Your browser doesn't support javascript.
loading
Mostrar: 20 | 50 | 100
Resultados 1 - 20 de 84
Filtrar
1.
PLoS Genet ; 16(12): e1009232, 2020 12.
Artigo em Inglês | MEDLINE | ID: mdl-33347437

RESUMO

Motile cilia can beat with distinct patterns, but how motility variations are regulated remain obscure. Here, we have studied the role of the coiled-coil protein CFAP53 in the motility of different cilia-types in the mouse. While node (9+0) cilia of Cfap53 mutants were immotile, tracheal and ependymal (9+2) cilia retained motility, albeit with an altered beat pattern. In node cilia, CFAP53 mainly localized at the base (centriolar satellites), whereas it was also present along the entire axoneme in tracheal cilia. CFAP53 associated tightly with microtubules and interacted with axonemal dyneins and TTC25, a dynein docking complex component. TTC25 and outer dynein arms (ODAs) were lost from node cilia, but were largely maintained in tracheal cilia of Cfap53-/- mice. Thus, CFAP53 at the base of node cilia facilitates axonemal transport of TTC25 and dyneins, while axonemal CFAP53 in 9+2 cilia stabilizes dynein binding to microtubules. Our study establishes how differential localization and function of CFAP53 contributes to the unique motion patterns of two important mammalian cilia-types.


Assuntos
Dineínas do Axonema/metabolismo , Axonema/metabolismo , Transporte Biológico Ativo/genética , Movimento Celular/genética , Cílios/metabolismo , Embrião de Mamíferos/metabolismo , Microtúbulos/metabolismo , Animais , Dineínas do Axonema/genética , Axonema/genética , Proteínas de Transporte/genética , Proteínas de Transporte/metabolismo , Cílios/genética , Embrião de Mamíferos/fisiologia , Embrião de Mamíferos/ultraestrutura , Epêndima/embriologia , Epêndima/metabolismo , Epêndima/fisiologia , Imunofluorescência , Genótipo , Imunoprecipitação , Camundongos , Camundongos Knockout , Microscopia Eletrônica de Transmissão , Microtúbulos/genética , Mutação , Fenótipo , Traqueia/embriologia , Traqueia/metabolismo , Traqueia/fisiologia , Traqueia/ultraestrutura
2.
PLoS Genet ; 16(8): e1008954, 2020 08.
Artigo em Inglês | MEDLINE | ID: mdl-32785227

RESUMO

The flagellum is essential for sperm motility and fertilization in vivo. The axoneme is the main component of the flagella, extending through its entire length. An axoneme is comprised of two central microtubules surrounded by nine doublets, the nexin-dynein regulatory complex, radial spokes, and dynein arms. Failure to properly assemble components of the axoneme in a sperm flagellum, leads to fertility alterations. To understand this process in detail, we have defined the function of an uncharacterized gene, Cfap97 domain containing 1 (Cfap97d1). This gene is evolutionarily conserved in mammals and multiple other species, including Chlamydomonas. We have used two independently generated Cfap97d1 knockout mouse models to study the gene function in vivo. Cfap97d1 is exclusively expressed in testes starting from post-natal day 20 and continuing throughout adulthood. Deletion of the Cfap97d1 gene in both mouse models leads to sperm motility defects (asthenozoospermia) and male subfertility. In vitro fertilization (IVF) of cumulus-intact oocytes with Cfap97d1 deficient sperm yielded few embryos whereas IVF with zona pellucida-free oocytes resulted in embryo numbers comparable to that of the control. Knockout spermatozoa showed abnormal motility characterized by frequent stalling in the anti-hook position. Uniquely, Cfap97d1 loss caused a phenotype associated with axonemal doublet heterogeneity linked with frequent loss of the fourth doublet in the sperm stored in the epididymis. This study demonstrates that Cfap97d1 is required for sperm flagellum ultra-structure maintenance, thereby playing a critical role in sperm function and male fertility in mice.


Assuntos
Axonema/genética , Proteínas do Citoesqueleto/genética , Dineínas/genética , Infertilidade Masculina/genética , Animais , Chlamydomonas/genética , Cílios/genética , Cílios/patologia , Fertilização In Vitro , Humanos , Infertilidade Masculina/patologia , Masculino , Camundongos , Camundongos Knockout , Motilidade Espermática/genética , Cauda do Espermatozoide/metabolismo , Cauda do Espermatozoide/patologia , Espermatozoides/crescimento & desenvolvimento , Espermatozoides/patologia , Testículo/crescimento & desenvolvimento , Testículo/patologia
3.
Am J Hum Genet ; 107(3): 514-526, 2020 09 03.
Artigo em Inglês | MEDLINE | ID: mdl-32791035

RESUMO

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.


Assuntos
Anormalidades Múltiplas/genética , Astenozoospermia/genética , Axonema/genética , Infertilidade Masculina/genética , Peptídeos e Proteínas de Sinalização Intercelular/genética , Anormalidades Múltiplas/patologia , Alelos , Animais , Astenozoospermia/fisiopatologia , Axonema/patologia , Sistemas CRISPR-Cas/genética , Proteínas de Ciclo Celular/genética , Homozigoto , Humanos , Infertilidade Masculina/patologia , Mutação com Perda de Função/genética , Perda de Heterozigosidade/genética , Masculino , Camundongos , Camundongos Knockout , Proteínas dos Microtúbulos/genética , Mitocôndrias/genética , Cauda do Espermatozoide/metabolismo , Cauda do Espermatozoide/patologia , Testículo/metabolismo , Testículo/patologia , Sequenciamento Completo do Exoma
4.
PLoS Genet ; 16(3): e1008664, 2020 03.
Artigo em Inglês | MEDLINE | ID: mdl-32203505

RESUMO

Motile cilia/flagella are essential for swimming and generating extracellular fluid flow in eukaryotes. Motile cilia harbor a 9+2 arrangement consisting of nine doublet microtubules with dynein arms at the periphery and a pair of singlet microtubules at the center (central pair). In the central system, the radial spoke has a T-shaped architecture and regulates the motility and motion pattern of cilia. Recent cryoelectron tomography data reveal three types of radial spokes (RS1, RS2, and RS3) in the 96 nm axoneme repeat unit; however, the molecular composition of the third radial spoke, RS3 is unknown. In human pathology, it is well known mutation of the radial spoke head-related genes causes primary ciliary dyskinesia (PCD) including respiratory defect and infertility. Here, we describe the role of the primary ciliary dyskinesia protein Rsph4a in the mouse motile cilia. Cryoelectron tomography reveals that the mouse trachea cilia harbor three types of radial spoke as with the other vertebrates and that all triplet spoke heads are lacking in the trachea cilia of Rsph4a-deficient mice. Furthermore, observation of ciliary movement and immunofluorescence analysis indicates that Rsph4a contributes to the generation of the planar beating of motile cilia by building the distal architecture of radial spokes in the trachea, the ependymal tissues, and the oviduct. Although detailed mechanism of RSs assembly remains unknown, our results suggest Rsph4a is a generic component of radial spoke heads, and could explain the severe phenotype of human PCD patients with RSPH4A mutation.


Assuntos
Cílios/metabolismo , Proteínas do Citoesqueleto/metabolismo , Proteínas do Tecido Nervoso/metabolismo , Animais , Axonema/genética , Axonema/metabolismo , Cílios/genética , Transtornos da Motilidade Ciliar/genética , Transtornos da Motilidade Ciliar/metabolismo , Proteínas do Citoesqueleto/genética , Dineínas/metabolismo , Feminino , Flagelos/genética , Flagelos/metabolismo , Humanos , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Microtúbulos/metabolismo , Mutação , Proteínas do Tecido Nervoso/genética
5.
PLoS Genet ; 16(1): e1008585, 2020 01.
Artigo em Inglês | MEDLINE | ID: mdl-31961863

RESUMO

Flagella and cilia are evolutionarily conserved cellular organelles. Abnormal formation or motility of these organelles in humans causes several syndromic diseases termed ciliopathies. The central component of flagella and cilia is the axoneme that is composed of the '9+2' microtubule arrangement, dynein arms, radial spokes, and the Nexin-Dynein Regulatory Complex (N-DRC). The N-DRC is localized between doublet microtubules and has been extensively studied in the unicellular flagellate Chlamydomonas. Recently, it has been reported that TCTE1 (DRC5), a component of the N-DRC, is essential for proper sperm motility and male fertility in mice. Further, TCTE1 has been shown to interact with FBXL13 (DRC6) and DRC7; however, functional roles of FBXL13 and DRC7 in mammals have not been elucidated. Here we show that Fbxl13 and Drc7 expression are testes-enriched in mice. Although Fbxl13 knockout (KO) mice did not show any obvious phenotypes, Drc7 KO male mice were infertile due to their short immotile spermatozoa. In Drc7 KO spermatids, the axoneme is disorganized and the '9+2' microtubule arrangement was difficult to detect. Further, other N-DRC components fail to incorporate into the flagellum without DRC7. These results indicate that Drc7, but not Fbxl13, is essential for the correct assembly of the N-DRC and flagella.


Assuntos
Dineínas/metabolismo , Flagelos/genética , Infertilidade Masculina/genética , Proteínas Associadas aos Microtúbulos/metabolismo , Espermatozoides/metabolismo , Animais , Axonema/genética , Axonema/metabolismo , Axonema/patologia , Feminino , Flagelos/metabolismo , Flagelos/patologia , Células HEK293 , Humanos , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Espermatogênese , Espermatozoides/citologia , Espermatozoides/patologia
6.
J Biol Chem ; 295(3): 729-742, 2020 01 17.
Artigo em Inglês | MEDLINE | ID: mdl-31819011

RESUMO

The basal body in the human parasite Trypanosoma brucei is structurally equivalent to the centriole in animals and functions in the nucleation of axonemal microtubules in the flagellum. T. brucei lacks many evolutionarily conserved centriolar protein homologs and constructs the basal body through unknown mechanisms. Two evolutionarily conserved centriole/basal body cartwheel proteins, TbSAS-6 and TbBLD10, and a trypanosome-specific protein, BBP65, play essential roles in basal body biogenesis in T. brucei, but how they cooperate in the regulation of basal body assembly remains elusive. Here using RNAi, endogenous epitope tagging, immunofluorescence microscopy, and 3D-structured illumination super-resolution microscopy, we identified a new trypanosome-specific protein named BBP164 and found that it has an essential role in basal body biogenesis in T. brucei Further investigation of the functional interplay among BBP164 and the other three regulators of basal body assembly revealed that BBP164 and BBP65 are interdependent for maintaining their stability and depend on TbSAS-6 and TbBLD10 for their stabilization in the basal body. Additionally, TbSAS-6 and TbBLD10 are independent from each other and from BBP164 and BBP65 for maintaining their stability in the basal body. These findings demonstrate that basal body cartwheel proteins are required for stabilizing other basal body components and uncover that regulation of protein stability is an unusual control mechanism for assembly of the basal body in T. brucei.


Assuntos
Corpos Basais/metabolismo , Microtúbulos/metabolismo , Proteínas de Protozoários/genética , Trypanosoma brucei brucei/genética , Animais , Axonema/química , Axonema/genética , Axonema/metabolismo , Corpos Basais/química , Corpos Basais/parasitologia , Centríolos/química , Centríolos/genética , Centríolos/parasitologia , Flagelos/química , Flagelos/genética , Flagelos/parasitologia , Humanos , Microtúbulos/química , Microtúbulos/parasitologia , Estabilidade Proteica , Proteínas de Protozoários/química , Interferência de RNA , Trypanosoma brucei brucei/química , Trypanosoma brucei brucei/patogenicidade
7.
Elife ; 82019 12 19.
Artigo em Inglês | MEDLINE | ID: mdl-31855176

RESUMO

With eight flagella of four different lengths, the parasitic protist Giardia is an ideal model to evaluate flagellar assembly and length regulation. To determine how four different flagellar lengths are maintained, we used live-cell quantitative imaging and mathematical modeling of conserved components of intraflagellar transport (IFT)-mediated assembly and kinesin-13-mediated disassembly in different flagellar pairs. Each axoneme has a long cytoplasmic region extending from the basal body, and transitions to a canonical membrane-bound flagellum at the 'flagellar pore'. We determined that each flagellar pore is the site of IFT accumulation and injection, defining a diffusion barrier functionally analogous to the transition zone. IFT-mediated assembly is length-independent, as train size, speed, and injection frequencies are similar for all flagella. We demonstrate that kinesin-13 localization to the flagellar tips is inversely correlated to flagellar length. Therefore, we propose a model where a length-dependent disassembly mechanism controls multiple flagellar lengths within the same cell.


Assuntos
Flagelos/fisiologia , Giardia/genética , Giardia/metabolismo , Cinesina/genética , Axonema/genética , Axonema/metabolismo , Chlamydomonas reinhardtii , Cílios/genética , Citoplasma/genética , Citoplasma/metabolismo , Difusão , Flagelos/genética , Giardia/crescimento & desenvolvimento , Cinesina/metabolismo , Modelos Teóricos , Transporte Proteico/genética
8.
Cell ; 179(4): 909-922.e12, 2019 10 31.
Artigo em Inglês | MEDLINE | ID: mdl-31668805

RESUMO

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.


Assuntos
Axonema/ultraestrutura , Cílios/ultraestrutura , Ciliopatias/patologia , Microtúbulos/ultraestrutura , Axonema/química , Axonema/genética , Movimento Celular/genética , Cílios/química , Cílios/genética , Ciliopatias/genética , Ciliopatias/metabolismo , Microscopia Crioeletrônica , Humanos , Proteínas dos Microtúbulos/química , Proteínas dos Microtúbulos/ultraestrutura , Microtúbulos/química , Microtúbulos/genética , Estresse Mecânico
9.
Am J Hum Genet ; 105(6): 1148-1167, 2019 12 05.
Artigo em Inglês | MEDLINE | ID: mdl-31735292

RESUMO

In humans, structural or functional defects of the sperm flagellum induce asthenozoospermia, which accounts for the main sperm defect encountered in infertile men. Herein we focused on morphological abnormalities of the sperm flagellum (MMAF), a phenotype also termed "short tails," which constitutes one of the most severe sperm morphological defects resulting in asthenozoospermia. In previous work based on whole-exome sequencing of a cohort of 167 MMAF-affected individuals, we identified bi-allelic loss-of-function mutations in more than 30% of the tested subjects. In this study, we further analyzed this cohort and identified five individuals with homozygous truncating variants in TTC29, a gene preferentially and highly expressed in the testis, and encoding a tetratricopeptide repeat-containing protein related to the intraflagellar transport (IFT). One individual carried a frameshift variant, another one carried a homozygous stop-gain variant, and three carried the same splicing variant affecting a consensus donor site. The deleterious effect of this last variant was confirmed on the corresponding transcript and protein product. In addition, we produced and analyzed TTC29 loss-of-function models in the flagellated protist T. brucei and in M. musculus. Both models confirmed the importance of TTC29 for flagellar beating. We showed that in T. brucei the TPR structural motifs, highly conserved between the studied orthologs, are critical for TTC29 axonemal localization and flagellar beating. Overall our work demonstrates that TTC29 is a conserved axonemal protein required for flagellar structure and beating and that TTC29 mutations are a cause of male sterility due to MMAF.


Assuntos
Astenozoospermia/etiologia , Axonema/patologia , Flagelos/patologia , Infertilidade Masculina/etiologia , Proteínas Associadas aos Microtúbulos/genética , Mutação , Animais , Astenozoospermia/metabolismo , Astenozoospermia/patologia , Axonema/genética , Axonema/metabolismo , Evolução Molecular , Feminino , Fertilização In Vitro , Flagelos/genética , Flagelos/metabolismo , Humanos , Infertilidade Masculina/metabolismo , Infertilidade Masculina/patologia , Masculino , Camundongos Endogâmicos C57BL , Trypanosoma brucei brucei/fisiologia , Tripanossomíase
10.
Mol Biol Cell ; 30(21): 2659-2680, 2019 10 01.
Artigo em Inglês | MEDLINE | ID: mdl-31483737

RESUMO

Ciliary motility depends on both the precise spatial organization of multiple dynein motors within the 96 nm axonemal repeat and the highly coordinated interactions between different dyneins and regulatory complexes located at the base of the radial spokes. Mutations in genes encoding cytoplasmic assembly factors, intraflagellar transport factors, docking proteins, dynein subunits, and associated regulatory proteins can all lead to defects in dynein assembly and ciliary motility. Significant progress has been made in the identification of dynein subunits and extrinsic factors required for preassembly of dynein complexes in the cytoplasm, but less is known about the docking factors that specify the unique binding sites for the different dynein isoforms on the surface of the doublet microtubules. We have used insertional mutagenesis to identify a new locus, IDA8/BOP2, required for targeting the assembly of a subset of inner dynein arms (IDAs) to a specific location in the 96 nm repeat. IDA8 encodes flagellar-associated polypeptide (FAP)57/WDR65, a highly conserved WD repeat, coiled coil domain protein. Using high resolution proteomic and structural approaches, we find that FAP57 forms a discrete complex. Cryo-electron tomography coupled with epitope tagging and gold labeling reveal that FAP57 forms an extended structure that interconnects multiple IDAs and regulatory complexes.


Assuntos
Proteínas de Algas/metabolismo , Axonema/metabolismo , Cílios/metabolismo , Dineínas/metabolismo , Flagelos/metabolismo , Proteômica/métodos , Proteínas de Algas/genética , Sequência de Aminoácidos , Axonema/genética , Chlamydomonas reinhardtii/genética , Chlamydomonas reinhardtii/metabolismo , Cílios/genética , Cílios/ultraestrutura , Microscopia Crioeletrônica/métodos , Dineínas/genética , Tomografia com Microscopia Eletrônica , Flagelos/genética , Flagelos/ultraestrutura , Microscopia de Fluorescência/métodos , Microtúbulos/metabolismo , Microtúbulos/ultraestrutura , Mutação , Subunidades Proteicas/genética , Subunidades Proteicas/metabolismo , Gravação de Videoteipe/métodos
11.
J Med Genet ; 56(11): 750-757, 2019 11.
Artigo em Inglês | MEDLINE | ID: mdl-31413122

RESUMO

BACKGROUND: The genetic causes for most male infertility due to severe asthenozoospermia remain unclear. OBJECTIVE: Our objective was to identify unknown genetic factors in 47 patients with severe asthenozoospermia from 45 unrelated Chinese families. METHODS: We performed whole exome sequencing of 47 individuals with severe asthenozoospermia from 45 unrelated families. Mutation screening was performed in a control cohort of 637 individuals, including 219 with oligoasthenospermia, 195 with non-obstructive azoospermia and 223 fertile controls. Ultrastructural and immunostaining analyses of patients' spermatozoa were performed to characterise the effect of variants. RESULTS: One homozygous non-sense mutation (NM_194302, c.G5341T:p.E1781X), two compound heterozygous mutations (c.C2284T:p.R762X and c.1751delC:p.P584fs) and two compound heterozygous mutations (c.5714_5721del:p.L1905fs and c.C3021A:p.N1007K) were identified in CFAP65 of three individuals with completely immotile spermatozoa, respectively. No biallelic deleterious variants of CFAP65 were detected in the control cohort of 637 individuals. Ultrastructural and immunostaining analyses of spermatozoa from two patients showed highly aberrant sperm morphology with severe defects such as acrosome hypoplasia, disruption of the mitochondrial sheath and absence of the central pair complex. CONCLUSION: To the best of our knowledge, we are the first to report that CFAP65 mutations may cause spermatozoa to be completely immotile.


Assuntos
Acrossomo/patologia , Astenozoospermia/genética , Proteínas do Citoesqueleto/genética , Flagelos/genética , Infertilidade Masculina/genética , Mutação/genética , Adulto , Alelos , Axonema/genética , Exoma/genética , Homozigoto , Humanos , Masculino , Cauda do Espermatozoide/patologia , Espermatozoides/patologia , Sequenciamento Completo do Exoma/métodos
12.
J Assist Reprod Genet ; 36(8): 1683-1700, 2019 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-31273583

RESUMO

PROPOSE: To study CCDC103 expression profiles and understand how pathogenic variants in CCDC103 affect its expression profile at mRNA and protein level. METHODS: To increase the knowledge about the CCDC103, we attempted genotype-phenotype correlations in two patients carrying novel homozygous (missense and frameshift) CCDC103 variants. Whole-exome sequencing, quantitative PCR, Western blot, electron microscopy, immunohistochemistry, immunocytochemistry, and immunogold labelling were performed to characterize CCDC103 expression profiles in reproductive and somatic cells. RESULTS: Our data demonstrate that pathogenic variants in CCDC103 gene negatively affect gene and protein expression in both patients who presented absence of DA on their axonemes. Further, we firstly report that CCDC103 is expressed at different levels in reproductive tissues and somatic cells and described that CCDC103 protein forms oligomers with tissue-specific sizes, which suggests that CCDC103 possibly undergoes post-translational modifications. Moreover, we reported that CCDC103 was restricted to the midpiece of sperm and is present at the cytoplasm of the other cells. CONCLUSIONS: Overall, our data support the CCDC103 involvement in PCD and suggest that CCDC103 may have different assemblies and roles in cilia and sperm flagella biology that are still unexplored.


Assuntos
Axonema/patologia , Transtornos da Motilidade Ciliar/genética , Infertilidade Masculina/patologia , Síndrome de Kartagener/genética , Proteínas Associadas aos Microtúbulos/genética , Mutação , Cauda do Espermatozoide/patologia , Axonema/genética , Transtornos da Motilidade Ciliar/patologia , Dineínas/metabolismo , Feminino , Humanos , Infertilidade Masculina/etiologia , Síndrome de Kartagener/patologia , Masculino , Pessoa de Meia-Idade , Reprodução , Situs Inversus/genética , Situs Inversus/patologia , Cauda do Espermatozoide/metabolismo
13.
Clin Genet ; 96(5): 385-393, 2019 11.
Artigo em Inglês | MEDLINE | ID: mdl-31278745

RESUMO

Male patients with multiple morphological abnormalities of the sperm flagella (MMAF) are infertile and exhibit absent, short, coiled, bent and/or irregular sperm flagella. Mutations in the SPEF2 gene reduce sperm motility and cause sperm tail defects in animal models and humans. In the present study, we performed a genetic analysis on an MMAF patient and identified novel biallelic mutations in the SPEF2 gene. The biallelic mutations were confirmed by Sanger sequencing and in silico analysis revealed that, these variations were deleterious. The expression of truncated SPEF2 protein was reduced significantly in the patient's spermatozoa. The spermatozoa harbored biallelic mutations and showed severe ultrastructural defects in the axoneme and mitochondrial sheath. Our data suggest that biallelic mutations in SPEF2 can cause severe sperm flagellum defects, thus providing a novel candidate genetic pathogen for the human MMAF phenotype.


Assuntos
Anormalidades Múltiplas/genética , Proteínas de Ciclo Celular/genética , Infertilidade Masculina/genética , Cauda do Espermatozoide/ultraestrutura , Anormalidades Múltiplas/patologia , Alelos , Axonema/genética , Axonema/ultraestrutura , Humanos , Infertilidade Masculina/patologia , Masculino , Mutação/genética , Linhagem , Fenótipo , Motilidade Espermática/genética , Cauda do Espermatozoide/metabolismo , Cauda do Espermatozoide/patologia , Espermatozoides/crescimento & desenvolvimento , Espermatozoides/patologia , Sequenciamento Completo do Exoma
14.
Am J Respir Cell Mol Biol ; 61(3): 312-321, 2019 09.
Artigo em Inglês | MEDLINE | ID: mdl-30896965

RESUMO

Primary ciliary dyskinesia (PCD) is a genetically and phenotypically heterogeneous disease caused by mutations in over 40 different genes. Individuals with PCD caused by mutations in RSPH1 (radial spoke head 1 homolog) have been reported to have a milder phenotype than other individuals with PCD, as evidenced by a lower incidence of neonatal respiratory distress, higher nasal nitric oxide concentrations, and better lung function. To better understand genotype-phenotype relationships in PCD, we have characterized a mutant mouse model with a deletion of Rsph1. Approximately 50% of cilia from Rsph1-/- cells appeared normal by transmission EM, whereas the remaining cilia revealed a range of defects, primarily transpositions or a missing central pair. Ciliary beat frequency in Rsph1-/- cells was significantly lower than in control cells (20.2 ± 0.8 vs. 25.0 ± 0.9 Hz), and the cilia exhibited an aberrant rotational waveform. Young Rsph1-/- animals demonstrated a low rate of mucociliary clearance in the nasopharynx that was reduced to zero by about 1 month of age. Rsph1-/- animals accumulated mucus in the nasal cavity but had a lower bacterial burden than animals with a deletion of dynein axonemal intermediate chain 1 (Dnaic1-/-). Thus, Rsph1-/- mice display a PCD phenotype similar to but less severe than that observed in Dnaic1-/- mice, similar to what has been observed in humans. The results suggest that some individuals with PCD may not have a complete loss of mucociliary clearance and further suggest that early diagnosis and intervention may be important to maintain this low amount of clearance.


Assuntos
Proteínas de Ligação a DNA/genética , Síndrome de Kartagener/genética , Depuração Mucociliar/genética , Fenótipo , Animais , Axonema/genética , Cílios/genética , Humanos , Camundongos , Mutação/genética , Deleção de Sequência/genética
15.
Nat Commun ; 10(1): 1143, 2019 03 08.
Artigo em Inglês | MEDLINE | ID: mdl-30850601

RESUMO

Motile cilia are microtubule-based organelles that play important roles in most eukaryotes. Although axonemal microtubules are sufficiently stable to withstand their beating motion, it remains unknown how they are stabilized while serving as tracks for axonemal dyneins. To address this question, we have identified two uncharacterized proteins, FAP45 and FAP52, as microtubule inner proteins (MIPs) in Chlamydomonas. These proteins are conserved among eukaryotes with motile cilia. Using cryo-electron tomography (cryo-ET) and high-speed atomic force microscopy (HS-AFM), we show that lack of these proteins leads to a loss of inner protrusions in B-tubules and less stable microtubules. These protrusions are located near the inner junctions of doublet microtubules and lack of both FAP52 and a known inner junction protein FAP20 results in detachment of the B-tubule from the A-tubule, as well as flagellar shortening. These results demonstrate that FAP45 and FAP52 bind to the inside of microtubules and stabilize ciliary axonemes.


Assuntos
Proteínas de Algas/química , Axonema/metabolismo , Chlamydomonas reinhardtii/metabolismo , Cílios/metabolismo , Flagelos/metabolismo , Proteínas de Algas/genética , Proteínas de Algas/metabolismo , Dineínas do Axonema/química , Dineínas do Axonema/genética , Dineínas do Axonema/metabolismo , Axonema/genética , Axonema/ultraestrutura , Chlamydomonas reinhardtii/genética , Chlamydomonas reinhardtii/ultraestrutura , Cílios/genética , Cílios/ultraestrutura , Microscopia Crioeletrônica , Tomografia com Microscopia Eletrônica , Flagelos/genética , Flagelos/ultraestrutura , Expressão Gênica , Microscopia de Força Atômica
16.
Nat Commun ; 9(1): 5277, 2018 12 11.
Artigo em Inglês | MEDLINE | ID: mdl-30538248

RESUMO

Defective ciliogenesis causes human developmental diseases termed ciliopathies. Microtubule (MT) asters originating from centrosomes in mitosis ensure the fidelity of cell division by positioning the spindle apparatus. However, the function of microtubule asters in interphase remains largely unknown. Here, we reveal an essential role of MT asters in transition zone (TZ) assembly during ciliogenesis. We demonstrate that the centrosome protein FSD1, whose biological function is largely unknown, anchors MT asters to interphase centrosomes by binding to microtubules. FSD1 knockdown causes defective ciliogenesis and affects embryonic development in vertebrates. We further show that disruption of MT aster anchorage by depleting FSD1 or other known anchoring proteins delocalizes the TZ assembly factor Cep290 from centriolar satellites, and causes TZ assembly defects. Thus, our study establishes FSD1 as a MT aster anchorage protein and reveals an important function of MT asters anchored by FSD1 in TZ assembly during ciliogenesis.


Assuntos
Axonema/metabolismo , Cílios/metabolismo , Microtúbulos/metabolismo , Proteínas do Tecido Nervoso/metabolismo , Animais , Axonema/genética , Centrossomo/metabolismo , Cílios/genética , Humanos , Mitose , Proteínas do Tecido Nervoso/genética , Fuso Acromático/genética , Fuso Acromático/metabolismo , Peixe-Zebra/embriologia , Peixe-Zebra/genética , Peixe-Zebra/metabolismo
17.
Am J Hum Genet ; 103(6): 995-1008, 2018 12 06.
Artigo em Inglês | MEDLINE | ID: mdl-30471718

RESUMO

Dysfunction of motile monocilia, altering the leftward flow at the embryonic node essential for determination of left-right body asymmetry, is a major cause of laterality defects. Laterality defects are also often associated with reduced mucociliary clearance caused by defective multiple motile cilia of the airway and are responsible for destructive airway disease. Outer dynein arms (ODAs) are essential for ciliary beat generation, and human respiratory cilia contain different ODA heavy chains (HCs): the panaxonemally distributed γ-HC DNAH5, proximally located ß-HC DNAH11 (defining ODA type 1), and the distally localized ß-HC DNAH9 (defining ODA type 2). Here we report loss-of-function mutations in DNAH9 in five independent families causing situs abnormalities associated with subtle respiratory ciliary dysfunction. Consistent with the observed subtle respiratory phenotype, high-speed video microscopy demonstrates distally impaired ciliary bending in DNAH9 mutant respiratory cilia. DNAH9-deficient cilia also lack other ODA components such as DNAH5, DNAI1, and DNAI2 from the distal axonemal compartment, demonstrating an essential role of DNAH9 for distal axonemal assembly of ODAs type 2. Yeast two-hybrid and co-immunoprecipitation analyses indicate interaction of DNAH9 with the ODA components DNAH5 and DNAI2 as well as the ODA-docking complex component CCDC114. We further show that during ciliogenesis of respiratory cilia, first proximally located DNAH11 and then distally located DNAH9 is assembled in the axoneme. We propose that the ß-HC paralogs DNAH9 and DNAH11 achieved specific functional roles for the distinct axonemal compartments during evolution with human DNAH9 function matching that of ancient ß-HCs such as that of the unicellular Chlamydomonas reinhardtii.


Assuntos
Dineínas do Axonema/genética , Cílios/genética , Dineínas/genética , Mutação/genética , Axonema/genética , Transtornos da Motilidade Ciliar/genética , Humanos , Síndrome de Kartagener/genética , Fenótipo
18.
Open Biol ; 8(11)2018 11 21.
Artigo em Inglês | MEDLINE | ID: mdl-30463910

RESUMO

Flagella have multiple functions that are associated with different axonemal structures. Motile flagella typically have a 9 + 2 arrangement of microtubules, whereas sensory flagella normally have a 9 + 0 arrangement. Leishmania exhibits both of these flagellum forms and differentiation between these two flagellum forms is associated with cytoskeletal and cell shape changes. We disrupted flagellum elongation in Leishmania by deleting the intraflagellar transport (IFT) protein IFT140 and examined the effects on cell morphogenesis. Δift140 cells have no external flagellum, having only a very short flagellum within the flagellar pocket. This short flagellum had a collapsed 9 + 0 (9v) axoneme configuration reminiscent of that in the amastigote and was not attached to the pocket membrane. Although amastigote-like changes occurred in the flagellar cytoskeleton, the cytoskeletal structures of Δift140 cells retained their promastigote configurations, as examined by fluorescence microscopy of tagged proteins and serial electron tomography. Thus, Leishmania promastigote cell morphogenesis does not depend on the formation of a long flagellum attached at the neck. Furthermore, our data show that disruption of the IFT system is sufficient to produce a switch from the 9 + 2 to the collapsed 9 + 0 (9v) axonemal structure, echoing the process that occurs during the promastigote to amastigote differentiation.


Assuntos
Axonema/metabolismo , Proteínas de Transporte/metabolismo , Flagelos/metabolismo , Leishmania mexicana/metabolismo , Proteínas de Protozoários/metabolismo , Axonema/genética , Proteínas de Transporte/genética , Flagelos/genética , Leishmania mexicana/citologia , Leishmania mexicana/genética , Proteínas de Protozoários/genética
19.
Am J Hum Genet ; 103(5): 727-739, 2018 11 01.
Artigo em Inglês | MEDLINE | ID: mdl-30388400

RESUMO

Primary defects in motile cilia result in dysfunction of the apparatus responsible for generating fluid flows. Defects in these mechanisms underlie disorders characterized by poor mucus clearance, resulting in susceptibility to chronic recurrent respiratory infections, often associated with infertility; laterality defects occur in about 50% of such individuals. Here we report biallelic variants in LRRC56 (known as oda8 in Chlamydomonas) identified in three unrelated families. The phenotype comprises laterality defects and chronic pulmonary infections. High-speed video microscopy of cultured epithelial cells from an affected individual showed severely dyskinetic cilia but no obvious ultra-structural abnormalities on routine transmission electron microscopy (TEM). Further investigation revealed that LRRC56 interacts with the intraflagellar transport (IFT) protein IFT88. The link with IFT was interrogated in Trypanosoma brucei. In this protist, LRRC56 is recruited to the cilium during axoneme construction, where it co-localizes with IFT trains and is required for the addition of dynein arms to the distal end of the flagellum. In T. brucei carrying LRRC56-null mutations, or a variant resulting in the p.Leu259Pro substitution corresponding to the p.Leu140Pro variant seen in one of the affected families, we observed abnormal ciliary beat patterns and an absence of outer dynein arms restricted to the distal portion of the axoneme. Together, our findings confirm that deleterious variants in LRRC56 result in a human disease and suggest that this protein has a likely role in dynein transport during cilia assembly that is evolutionarily important for cilia motility.


Assuntos
Transporte Biológico/genética , Flagelos/genética , Depuração Mucociliar/genética , Mutação/genética , Proteínas/genética , Adulto , Alelos , Axonema/genética , Linhagem Celular , Chlamydomonas/genética , Cílios/genética , Dineínas/genética , Células Epiteliais/patologia , Feminino , Células HEK293 , Humanos , Lactente , Masculino , Fenótipo , Trypanosoma brucei brucei/genética
20.
Am J Hum Genet ; 103(3): 413-420, 2018 09 06.
Artigo em Inglês | MEDLINE | ID: mdl-30122541

RESUMO

Flagella and motile cilia share a 9 + 2 microtubule-doublet axoneme structure, and asthenozoospermia (reduced spermatozoa motility) is found in 76% of men with primary ciliary dyskinesia (PCD). Nevertheless, causal genetic variants in a conserved axonemal component have been found in cases of isolated asthenozoospermia: 30% of men with multiple morphological anomalies of sperm flagella (MMAF) carry bi-allelic mutations in DNAH1, encoding one of the seven inner-arm dynein heavy chains of the 9 + 2 axoneme. To further understand the basis for isolated asthenozoospermia, we used whole-exome and Sanger sequencing to study two brothers and two independent men with MMAF. In three men, we found bi-allelic loss-of-function mutations in WDR66, encoding cilia- and flagella-associated protein 251 (CFAP251): the two brothers were homozygous for the frameshift chr12: g.122359334delA (p.Asp42Metfs∗4), and the third individual was compound heterozygous for chr12: g.122359542G>T (p.Glu111∗) and chr12: g.122395032_122395033delCT (p.Leu530Valfs∗4). We show that CFAP251 is normally located along the flagellum but is absent in men carrying WDR66 mutations and reveal a spermatozoa-specific isoform probably generated during spermatozoon maturation. CFAP251 is a component of the calmodulin- and radial-spoke- associated complex, located adjacent to DNAH1, on the inner surface of the peripheral microtubule doublets of the axoneme. In Tetrahymena, the CFAP251 ortholog is necessary for efficient coordinated ciliary beating. Using immunofluorescent and transmission electron microscopy, we provide evidence that loss of CFAP251 affects the formation of the mitochondrial sheath. We propose that CFAP251 plays a structural role during biogenesis of the spermatozoon flagellum in vertebrates.


Assuntos
Proteínas de Ligação a Calmodulina/genética , Calmodulina/genética , Infertilidade Masculina/genética , Mitocôndrias/genética , Mutação/genética , Motilidade Espermática/genética , Espermatozoides/patologia , Axonema/genética , Proteínas de Ligação ao Cálcio/genética , Linhagem Celular Tumoral , Cílios/genética , Dineínas/genética , Exoma/genética , Feminino , Células HeLa , Humanos , Masculino , Cauda do Espermatozoide/patologia , Tetrahymena/genética
SELEÇÃO DE REFERÊNCIAS
DETALHE DA PESQUISA
...