and is essential for the normal movement of '9+2' cilia. Research using the green alga Chlamydomonas reinhardtii, an important model system for studying cilia, has provided most of our knowledge of the protein composition of the CA, and recent work using this organism has expanded the number of known and candidate CA proteins nearly threefold. Here we take advantage of this enhanced proteome to examine the genomes of a wide range of eukaryotic organisms, representing all of the major phylogenetic groups, to identify predicted orthologues of the C. reinhardtii CA proteins and explore how widely the proteins are conserved and whether there are patterns to this conservation. We also discuss in detail two contrasting groups of CA proteins-the ASH-domain proteins, which are broadly conserved, and the PAS proteins, which are restricted primarily to the volvocalean algae. This article is part of the Theo Murphy meeting issue 'Unity and diversity of cilia in locomotion and transport'.
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Project description:Dysfunction of motile cilia can impair mucociliary clearance in the airway and result in primary ciliary dyskinesia (PCD), a pediatric syndrome characterized by chronic respiratory infection and bronchiectasis, as well as infertility, laterality defects, and occasionally hydrocephalus due to defects in cilia in other organ systems. We have previously shown that mutations in genes encoding components of the ciliary central pair apparatus (CPA) perturb ciliary motility and result in PCD in mouse models. However, little is known about how epithelial cell types in the ciliary microenvironment of the upper airway respond to defects in ciliary motility and mucociliary clearance. In this study, we have used a single-cell RNA sequencing (scRNA-seq) approach to investigate the effects of ciliary dysfunction on the tracheal epithelial cells from mouse models with mutations in CPA genes Cfap221 (also known as Pcdp1), Cfap54, and Spef2. Expected cell types were identified in the tracheal epithelial samples, including basal cells, suprabasal cells, secretory cells, deuterosomal cells, ciliated cells, tuft cells, ionocytes, and neuroendocrine cells, as well as an unidentified cell type that does not express markers of typical airway cells. The deuterosomal cells were found to exist in two states that differ largely in expression of genes involved in differentiation into the ciliated cell type. Differentially expressed genes (DEGs) were identified for each cell type, and functional enrichment analysis revealed a variety of important cellular functions altered in mutant cells. Overlapping DEGs shed light on general cellular responses to cilia dysfunction, while unique DEGs indicate that some responses may be specific to the individual mutation and ciliary defect. These analyses uncover new information about cellular responses to mucociliary clearance defects in the airway and pathogenesis of PCD.
2024-11-14 | GSE254100 | GEO
Project description:Motile cilia are ultrastructurally complex cell organelles with the ability to actively move. The highly conserved central apparatus of motile 9 × 2 + 2 cilia is composed of two microtubules and several large microtubule-bound projections, including the C1b/C1f supercomplex. The composition and function of C1b/C1f subunits has only recently started to emerge. We show that in the model ciliate Tetrahymena thermophila, C1b/C1f contains several evolutionarily conserved proteins: Spef2A, Cfap69, Cfap246/LRGUK, Adgb/androglobin, and a ciliate-specific protein Tt170/TTHERM_00205170. Deletion of genes encoding either Spef2A or Cfap69 led to a loss of the entire C1b projection and resulted in an abnormal vortex motion of cilia. Loss of either Cfap246 or Adgb caused only minor alterations in ciliary motility. Comparative analyses of wild-type and C1b-deficient mutant ciliomes revealed that the levels of subunits forming the adjacent C2b projection but not C1d projection are greatly reduced, indicating that C1b stabilizes C2b. Moreover, the levels of several IFT and BBS proteins, HSP70, and enzymes that catalyze the final steps of the glycolytic pathway: enolase ENO1 and pyruvate kinase PYK1, are also reduced in the C1b-less mutants.
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Project description:Cilia are essential organelles required for cell signaling and motility. Nearly all motile cilia have a '9+2' axoneme composed of nine outer doublet microtubules plus two central microtubules; the central microtubules together with their projections are termed the central apparatus (CA). In Chlamydomonas reinhardtii, a model organism for studying cilia, 30 proteins are known CA components, and ∼36 more are predicted to be CA proteins. Among the candidate CA proteins is the highly conserved FAP70 (CFAP70 in humans), which also has been reported to be associated with the doublet microtubules. Here, we determined by super-resolution structured illumination microscopy that FAP70 is located exclusively in the CA, and show by cryo-electron microscopy that its N-terminus is located at the base of the C2a projection of the CA. We also found that fap70-1 mutant axonemes lack most of the C2a projection. Mass spectrometry revealed that fap70-1 axonemes lack not only FAP70 but two other conserved candidate CA proteins, FAP65 (CFAP65 in humans) and FAP147 (MYCBPAP in humans). Finally, FAP65 and FAP147 co-immunoprecipitated with HA-tagged FAP70. Taken together, these data identify FAP70, FAP65 and FAP147 as the first defining components of the C2a projection.
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Project description:For all motile eukaryotic cilia and flagella, beating is regulated by changes in intraciliary calcium concentration. Although the mechanism for calcium regulation is not understood, numerous studies have shown that calmodulin (CaM) is a key axonemal calcium sensor. Using anti-CaM antibodies and Chlamydomonas reinhardtii axonemal extracts, we precipitated a complex that includes four polypeptides and that specifically interacts with CaM in high [Ca(2+)]. One of the complex members, FAP221, is an orthologue of mammalian Pcdp1 (primary ciliary dyskinesia protein 1). Both FAP221 and mammalian Pcdp1 specifically bind CaM in high [Ca(2+)]. Reduced expression of Pcdp1 complex members in C. reinhardtii results in failure of the C1d central pair projection to assemble and significant impairment of motility including uncoordinated bends, severely reduced beat frequency, and altered waveforms. These combined results reveal that the central pair Pcdp1 (FAP221) complex is essential for control of ciliary motility.
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Project description:Motile cilia and flagella play critical roles in fluid clearance and cell motility, and dysfunction commonly results in the pediatric syndrome primary ciliary dyskinesia (PCD). CFAP221, also known as PCDP1, is required for ciliary and flagellar function in mice and Chlamydomonas reinhardtii, where it localizes to the C1d projection of the central microtubule apparatus and functions in a complex that regulates flagellar motility in a calcium-dependent manner. We demonstrate that the genes encoding the mouse homologues of the other C. reinhardtii C1d complex members are primarily expressed in motile ciliated tissues, suggesting a conserved function in mammalian motile cilia. The requirement for one of these C1d complex members, CFAP54, was identified in a mouse line with a gene-trapped allele. Homozygous mice have PCD characterized by hydrocephalus, male infertility, and mucus accumulation. The infertility results from defects in spermatogenesis. Motile cilia have a structural defect in the C1d projection, indicating that the C1d assembly mechanism requires CFAP54. This structural defect results in decreased ciliary beat frequency and perturbed cilia-driven flow. This study identifies a critical role for CFAP54 in proper assembly and function of mammalian cilia and flagella and establishes the gene-trapped allele as a new model of PCD.
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Project description:Primary ciliary dyskinesia (PCD) is a genetically heterogeneous syndrome that results from defects in motile cilia. The ciliary axoneme has a 9?+?2 microtubule structure consisting of nine peripheral doublets surrounding a central pair apparatus (CPA), which plays a critical role in regulating proper ciliary function. We have previously shown that mouse models with mutations in CPA genes CFAP221, CFAP54, and SPEF2 have a PCD phenotype with defects in ciliary motility. In this study, we investigated potential genetic interaction between these CPA genes by generating each combination of double heterozygous and double homozygous mutants. No detectable cilia-related phenotypes were observed in double heterozygotes, but all three double homozygous mutant lines exhibit early mortality and typically develop severe PCD-associated phenotypes of hydrocephalus, mucociliary clearance defects in the upper airway, and abnormal spermatogenesis. Double homozygous cilia are generally intact and display a normal morphology and distribution. Spermiogenesis is aborted in double homozygotes, with an absence of mature flagella on elongating spermatids and epididymal sperm. These findings identify genetic interactions between CPA genes and genetic mechanisms regulating the CPA and motile cilia function.
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