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1.
Annu Rev Cell Dev Biol ; 38: 103-123, 2022 10 06.
Artigo em Inglês | MEDLINE | ID: mdl-35767872

RESUMO

Cilia are ubiquitous microtubule-based eukaryotic organelles that project from the cell to generate motility or function in cellular signaling. Motile cilia or flagella contain axonemal dynein motors and other complexes to achieve beating. Primary cilia are immotile and act as signaling hubs, with receptors shuttling between the cytoplasm and ciliary compartment. In both cilia types, an intraflagellar transport (IFT) system powered by unique kinesin and dynein motors functions to deliver the molecules required to build cilia and maintain their functions. Cryo-electron tomography has helped to reveal the organization of protein complex arrangement along the axoneme and the structure of anterograde IFT trains as well as the structure of primary cilia. Only recently, single-particle analysis (SPA) cryo-electron microscopy has provided molecular details of the protein organization of ciliary components, helping us to understand how they bind to microtubule doublets and how mechanical force propagated by dynein conformational changes is converted into ciliary beating. Here we highlight recent structural advances that are leading to greater knowledge of ciliary function.


Assuntos
Dineínas do Axonema , Cílios , Dineínas do Axonema/genética , Dineínas do Axonema/metabolismo , Transporte Biológico/fisiologia , Biologia , Cílios/metabolismo , Microscopia Crioeletrônica , Flagelos/metabolismo , Cinesinas
2.
Nat Methods ; 19(2): 216-222, 2022 02.
Artigo em Inglês | MEDLINE | ID: mdl-35027766

RESUMO

Cryofixation has proven to be the gold standard for efficient preservation of native cell ultrastructure compared to chemical fixation, but this approach is not widely used in fluorescence microscopy owing to implementation challenges. Here, we develop Cryo-ExM, a method that preserves native cellular organization by coupling cryofixation with expansion microscopy. This method bypasses artifacts associated with chemical fixation and its simplicity will contribute to its widespread use in super-resolution microscopy.


Assuntos
Criopreservação/métodos , Microscopia de Fluorescência/métodos , Animais , Linhagem Celular , Chlamydomonas reinhardtii/citologia , Criopreservação/instrumentação , Citoesqueleto , Epitopos , Proteínas de Fluorescência Verde/genética , Proteínas de Fluorescência Verde/metabolismo , Humanos , Camundongos
3.
EMBO J ; 39(22): e106246, 2020 11 16.
Artigo em Inglês | MEDLINE | ID: mdl-32954513

RESUMO

Centrioles are evolutionarily conserved barrels of microtubule triplets that form the core of the centrosome and the base of the cilium. While the crucial role of the proximal region in centriole biogenesis has been well documented, its native architecture and evolutionary conservation remain relatively unexplored. Here, using cryo-electron tomography of centrioles from four evolutionarily distant species, we report on the architectural diversity of the centriole's proximal cartwheel-bearing region. Our work reveals that the cartwheel central hub is constructed from a stack of paired rings with cartwheel inner densities inside. In both Paramecium and Chlamydomonas, the repeating structural unit of the cartwheel has a periodicity of 25 nm and consists of three ring pairs, with 6 radial spokes emanating and merging into a single bundle that connects to the microtubule triplet via the D2-rod and the pinhead. Finally, we identified that the cartwheel is indirectly connected to the A-C linker through the triplet base structure extending from the pinhead. Together, our work provides unprecedented evolutionary insights into the architecture of the centriole proximal region, which underlies centriole biogenesis.


Assuntos
Centríolos/fisiologia , Centríolos/ultraestrutura , Tomografia com Microscopia Eletrônica/métodos , Centrossomo , Chlamydomonas reinhardtii/fisiologia , Cílios , Humanos , Microtúbulos , Modelos Moleculares , Naegleria/fisiologia , Paramecium tetraurellia/fisiologia
4.
Nature ; 521(7553): 520-4, 2015 May 28.
Artigo em Inglês | MEDLINE | ID: mdl-25807483

RESUMO

Congenital heart disease (CHD) is the most prevalent birth defect, affecting nearly 1% of live births; the incidence of CHD is up to tenfold higher in human fetuses. A genetic contribution is strongly suggested by the association of CHD with chromosome abnormalities and high recurrence risk. Here we report findings from a recessive forward genetic screen in fetal mice, showing that cilia and cilia-transduced cell signalling have important roles in the pathogenesis of CHD. The cilium is an evolutionarily conserved organelle projecting from the cell surface with essential roles in diverse cellular processes. Using echocardiography, we ultrasound scanned 87,355 chemically mutagenized C57BL/6J fetal mice and recovered 218 CHD mouse models. Whole-exome sequencing identified 91 recessive CHD mutations in 61 genes. This included 34 cilia-related genes, 16 genes involved in cilia-transduced cell signalling, and 10 genes regulating vesicular trafficking, a pathway important for ciliogenesis and cell signalling. Surprisingly, many CHD genes encoded interacting proteins, suggesting that an interactome protein network may provide a larger genomic context for CHD pathogenesis. These findings provide novel insights into the potential Mendelian genetic contribution to CHD in the fetal population, a segment of the human population not well studied. We note that the pathways identified show overlap with CHD candidate genes recovered in CHD patients, suggesting that they may have relevance to the more complex genetics of CHD overall. These CHD mouse models and >8,000 incidental mutations have been sperm archived, creating a rich public resource for human disease modelling.


Assuntos
Cílios/patologia , Cardiopatias Congênitas/genética , Cardiopatias Congênitas/patologia , Animais , Cílios/diagnóstico por imagem , Cílios/genética , Cílios/fisiologia , Análise Mutacional de DNA , Eletrocardiografia , Exoma/genética , Genes Recessivos , Testes Genéticos , Cardiopatias Congênitas/diagnóstico por imagem , Humanos , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Mutação/genética , Transdução de Sinais , Ultrassonografia
5.
Am J Hum Genet ; 95(3): 257-74, 2014 Sep 04.
Artigo em Inglês | MEDLINE | ID: mdl-25192045

RESUMO

A diverse family of cytoskeletal dynein motors powers various cellular transport systems, including axonemal dyneins generating the force for ciliary and flagellar beating essential to movement of extracellular fluids and of cells through fluid. Multisubunit outer dynein arm (ODA) motor complexes, produced and preassembled in the cytosol, are transported to the ciliary or flagellar compartment and anchored into the axonemal microtubular scaffold via the ODA docking complex (ODA-DC) system. In humans, defects in ODA assembly are the major cause of primary ciliary dyskinesia (PCD), an inherited disorder of ciliary and flagellar dysmotility characterized by chronic upper and lower respiratory infections and defects in laterality. Here, by combined high-throughput mapping and sequencing, we identified CCDC151 loss-of-function mutations in five affected individuals from three independent families whose cilia showed a complete loss of ODAs and severely impaired ciliary beating. Consistent with the laterality defects observed in these individuals, we found Ccdc151 expressed in vertebrate left-right organizers. Homozygous zebrafish ccdc151(ts272a) and mouse Ccdc151(Snbl) mutants display a spectrum of situs defects associated with complex heart defects. We demonstrate that CCDC151 encodes an axonemal coiled coil protein, mutations in which abolish assembly of CCDC151 into respiratory cilia and cause a failure in axonemal assembly of the ODA component DNAH5 and the ODA-DC-associated components CCDC114 and ARMC4. CCDC151-deficient zebrafish, planaria, and mice also display ciliary dysmotility accompanied by ODA loss. Furthermore, CCDC151 coimmunoprecipitates CCDC114 and thus appears to be a highly evolutionarily conserved ODA-DC-related protein involved in mediating assembly of both ODAs and their axonemal docking machinery onto ciliary microtubules.


Assuntos
Dineínas do Axonema/metabolismo , Cílios/patologia , Síndrome de Kartagener/genética , Proteínas Associadas aos Microtúbulos/fisiologia , Mutação/genética , Animais , Dineínas do Axonema/genética , Axonema/genética , Células Cultivadas , Cílios/metabolismo , Embrião de Mamíferos/citologia , Embrião de Mamíferos/metabolismo , Exoma/genética , Feminino , Imunofluorescência , Humanos , Immunoblotting , Imunoprecipitação , Hibridização In Situ , Síndrome de Kartagener/metabolismo , Síndrome de Kartagener/patologia , Masculino , Camundongos , Camundongos Knockout , Proteínas Associadas aos Microtúbulos/genética , Proteínas Associadas aos Microtúbulos/metabolismo , Linhagem , Fenótipo , Técnicas do Sistema de Duplo-Híbrido , Peixe-Zebra/genética , Peixe-Zebra/crescimento & desenvolvimento , Peixe-Zebra/metabolismo
6.
Am J Med Genet A ; 167A(9): 2188-96, 2015 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-25914204

RESUMO

Ciliopathies such as cranioectodermal dysplasia, Sensenbrenner syndrome, short-rib polydactyly, and Jeune syndrome are associated with respiratory complications arising from rib cage dysplasia. While such ciliopathies have been demonstrated to involve primary cilia defects, we show motile cilia dysfunction in the airway of a patient diagnosed with cranioectodermal dysplasia. While this patient had mild thoracic dystrophy not requiring surgical treatment, there was nevertheless newborn respiratory distress, restrictive airway disease with possible obstructive airway involvement, repeated respiratory infections, and atelectasis. High-resolution videomicroscopy of nasal epithelial biopsy showed immotile/dyskinetic cilia and nasal nitric oxide was reduced, both of which are characteristics of primary ciliary dyskinesia, a sinopulmonary disease associated with mucociliary clearance defects due to motile cilia dysfunction in the airway. Exome sequencing analysis of this patient identified compound heterozygous mutations in WDR35, but no mutations in any of the 30 known primary ciliary dyskinesia genes or other cilia-related genes. Given that WDR35 is only known to be required for primary cilia function, we carried out WDR35 siRNA knockdown in human respiratory epithelia to assess the role of WDR35 in motile cilia function. This showed WDR35 deficiency disrupted ciliogenesis in the airway, indicating WDR35 is also required for formation of motile cilia. Together, these findings suggest patients with WDR35 mutations have an airway mucociliary clearance defect masked by their restrictive airway disease.


Assuntos
Osso e Ossos/anormalidades , Cílios/genética , Craniossinostoses/genética , Displasia Ectodérmica/genética , Doenças Respiratórias/genética , Criança , Proteínas do Citoesqueleto , Proteínas Hedgehog , Heterozigoto , Humanos , Peptídeos e Proteínas de Sinalização Intracelular , Masculino , Mutação/genética , Proteínas/genética
7.
Birth Defects Res C Embryo Today ; 102(2): 115-25, 2014 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-24975753

RESUMO

Structural birth defect (SBD) is a major cause of morbidity and mortality in the newborn period. Although the etiology of SBD is diverse, a wide spectrum of SBD associated with ciliopathies points to the cilium as having a central role in the pathogenesis of SBDs. Ciliopathies are human diseases arising from disruption of cilia structure and/or function. They are associated with developmental anomalies in one or more organ systems and can involve defects in motile cilia, such as those in the airway epithelia or from defects in nonmotile (primary cilia) that have sensory and cell signaling function. Availability of low cost next generation sequencing has allowed for explosion of new knowledge in genetic etiology of ciliopathies. This has led to the appreciation that many genes are shared in common between otherwise clinically distinct ciliopathies. Further insights into the relevance of the cilium in SBD has come from recovery of pathogenic mutations in cilia-related genes from many large-scale mouse forward genetic screens with differing developmental phenotyping focus. Our mouse mutagenesis screen for congenital heart disease (CHD) using noninvasive fetal echocardiography has yielded a marked enrichment for pathogenic mutations in genes required for motile or primary cilia function. These novel mutant mouse models will be invaluable for modeling human ciliopathies and further interrogating the role of the cilium in the pathogenesis of SBD and CHD. Overall, these findings suggest a central role for the cilium in the pathogenesis of a wide spectrum of developmental anomalies associated with CHD and SBDs.


Assuntos
Cílios/patologia , Anormalidades Congênitas/genética , Modelos Animais de Doenças , Animais , Encéfalo/anormalidades , Anormalidades Congênitas/patologia , Discinesias/genética , Discinesias/patologia , Cardiopatias Congênitas/genética , Cardiopatias Congênitas/patologia , Sequenciamento de Nucleotídeos em Larga Escala , Camundongos , Mutação , Organelas/patologia , Transdução de Sinais
8.
Elife ; 122024 Mar 05.
Artigo em Inglês | MEDLINE | ID: mdl-38441556

RESUMO

From a cohort of 167 infertile patients suffering from multiple morphological abnormalities of the flagellum (MMAF), pathogenic bi-allelic mutations were identified in the CCDC146 gene. In somatic cells, CCDC146 is located at the centrosome and at multiple microtubule-related organelles during mitotic division, suggesting that it is a microtubule-associated protein (MAP). To decipher the molecular pathogenesis of infertility associated with CCDC146 mutations, a Ccdc146 knock-out (KO) mouse line was created. KO male mice were infertile, and sperm exhibited a phenotype identical to CCDC146 mutated patients. CCDC146 expression starts during late spermiogenesis. In the spermatozoon, the protein is conserved but is not localized to centrioles, unlike in somatic cells, rather it is present in the axoneme at the level of microtubule doublets. Expansion microscopy associated with the use of the detergent sarkosyl to solubilize microtubule doublets suggests that the protein may be a microtubule inner protein (MIP). At the subcellular level, the absence of CCDC146 impacted all microtubule-based organelles such as the manchette, the head-tail coupling apparatus (HTCA), and the axoneme. Through this study, a new genetic cause of infertility and a new factor in the formation and/or structure of the sperm axoneme were characterized.


Assuntos
Anormalidades Múltiplas , Infertilidade Masculina , Animais , Humanos , Masculino , Camundongos , Centríolos , Infertilidade Masculina/genética , Camundongos Knockout , Proteínas Associadas aos Microtúbulos/genética , Sêmen
9.
Bio Protoc ; 13(17): e4792, 2023 Sep 05.
Artigo em Inglês | MEDLINE | ID: mdl-37719077

RESUMO

Expansion microscopy is an innovative method that enables super-resolution imaging of biological materials using a simple confocal microscope. The principle of this method relies on the physical isotropic expansion of a biological specimen cross-linked to a swellable polymer, stained with antibodies, and imaged. Since its first development, several improved versions of expansion microscopy and adaptations for different types of samples have been produced. Here, we show the application of ultrastructure expansion microscopy (U-ExM) to investigate the 3D organization of the green algae Chlamydomonas reinhardtii cellular ultrastructure, with a particular emphasis on the different types of sample fixation that can be used, as well as compatible staining procedures including membranes. Graphical overview.

10.
J Bacteriol ; 194(17): 4642-51, 2012 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-22753061

RESUMO

Cyclic dimeric GMP (c-di-GMP) regulates numerous processes in Gram-negative bacteria, yet little is known about its role in Gram-positive bacteria. Here we characterize two c-di-GMP phosphodiesterases from the filamentous high-GC Gram-positive actinobacterium Streptomyces coelicolor, involved in controlling colony morphology and development. A transposon mutation in one of the two phosphodiesterase genes, SCO0928, hereby designated rmdA (regulator of morphology and development A), resulted in decreased levels of spore-specific gray pigment and a delay in spore formation. The RmdA protein contains GGDEF-EAL domains arranged in tandem and possesses c-di-GMP phosphodiesterase activity, as is evident from in vitro enzymatic assays using the purified protein. RmdA contains a PAS9 domain and is a hemoprotein. Inactivation of another GGDEF-EAL-encoding gene, SCO5495, designated rmdB, resulted in a phenotype identical to that of the rmdA mutant. Purified soluble fragment of RmdB devoid of transmembrane domains also possesses c-di-GMP phosphodiesterase activity. The rmdA rmdB double mutant has a bald phenotype and is impaired in aerial mycelium formation. This suggests that RmdA and RmdB functions are additive and at least partially overlapping. The rmdA and rmdB mutations likely result in increased local pools of intracellular c-di-GMP, because intracellular c-di-GMP levels in the single mutants did not differ significantly from those of the wild type, whereas in the double rmdA rmdB mutant, c-di-GMP levels were 3-fold higher than those in the wild type. This study highlights the importance of c-di-GMP-dependent signaling in actinomycete colony morphology and development and identifies two c-di-GMP phosphodiesterases controlling these processes.


Assuntos
GMP Cíclico/análogos & derivados , Diester Fosfórico Hidrolases/metabolismo , Streptomyces coelicolor/genética , Streptomyces coelicolor/metabolismo , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , GMP Cíclico/metabolismo , Elementos de DNA Transponíveis , Regulação Bacteriana da Expressão Gênica , Mutação , Diester Fosfórico Hidrolases/genética , Esporos Bacterianos/genética , Esporos Bacterianos/metabolismo , Streptomyces coelicolor/citologia , Streptomyces coelicolor/crescimento & desenvolvimento
11.
Science ; 377(6605): 543-548, 2022 07 29.
Artigo em Inglês | MEDLINE | ID: mdl-35901159

RESUMO

The cilium is an antenna-like organelle that performs numerous cellular functions, including motility, sensing, and signaling. The base of the cilium contains a selective barrier that regulates the entry of large intraflagellar transport (IFT) trains, which carry cargo proteins required for ciliary assembly and maintenance. However, the native architecture of the ciliary base and the process of IFT train assembly remain unresolved. In this work, we used in situ cryo-electron tomography to reveal native structures of the transition zone region and assembling IFT trains at the ciliary base in Chlamydomonas. We combined this direct cellular visualization with ultrastructure expansion microscopy to describe the front-to-back stepwise assembly of IFT trains: IFT-B forms the backbone, onto which bind IFT-A, dynein-1b, and finally kinesin-2 before entry into the cilium.


Assuntos
Chlamydomonas , Cílios , Flagelos , Chlamydomonas/metabolismo , Cílios/metabolismo , Microscopia Crioeletrônica/métodos , Dineínas/metabolismo , Tomografia com Microscopia Eletrônica/métodos , Flagelos/metabolismo , Flagelos/ultraestrutura , Cinesinas/metabolismo , Transporte Proteico , Transdução de Sinais
12.
Curr Opin Struct Biol ; 66: 58-65, 2021 02.
Artigo em Inglês | MEDLINE | ID: mdl-33176264

RESUMO

The centriole is a magnificent molecular assembly of several giga-daltons, one of the largest of the eukaryotic cell, and whose atomic structure remains unsolved to date. However, numerous electron microscopy, cryo-tomography, and super-resolution studies now make it possible to establish a global architectural view of it with its different sub-regions. These analyses broaden our understanding by providing additional informations to cell biology and structural biology approaches. In this review, we describe current knowledge on the overall organization of the centriole. We will highlight each sub-structural element, their differences between species and their putative protein composition. We will conclude on the current limitations that still take us away from a complete atomic view of the centriole architecture.


Assuntos
Centríolos
13.
Nat Commun ; 12(1): 1463, 2021 03 05.
Artigo em Inglês | MEDLINE | ID: mdl-33674590

RESUMO

Kinesin-1 is a processive motor protein that uses ATP-derived energy to transport a variety of intracellular cargoes toward the cell periphery. The ability to visualize and monitor kinesin transport in live cells is critical to study the myriad of functions associated with cargo trafficking. Herein we report the discovery of a fluorogenic small molecule substrate (QPD-OTf) for kinesin-1 that yields a precipitating dye along its walking path on microtubules (MTs). QPD-OTf enables to monitor native kinesin-1 transport activity in cellulo without external modifications. In vitro assays show that kinesin-1 and MTs are sufficient to yield fluorescent crystals; in cells, kinesin-1 specific transport of cargo from the Golgi appears as trails of fluorescence over time. These findings are further supported by docking studies, which suggest the binding of the activity-based substrate in the nucleotide binding site of kinesin-1.


Assuntos
Cinesinas/química , Cinesinas/metabolismo , Microtúbulos/metabolismo , Trifosfato de Adenosina , Animais , Sítios de Ligação , Complexo de Golgi/metabolismo , Células HEK293 , Células HeLa , Humanos , Cinesinas/genética , Camundongos , Paclitaxel , Transporte Proteico , Células RAW 264.7
14.
Sci Adv ; 6(7): eaaz4137, 2020 02.
Artigo em Inglês | MEDLINE | ID: mdl-32110738

RESUMO

The ninefold radial arrangement of microtubule triplets (MTTs) is the hallmark of the centriole, a conserved organelle crucial for the formation of centrosomes and cilia. Although strong cohesion between MTTs is critical to resist forces applied by ciliary beating and the mitotic spindle, how the centriole maintains its structural integrity is not known. Using cryo-electron tomography and subtomogram averaging of centrioles from four evolutionarily distant species, we found that MTTs are bound together by a helical inner scaffold covering ~70% of the centriole length that maintains MTTs cohesion under compressive forces. Ultrastructure Expansion Microscopy (U-ExM) indicated that POC5, POC1B, FAM161A, and Centrin-2 localize to the scaffold structure along the inner wall of the centriole MTTs. Moreover, we established that these four proteins interact with each other to form a complex that binds microtubules. Together, our results provide a structural and molecular basis for centriole cohesion and geometry.


Assuntos
Centríolos/química , Centríolos/metabolismo , Centríolos/ultraestrutura , Chlamydomonas/metabolismo , Chlamydomonas/ultraestrutura , Microtúbulos/metabolismo , Microtúbulos/ultraestrutura , Complexos Multiproteicos/metabolismo , Paramecium tetraurellia/metabolismo , Paramecium tetraurellia/ultraestrutura , Ligação Proteica , Combinação Trimetoprima e Sulfametoxazol/metabolismo
15.
Front Physiol ; 11: 538701, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-33192543

RESUMO

BACKGROUND/AIMS: Infectious and genetic factors are invoked, respectively in isolated biliary atresia (BA), or syndromic BA, with major extrahepatic anomalies. However, isolated BA is also associated with minor extrahepatic gut and cardiovascular anomalies and multiple susceptibility genes, suggesting common origins. METHODS: We investigated novel susceptibility genes with genome-wide association, targeted sequencing and tissue staining in BA requiring liver transplantation, independent of BA subtype. Candidate gene effects on morphogenesis, developmental pathways, and ciliogenesis, which regulates left-right patterning were investigated with zebrafish knockdown and mouse knockout models, mouse airway cell cultures, and liver transcriptome analysis. RESULTS: Single nucleotide polymorphisms in Mannosidase-1-α-2 (MAN1A2) were significantly associated with BA and with other polymorphisms known to affect MAN1A2 expression but were not differentially enriched in either BA subtype. In zebrafish embryos, man1a2 knockdown caused poor biliary network formation, ciliary dysgenesis in Kupffer's vesicle, cardiac and liver heterotaxy, and dysregulated egfra and other developmental genes. Suboptimal man1a2 knockdown synergized with suboptimal EGFR signaling or suboptimal knockdown of the EGFR pathway gene, adenosine-ribosylation-factor-6, which had minimal effects individually, to reproduce biliary defects but not heterotaxy. In cultured mouse airway epithelium, Man1a2 knockdown arrested ciliary development and motility. Man1a2 -/- mice, which experience respiratory failure, also demonstrated portal and bile ductular inflammation. Human BA liver and Man1a2 -/- liver exhibited reduced Man1a2 expression and dysregulated ciliary genes, known to cause multisystem human laterality defects. CONCLUSION: BA requiring transplantation associates with sequence variants in MAN1A2. man1a2 regulates laterality, in addition to hepatobiliary morphogenesis, by regulating ciliogenesis in zebrafish and mice, providing a novel developmental basis for multisystem defects in BA.

16.
J Vis Exp ; (139)2018 09 21.
Artigo em Inglês | MEDLINE | ID: mdl-30295659

RESUMO

Centrioles are large macromolecular assemblies important for the proper execution of fundamental cell biological processes such as cell division, cell motility, or cell signaling. The green algae Chlamydomonas reinhardtii has proven to be an insightful model in the study of centriole architecture, function, and protein composition. Despite great advances toward understanding centriolar architecture, one of the current challenges is to determine the precise localization of centriolar components within structural regions of the centriole in order to better understand their role in centriole biogenesis. A major limitation lies in the resolution of fluorescence microscopy, which complicates the interpretation of protein localization in this organelle with dimensions close to the diffraction limit. To tackle this question, we are providing a method to purify and image a large number of C. reinhardtii centrioles with different orientations using super-resolution microscopy. This technique allows further processing of data through fluorescent single-particle averaging (Fluo-SPA) owing to the large number of centrioles acquired. Fluo-SPA generates averages of stained C. reinhardtii centrioles in different orientations, thus facilitating the localization of distinct proteins in centriolar sub-regions. Importantly, this method can be applied to image centrioles from other species or other large macromolecular assemblies.


Assuntos
Centríolos/metabolismo , Chlamydomonas reinhardtii/citologia , Imagem Óptica/métodos , Animais , Divisão Celular , Proteínas de Plantas/metabolismo , Transporte Proteico
17.
Genome Biol ; 19(1): 87, 2018 07 17.
Artigo em Inglês | MEDLINE | ID: mdl-30012220

RESUMO

BACKGROUND: Genome-wide association studies conducted on QRS duration, an electrocardiographic measurement associated with heart failure and sudden cardiac death, have led to novel biological insights into cardiac function. However, the variants identified fall predominantly in non-coding regions and their underlying mechanisms remain unclear. RESULTS: Here, we identify putative functional coding variation associated with changes in the QRS interval duration by combining Illumina HumanExome BeadChip genotype data from 77,898 participants of European ancestry and 7695 of African descent in our discovery cohort, followed by replication in 111,874 individuals of European ancestry from the UK Biobank and deCODE cohorts. We identify ten novel loci, seven within coding regions, including ADAMTS6, significantly associated with QRS duration in gene-based analyses. ADAMTS6 encodes a secreted metalloprotease of currently unknown function. In vitro validation analysis shows that the QRS-associated variants lead to impaired ADAMTS6 secretion and loss-of function analysis in mice demonstrates a previously unappreciated role for ADAMTS6 in connexin 43 gap junction expression, which is essential for myocardial conduction. CONCLUSIONS: Our approach identifies novel coding and non-coding variants underlying ventricular depolarization and provides a possible mechanism for the ADAMTS6-associated conduction changes.


Assuntos
Proteínas ADAMTS/genética , Conexina 43/genética , Exoma , Loci Gênicos , Sistema de Condução Cardíaco/metabolismo , Miocárdio/metabolismo , Animais , População Negra , Eletrocardiografia , Feminino , Expressão Gênica , Perfilação da Expressão Gênica , Estudo de Associação Genômica Ampla , Sistema de Condução Cardíaco/fisiopatologia , Humanos , Masculino , Camundongos , Pessoa de Meia-Idade , Miocárdio/patologia , Fases de Leitura Aberta , Polimorfismo de Nucleotídeo Único , População Branca , Sequenciamento do Exoma
18.
Artigo em Inglês | MEDLINE | ID: mdl-28159874

RESUMO

A central role for cilia in congenital heart disease (CHD) was recently identified in a large-scale mouse mutagenesis screen. Although the screen was phenotype-driven, the majority of genes recovered were cilia-related, suggesting that cilia play a central role in CHD pathogenesis. This partly reflects the role of cilia as a hub for cell signaling pathways regulating cardiovascular development. Consistent with this, many cilia-transduced cell signaling genes were also recovered, and genes regulating vesicular trafficking, a pathway essential for ciliogenesis and cell signaling. Interestingly, among CHD-cilia genes recovered, some regulate left-right patterning, indicating cardiac left-right asymmetry disturbance may play significant roles in CHD pathogenesis. Clinically, CHD patients show a high prevalence of ciliary dysfunction and show enrichment for de novo mutations in cilia-related pathways. Combined with the mouse findings, this would suggest CHD may be a new class of ciliopathy.


Assuntos
Cílios/fisiologia , Ciliopatias/complicações , Cardiopatias Congênitas/etiologia , Animais , Humanos
19.
JCI Insight ; 2(5): e91702, 2017 03 09.
Artigo em Inglês | MEDLINE | ID: mdl-28289722

RESUMO

Ciliary motion defects cause defective mucociliary transport (MCT) in primary ciliary dyskinesia (PCD). Current diagnostic tests do not assess how MCT is affected by perturbation of ciliary motion. In this study, we sought to use micro-optical coherence tomography (µOCT) to delineate the mechanistic basis of cilia motion defects of PCD genes by functional categorization of cilia motion. Tracheae from three PCD mouse models were analyzed using µOCT to characterize ciliary motion and measure MCT. We developed multiple measures of ciliary activity, integrated these measures, and quantified dyskinesia by the angular range of the cilia effective stroke (ARC). Ccdc39-/- mice, with a known severe PCD mutation of ciliary axonemal organization, had absent motile ciliary regions, resulting in abrogated MCT. In contrast, Dnah5-/- mice, with a missense mutation of the outer dynein arms, had reduced ciliary beat frequency (CBF) but preserved motile area and ciliary stroke, maintaining some MCT. Wdr69-/- PCD mice exhibited normal motile area and CBF and partially delayed MCT due to abnormalities of ciliary ARC. Visualization of ciliary motion using µOCT provides quantitative assessment of ciliary motion and MCT. Comprehensive ciliary motion investigation in situ classifies ciliary motion defects and quantifies their contribution to delayed mucociliary clearance.


Assuntos
Cílios/fisiologia , Tomografia de Coerência Óptica/métodos , Animais , Modelos Animais de Doenças , Feminino , Humanos , Síndrome de Kartagener/fisiopatologia , Masculino , Camundongos , Fenótipo
20.
Nat Genet ; 49(7): 1152-1159, 2017 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-28530678

RESUMO

Congenital heart disease (CHD) affects up to 1% of live births. Although a genetic etiology is indicated by an increased recurrence risk, sporadic occurrence suggests that CHD genetics is complex. Here, we show that hypoplastic left heart syndrome (HLHS), a severe CHD, is multigenic and genetically heterogeneous. Using mouse forward genetics, we report what is, to our knowledge, the first isolation of HLHS mutant mice and identification of genes causing HLHS. Mutations from seven HLHS mouse lines showed multigenic enrichment in ten human chromosome regions linked to HLHS. Mutations in Sap130 and Pcdha9, genes not previously associated with CHD, were validated by CRISPR-Cas9 genome editing in mice as being digenic causes of HLHS. We also identified one subject with HLHS with SAP130 and PCDHA13 mutations. Mouse and zebrafish modeling showed that Sap130 mediates left ventricular hypoplasia, whereas Pcdha9 increases penetrance of aortic valve abnormalities, both signature HLHS defects. These findings show that HLHS can arise genetically in a combinatorial fashion, thus providing a new paradigm for the complex genetics of CHD.


Assuntos
Heterogeneidade Genética , Síndrome do Coração Esquerdo Hipoplásico/genética , Sequência de Aminoácidos , Animais , Aorta/embriologia , Sistemas CRISPR-Cas , Mapeamento Cromossômico , Cromossomos Humanos/genética , Modelos Animais de Doenças , Exoma , Feminino , Edição de Genes , Técnicas de Inativação de Genes , Ventrículos do Coração/embriologia , Humanos , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Mutantes , Mutação , Mutação de Sentido Incorreto , Miócitos Cardíacos/patologia , Penetrância , Alinhamento de Sequência , Homologia de Sequência de Aminoácidos , Obstrução do Fluxo Ventricular Externo/genética , Peixe-Zebra/genética
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