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Genetics in Medicine : Official Journal... May 2023Primary ciliary dyskinesia (PCD) is a heterogeneous disorder that includes respiratory symptoms, laterality defects, and infertility caused by dysfunction of motile...
PURPOSE
Primary ciliary dyskinesia (PCD) is a heterogeneous disorder that includes respiratory symptoms, laterality defects, and infertility caused by dysfunction of motile cilia. Most PCD-causing variants result in abnormal outer dynein arms (ODAs), which provide the generative force for respiratory ciliary beating and proper mucociliary clearance.
METHODS
In addition to studies in mouse and planaria, clinical exome sequencing and functional analyses in human were performed.
RESULTS
In this study, we identified homozygous pathogenic variants in CLXN (EFCAB1/ODAD5) in 3 individuals with laterality defects and respiratory symptoms. Consistently, we found that Clxn is expressed in mice left-right organizer. Transmission electron microscopy depicted ODA defects in distal ciliary axonemes. Immunofluorescence microscopy revealed absence of CLXN from the ciliary axonemes, absence of the ODA components DNAH5, DNAI1, and DNAI2 from the distal axonemes, and mislocalization or absence of DNAH9. In addition, CLXN was undetectable in ciliary axonemes of individuals with defects in the ODA-docking machinery: ODAD1, ODAD2, ODAD3, and ODAD4. Furthermore, SMED-EFCAB1-deficient planaria displayed ciliary dysmotility.
CONCLUSION
Our results revealed that pathogenic variants in CLXN cause PCD with defects in the assembly of distal ODAs in the respiratory cilia. CLXN should be referred to as ODA-docking complex-associated protein ODAD5.
Topics: Humans; Animals; Mice; Cilia; Kartagener Syndrome; Calcium-Binding Proteins; Axoneme; Mutation; Axonemal Dyneins
PubMed: 36727596
DOI: 10.1016/j.gim.2023.100798 -
Experimental Animals Nov 2020Infertility is a global health issue that affects 1 in 6 couples, with male factors contributing to 50% of cases. The flagellar axoneme is a motility apparatus of... (Review)
Review
Infertility is a global health issue that affects 1 in 6 couples, with male factors contributing to 50% of cases. The flagellar axoneme is a motility apparatus of spermatozoa, and disruption of its structure or function could lead to male infertility. The axoneme consists of a "9+2" structure that contains a central pair of two singlet microtubules surrounded by nine doublet microtubules, in addition to several macromolecular complexes such as dynein arms, radial spokes, and nexin-dynein regulatory complexes. Molecular components of the flagellar axoneme are evolutionally conserved from unicellular flagellates to mammals, including mice. Although knockout (KO) mice have been generated to understand their function in the formation and motility regulation of sperm flagella, the majority of KO mice die before sexual maturation due to impaired ciliary motility, which makes it challenging to analyze mature spermatozoa. In this review, we introduce methods that have been used to overcome premature lethality, focusing on KO mouse lines of central pair components.
Topics: Animals; Axoneme; Dyneins; Infertility, Male; Male; Mice, Knockout; Microtubule-Associated Proteins; Microtubules; Sperm Motility; Sperm Tail
PubMed: 32554934
DOI: 10.1538/expanim.20-0064 -
Proceedings of the National Academy of... Jul 2017Flagella and cilia are critical cellular organelles that provide a means for cells to sense and progress through their environment. The central component of flagella and...
Flagella and cilia are critical cellular organelles that provide a means for cells to sense and progress through their environment. The central component of flagella and cilia is the axoneme, which comprises the "9+2" microtubule arrangement, dynein arms, radial spokes, and the nexin-dynein regulatory complex (-DRC). Failure to properly assemble components of the axoneme leads to defective flagella and in humans leads to a collection of diseases referred to as ciliopathies. Ciliopathies can manifest as severe syndromic diseases that affect lung and kidney function, central nervous system development, bone formation, visceral organ organization, and reproduction. T-Complex-Associated-Testis-Expressed 1 (TCTE1) is an evolutionarily conserved axonemal protein present from (DRC5) to mammals that localizes to the -DRC. Here, we show that mouse TCTE1 is testis-enriched in its expression, with its mRNA appearing in early round spermatids and protein localized to the flagellum. TCTE1 is 498 aa in length with a leucine rich repeat domain at the C terminus and is present in eukaryotes containing a flagellum. Knockout of results in male sterility because -null spermatozoa show aberrant motility. Although the axoneme is structurally normal in mutant spermatozoa, -null sperm demonstrate a significant decrease of ATP, which is used by dynein motors to generate the bending force of the flagellum. These data provide a link to defining the molecular intricacies required for axoneme function, sperm motility, and male fertility.
Topics: Adenosine Triphosphate; Animals; Axoneme; Chlamydomonas; Cilia; Crosses, Genetic; Cytoskeleton; Dyneins; Female; Flagella; Green Fluorescent Proteins; HEK293 Cells; Homozygote; Humans; Male; Mice; Microtubules; Mutation; Proteins; Sperm Motility; Spermatids; Spermatozoa; Testis
PubMed: 28630322
DOI: 10.1073/pnas.1621279114 -
Nature Communications Mar 2024Centrosomes and cilia are microtubule-based superstructures vital for cell division, signaling, and motility. The once thought hollow lumen of their microtubule core...
Centrosomes and cilia are microtubule-based superstructures vital for cell division, signaling, and motility. The once thought hollow lumen of their microtubule core structures was recently found to hold a rich meshwork of microtubule inner proteins (MIPs). To address the outstanding question of how distinct MIPs evolved to recognize microtubule inner surfaces, we applied computational sequence analyses, structure predictions, and experimental validation to uncover evolutionarily conserved microtubule- and MIP-binding modules named NWE, SNYG, and ELLEn, and PYG and GFG-repeat by their signature motifs. These modules intermix with MT-binding DM10-modules and Mn-repeats in 24 Chlamydomonas and 33 human proteins. The modules molecular characteristics provided keys to identify elusive cross-species homologs, hitherto unknown human MIP candidates, and functional properties for seven protein subfamilies, including the microtubule seam-binding NWE and ELLEn families. Our work defines structural innovations that underpin centriole and axoneme assembly and demonstrates that MIPs co-evolved with centrosomes and cilia.
Topics: Humans; Cilia; Microtubule Proteins; Axoneme; Microtubules; Centrioles
PubMed: 38538594
DOI: 10.1038/s41467-024-46737-3 -
Nature Communications Jun 2024Differentiation of male gametocytes into flagellated fertile male gametes relies on the assembly of axoneme, a major component of male development for mosquito...
Differentiation of male gametocytes into flagellated fertile male gametes relies on the assembly of axoneme, a major component of male development for mosquito transmission of the malaria parasite. RNA-binding protein (RBP)-mediated post-transcriptional regulation of mRNA plays important roles in eukaryotic sexual development, including the development of female Plasmodium. However, the role of RBP in defining the Plasmodium male transcriptome and its function in male gametogenesis remains incompletely understood. Here, we performed genome-wide screening for gender-specific RBPs and identified an undescribed male-specific RBP gene Rbpm1 in the Plasmodium. RBPm1 is localized in the nucleus of male gametocytes. RBPm1-deficient parasites fail to assemble the axoneme for male gametogenesis and thus mosquito transmission. RBPm1 interacts with the spliceosome E complex and regulates the splicing initiation of certain introns in a group of 26 axonemal genes. RBPm1 deficiency results in intron retention and protein loss of these axonemal genes. Intron deletion restores axonemal protein expression and partially rectifies axonemal defects in RBPm1-null gametocytes. Further splicing assays in both reporter and endogenous genes exhibit stringent recognition of the axonemal introns by RBPm1. The splicing activator RBPm1 and its target introns constitute an axonemal intron splicing program in the post-transcriptional regulation essential for Plasmodium male development.
Topics: Introns; RNA Splicing; RNA-Binding Proteins; Animals; Protozoan Proteins; Male; Axoneme; Female; Gametogenesis; Spliceosomes; Plasmodium berghei; Malaria; Plasmodium
PubMed: 38824128
DOI: 10.1038/s41467-024-49002-9 -
American Journal of Human Genetics Sep 2020Multiple morphological abnormalities of the sperm flagella (MMAF) is a severe form of asthenoteratozoospermia. Although recent studies have revealed several...
Multiple morphological abnormalities of the sperm flagella (MMAF) is a severe form of asthenoteratozoospermia. Although recent studies have revealed several MMAF-associated genes and demonstrated MMAF to be a genetically heterogeneous disease, at least one-third of the cases are still not well understood for their etiology. Here, we identified bi-allelic loss-of-function variants in CFAP58 by using whole-exome sequencing in five (5.6%) unrelated individuals from a cohort of 90 MMAF-affected Chinese men. Each of the men harboring bi-allelic CFAP58 variants presented typical MMAF phenotypes. Transmission electron microscopy demonstrated striking flagellar defects with axonemal and mitochondrial sheath malformations. CFAP58 is predominantly expressed in the testis and encodes a cilia- and flagella-associated protein. Immunofluorescence assays showed that CFAP58 localized at the entire flagella of control sperm and predominantly concentrated in the mid-piece. Immunoblotting and immunofluorescence assays showed that the abundances of axoneme ultrastructure markers SPAG6 and SPEF2 and a mitochondrial sheath protein, HSP60, were significantly reduced in the spermatozoa from men harboring bi-allelic CFAP58 variants. We generated Cfap58-knockout mice via CRISPR/Cas9 technology. The male mice were infertile and presented with severe flagellar defects, consistent with the sperm phenotypes in MMAF-affected men. Overall, our findings in humans and mice strongly suggest that CFAP58 plays a vital role in sperm flagellogenesis and demonstrate that bi-allelic loss-of-function variants in CFAP58 can cause axoneme and peri-axoneme malformations leading to male infertility. This study provides crucial insights for understanding and counseling of MMAF-associated asthenoteratozoospermia.
Topics: Abnormalities, Multiple; Alleles; Animals; Asthenozoospermia; Axoneme; CRISPR-Cas Systems; Cell Cycle Proteins; Homozygote; Humans; Infertility, Male; Intercellular Signaling Peptides and Proteins; Loss of Function Mutation; Loss of Heterozygosity; Male; Mice; Mice, Knockout; Microtubule Proteins; Mitochondria; Sperm Tail; Testis; Exome Sequencing
PubMed: 32791035
DOI: 10.1016/j.ajhg.2020.07.010 -
The FEBS Journal Sep 2017Intraflagellar transport (IFT) is a form of motor-dependent cargo transport that is essential for the assembly, maintenance, and length control of cilia, which play... (Review)
Review
Intraflagellar transport (IFT) is a form of motor-dependent cargo transport that is essential for the assembly, maintenance, and length control of cilia, which play critical roles in motility, sensory reception, and signal transduction in virtually all eukaryotic cells. During IFT, anterograde kinesin-2 and retrograde IFT dynein motors drive the bidirectional transport of IFT trains that deliver cargo, for example, axoneme precursors such as tubulins as well as molecules of the signal transduction machinery, to their site of assembly within the cilium. Following its discovery in Chlamydomonas, IFT has emerged as a powerful model system for studying general principles of motor-dependent cargo transport and we now appreciate the diversity that exists in the mechanism of IFT within cilia of different cell types. The absence of heterotrimeric kinesin-2 function, for example, causes a complete loss of both IFT and cilia in Chlamydomonas, but following its loss in Caenorhabditis elegans, where its primary function is loading the IFT machinery into cilia, homodimeric kinesin-2-driven IFT persists and assembles a full-length cilium. Generally, heterotrimeric kinesin-2 and IFT dynein motors are thought to play widespread roles as core IFT motors, whereas homodimeric kinesin-2 motors are accessory motors that mediate different functions in a broad range of cilia, in some cases contributing to axoneme assembly or the delivery of signaling molecules but in many other cases their ciliary functions, if any, remain unknown. In this review, we focus on mechanisms of motor action, motor cooperation, and motor-dependent cargo delivery during IFT.
Topics: Animals; Axoneme; Basal Bodies; Biological Transport; Caenorhabditis elegans; Chlamydomonas; Cilia; Dyneins; Flagella; Gene Expression Regulation; Kinesins; Protein Multimerization; Signal Transduction; Tubulin
PubMed: 28342295
DOI: 10.1111/febs.14068 -
Journal of Cell Science Feb 2010Cilia are specialized surface regions of eukaryotic cells that serve a variety of functions, ranging from motility to sensation and to regulation of cell growth and... (Review)
Review
Cilia are specialized surface regions of eukaryotic cells that serve a variety of functions, ranging from motility to sensation and to regulation of cell growth and differentiation. The discovery that a number of human diseases, collectively known as ciliopathies, result from defective cilium function has expanded interest in these structures. Among the many properties of cilia, motility and intraflagellar transport have been most extensively studied. The latter is the process by which multiprotein complexes associate with microtubule motors to transport structural subunits along the axoneme to and from the ciliary tip. By contrast, the mechanisms by which membrane proteins and lipids are specifically targeted to the cilium are still largely unknown. In this Commentary, we review the current knowledge of protein and lipid targeting to ciliary membranes and outline important issues for future study. We also integrate this information into a proposed model of how the cell specifically targets proteins and lipids to the specialized membrane of this unique organelle.
Topics: Animals; Axoneme; Cilia; Golgi Apparatus; Humans; Membrane Fusion; Membrane Lipids; Membrane Proteins; Models, Biological; Movement; Protein Transport
PubMed: 20145001
DOI: 10.1242/jcs.062968 -
PloS One 2016Mutations in the EYS gene are a common cause of autosomal recessive retinitis pigmentosa (arRP), yet the role of the EYS protein in humans is presently unclear. The aim...
PURPOSE
Mutations in the EYS gene are a common cause of autosomal recessive retinitis pigmentosa (arRP), yet the role of the EYS protein in humans is presently unclear. The aim of this study was to investigate the isoform structure, expression and potential function of EYS in the mammalian retina in order to better understand its involvement in the pathogenesis of arRP.
METHODS
To achieve the objective, we examined the expression of mRNA transcripts of EYS isoforms in human tissues and cell lines by RT-PCR. We also investigated the localisation of EYS in cultured cells and retinal cryo-sections by confocal fluorescence microscopy and Western blot analysis.
RESULTS
RT-PCR analysis confirmed that EYS has at least four isoforms. In addition to the previously reported EYS isoforms 1 and 4, we present the experimental validation of two smaller variants referred to as EYS isoforms 2 and 3. All four isoforms are expressed in the human retina and Y79 cells and the short variants were additionally detected in the testis. Immunofluorescent confocal microscopy and Western blot analysis revealed that all EYS isoforms preferentially localise to the cytoplasm of Y79 and HeLa cells. Moreover, an enrichment of the endogenous protein was observed near the centrosomes in Y79 cells. Interestingly, EYS was observed at the ciliary axoneme in Y79 ciliated cells. In macaque retinal cryosections, EYS was found to localise in the region of the photoreceptor ciliary axoneme in both rods and cones as well as in the cytoplasm of the ganglion cells.
CONCLUSION
The results obtained in this study lead us to speculate that, in photoreceptor cells, EYS could be a protein involved in maintaining the stability of the ciliary axoneme in both rods and cones. The variability of its isoform structure suggests that other roles are also possible and yet to be established.
Topics: Animals; Axoneme; Cilia; Cytoplasm; DNA Mutational Analysis; Electroretinography; Eye Proteins; HeLa Cells; Humans; Indoles; Macaca; Mutation; Protein Isoforms; Retina; Retinal Cone Photoreceptor Cells; Retinal Rod Photoreceptor Cells; Retinitis Pigmentosa
PubMed: 27846257
DOI: 10.1371/journal.pone.0166397 -
Current Opinion in Microbiology Aug 2010Motility of the sleeping sickness parasite, Trypanosoma brucei, impacts disease transmission and pathogenesis. Trypanosome motility is driven by a flagellum that harbors... (Review)
Review
Motility of the sleeping sickness parasite, Trypanosoma brucei, impacts disease transmission and pathogenesis. Trypanosome motility is driven by a flagellum that harbors a canonical 9+2 axoneme, together with trypanosome-specific elaborations. Trypanosome flagellum biology and motility have been the object of intense research over the last two years. These studies have led to the discovery of a novel form of motility, termed social motility, and provided revision of long-standing models for cell propulsion. Recent work has also uncovered novel structural features and motor proteins associated with the flagellar apparatus and has identified candidate signaling molecules that are predicted to regulate flagellar motility. Together with earlier inventories of flagellar proteins from proteomic and genomic studies, the stage is now set to move forward with functional studies to elucidate molecular mechanisms and investigate parasite motility in the context of host-parasite interactions.
Topics: Axoneme; Cell Movement; Flagella; Humans; Protozoan Proteins; Trypanosoma brucei brucei; Trypanosomiasis, African
PubMed: 20591724
DOI: 10.1016/j.mib.2010.05.015