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1.
Yonsei Med J ; 62(3): 224-230, 2021 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-33635012

RESUMO

PURPOSE: Nontuberculous mycobacteria (NTM) is ubiquitous in the environment, but NTM lung disease (NTM-LD) is uncommon. Since exposure to NTM is inevitable, patients who develop NTM-LD are likely to have specific susceptibility factors, such as primary ciliary dyskinesia (PCD). PCD is a genetically heterogeneous disorder of motile cilia and is characterized by chronic respiratory tract infection, organ laterality defect, and infertility. In this study, we performed whole exome sequencing (WES) and investigated the genetic characteristics of adult NTM patients with suspected PCD. MATERIALS AND METHODS: WES was performed in 13 NTM-LD patients who were suspected of having PCD by clinical symptoms and/or ultrastructural ciliary defect observed by transmission electron microscopy. A total of 45 PCD-causing genes, 23 PCD-candidate genes, and 990 ciliome genes were analyzed. RESULTS: Four patients were found to have biallelic loss-of-function (LoF) variants in the following PCD-causing genes: CCDC114, DNAH5, HYDIN, and NME5. In four other patients, only one LoF variant was identified, while the remaining five patients did not have any LoF variants. CONCLUSION: At least 30.8% of NTM-LD patients who were suspected of having PCD had biallelic LoF variants, and an additional 30.8% of patients had one LoF variant. Therefore, PCD should be considered in patients with NTM-LD with symptoms or signs suspicious of PCD.


Assuntos
Transtornos da Motilidade Ciliar/genética , Transtornos da Motilidade Ciliar/microbiologia , Micobactérias não Tuberculosas/genética , Sequenciamento Completo do Exoma , Adolescente , Adulto , Cílios/metabolismo , Cílios/ultraestrutura , Transtornos da Motilidade Ciliar/diagnóstico , Feminino , Estudos de Associação Genética , Humanos , Masculino , Pessoa de Meia-Idade , Mutação/genética , República da Coreia , Adulto Jovem
2.
Nat Commun ; 12(1): 1251, 2021 02 23.
Artigo em Inglês | MEDLINE | ID: mdl-33623007

RESUMO

Dysfunction of embryo transport causes ectopic pregnancy which affects approximately 2% of conceptions in the US and Europe, and is the most common cause of pregnancy-related death in the first trimester. Embryo transit involves a valve-like tubal-locking phenomenon that temporarily arrests oocytes at the ampullary-isthmic junction (AIJ) where fertilisation occurs, but the mechanisms involved are unknown. Here we show that female mice lacking the orphan adhesion G-protein coupled receptor Adgrd1 are sterile because they do not relieve the AIJ restraining mechanism, inappropriately retaining embryos within the oviduct. Adgrd1 is expressed on the oviductal epithelium and the post-ovulatory attenuation of tubal fluid flow is dysregulated in Adgrd1-deficient mice. Using a large-scale extracellular protein interaction screen, we identified Plxdc2 as an activating ligand for Adgrd1 displayed on cumulus cells. Our findings demonstrate that regulating oviductal fluid flow by Adgrd1 controls embryo transit and we present a model where embryo arrest at the AIJ is due to the balance of abovarial ciliary action and the force of adovarial tubal fluid flow, and in wild-type oviducts, fluid flow is gradually attenuated through Adgrd1 activation to enable embryo release. Our findings provide important insights into the molecular mechanisms involved in embryo transport in mice.


Assuntos
Líquidos Corporais/fisiologia , Embrião de Mamíferos/metabolismo , Oviductos/metabolismo , Receptores Acoplados a Proteínas-G/metabolismo , Reologia , Animais , Membrana Celular/metabolismo , Membrana Celular/ultraestrutura , Cílios/metabolismo , Cílios/ultraestrutura , Células do Cúmulo/metabolismo , Epitélio/metabolismo , Feminino , Genótipo , Infertilidade Feminina/metabolismo , Infertilidade Feminina/patologia , Ligantes , Masculino , Camundongos , Modelos Biológicos , Músculos/metabolismo , Mutação/genética , Oviductos/patologia , Oviductos/ultraestrutura , Regiões Promotoras Genéticas/genética , Ligação Proteica , Receptores de Superfície Celular/metabolismo , Receptores Acoplados a Proteínas-G/deficiência
3.
Nat Commun ; 12(1): 612, 2021 01 27.
Artigo em Inglês | MEDLINE | ID: mdl-33504787

RESUMO

The motile cilia of ependymal cells coordinate their beats to facilitate a forceful and directed flow of cerebrospinal fluid (CSF). Each cilium originates from a basal body with a basal foot protruding from one side. A uniform alignment of these basal feet is crucial for the coordination of ciliary beating. The process by which the basal foot originates from subdistal appendages of the basal body, however, is unresolved. Here, we show FGFR1 Oncogene Partner (FOP) is a useful marker for delineating the transformation of a circular, unpolarized subdistal appendage into a polarized structure with a basal foot. Ankyrin repeat and SAM domain-containing protein 1A (ANKS1A) interacts with FOP to assemble region I of the basal foot. Importantly, disruption of ANKS1A reduces the size of region I. This produces an unstable basal foot, which disrupts rotational polarity and the coordinated beating of cilia in young adult mice. ANKS1A deficiency also leads to severe degeneration of the basal foot in aged mice and the detachment of cilia from their basal bodies. This role of ANKS1A in the polarization of the basal foot is evolutionarily conserved in vertebrates. Thus, ANKS1A regulates FOP to build and maintain the polarity of subdistal appendages.


Assuntos
Cílios/metabolismo , Simulação de Dinâmica Molecular , Proteínas Adaptadoras de Transdução de Sinal/deficiência , Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Envelhecimento/patologia , Animais , Corpos Basais/metabolismo , Evolução Biológica , Cílios/ultraestrutura , Embrião não Mamífero/metabolismo , Camundongos Endogâmicos C57BL , Camundongos Knockout , Ligação Proteica , Fatores de Transcrição/metabolismo , Xenopus/embriologia , Xenopus/metabolismo
4.
Nat Commun ; 11(1): 5520, 2020 11 02.
Artigo em Inglês | MEDLINE | ID: mdl-33139725

RESUMO

Axonemal dynein ATPases direct ciliary and flagellar beating via adenosine triphosphate (ATP) hydrolysis. The modulatory effect of adenosine monophosphate (AMP) and adenosine diphosphate (ADP) on flagellar beating is not fully understood. Here, we describe a deficiency of cilia and flagella associated protein 45 (CFAP45) in humans and mice that presents a motile ciliopathy featuring situs inversus totalis and asthenospermia. CFAP45-deficient cilia and flagella show normal morphology and axonemal ultrastructure. Proteomic profiling links CFAP45 to an axonemal module including dynein ATPases and adenylate kinase as well as CFAP52, whose mutations cause a similar ciliopathy. CFAP45 binds AMP in vitro, consistent with structural modelling that identifies an AMP-binding interface between CFAP45 and AK8. Microtubule sliding of dyskinetic sperm from Cfap45-/- mice is rescued with the addition of either AMP or ADP with ATP, compared to ATP alone. We propose that CFAP45 supports mammalian ciliary and flagellar beating via an adenine nucleotide homeostasis module.


Assuntos
Nucleotídeos de Adenina/metabolismo , Astenozoospermia/genética , Proteínas do Citoesqueleto/deficiência , Situs Inversus/genética , Adolescente , Adulto , Animais , Astenozoospermia/patologia , Axonema/ultraestrutura , Sistemas CRISPR-Cas/genética , Cílios/metabolismo , Cílios/ultraestrutura , Proteínas do Citoesqueleto/genética , Análise Mutacional de DNA , Modelos Animais de Doenças , Epididimo/patologia , Feminino , Flagelos/metabolismo , Flagelos/ultraestrutura , Humanos , Mutação com Perda de Função , Masculino , Camundongos , Camundongos Knockout , Pessoa de Meia-Idade , Planárias/citologia , Planárias/genética , Planárias/metabolismo , Mucosa Respiratória/citologia , Mucosa Respiratória/patologia , Situs Inversus/diagnóstico por imagem , Situs Inversus/patologia , Motilidade Espermática/genética , Tomografia Computadorizada por Raios X , Sequenciamento Completo do Exoma
5.
Zhonghua Yi Xue Yi Chuan Xue Za Zhi ; 37(9): 1021-1024, 2020 Sep 10.
Artigo em Chinês | MEDLINE | ID: mdl-32820521

RESUMO

OBJECTIVE: To detect variant of PIH1D3 gene in a Chinese pedigree affected with primary ciliary dyskinesia (PCD) and explore its genotype-relationship correlation. METHODS: PCD patients from the pedigree were analyzed. Ultrastructures of the cilia and flagella of the nasal mucosa were analyzed. DNA samples of the patients were sequenced. RESULTS: The proband and all other affected members of his pedigree had a history of various degree of respiratory tract infection. Two patients had visceral heterotopia, and one was infertile. Electronic microscopy revealed abnormal structures of cilia and flagella. The inner and outer dynein arms were missing, and the arrangement of cilia was disordered. DNA sequencing showed that all patients have carried a c.355C>T variant of the PIH1D3 gene. The corresponding nucleotide was located in a key PIH1 domain, and the site is highly conserved among human, macaque, domestic dog, mouse, xenopus and zebrafish. CONCLUSION: Deletion of the PIH1D3 gene can lead to failure of assembly of inner and outer dynein arms in nasal cilia and sperm flagella, and failure of normal swimming of cilia and sperm. The diagnosis rate of PCD can be validated by genetic testing.


Assuntos
Transtornos da Motilidade Ciliar , Peptídeos e Proteínas de Sinalização Intracelular/genética , Cílios/patologia , Cílios/ultraestrutura , Transtornos da Motilidade Ciliar/genética , Flagelos/patologia , Flagelos/ultraestrutura , Genótipo , Humanos , Mucosa Nasal , Linhagem
6.
Nat Commun ; 11(1): 2637, 2020 05 26.
Artigo em Inglês | MEDLINE | ID: mdl-32457457

RESUMO

Metachronal waves commonly exist in natural cilia carpets. These emergent phenomena, which originate from phase differences between neighbouring self-beating cilia, are essential for biological transport processes including locomotion, liquid pumping, feeding, and cell delivery. However, studies of such complex active systems are limited, particularly from the experimental side. Here we report magnetically actuated, soft, artificial cilia carpets. By stretching and folding onto curved templates, programmable magnetization patterns can be encoded into artificial cilia carpets, which exhibit metachronal waves in dynamic magnetic fields. We have tested both the transport capabilities in a fluid environment and the locomotion capabilities on a solid surface. This robotic system provides a highly customizable experimental platform that not only assists in understanding fundamental rules of natural cilia carpets, but also paves a path to cilia-inspired soft robots for future biomedical applications.


Assuntos
Células Artificiais , Cílios/fisiologia , Células Artificiais/ultraestrutura , Cílios/ultraestrutura , Simulação por Computador , Hidrodinâmica , Magnetismo , Modelos Biológicos , Movimento (Física) , Impressão Tridimensional/instrumentação , Robótica/instrumentação
8.
Exp Parasitol ; 212: 107886, 2020 May.
Artigo em Inglês | MEDLINE | ID: mdl-32209315

RESUMO

In aquaculture of the swimming crab Portunus trituberculatus, massive deaths have been recorded in the winter months due to infection with a novel emerging parasite, Mesanophrys sp. However, no information was available regarding the prevention and control of this particular parasite. Therefore, the present study was conducted to evaluate the anti-parasitic efficacy and toxicity of formalin against the Mesanophrys sp. In vitro results showed that the anti-parasitic efficacy of formalin improved with concentration increasing from 0.0 to 20.0 ppm within 24 h. In particular, when treated with formalin at 16.0, 15.0, 11.0, 10.0, 9.0, and 6.0 ppm for 0.5, 1, 2, 4, 6, 12, and 24 h respectively, the Mesanophrys sp. mortality rate reached 100%. To gain insights into the effects the formalin treatment had on the parasite, cell micro- and ultra-structure were investigated. It was determined that the cells contracted gradually and became rounded, intracellular vacuoles were observed at early time points (Ф≤4.83 ± 1.26 µm) and then disappeared. Cilia were shed and macronuclear chromatin became condensed and agglutinated. Small holes and bubbles appeared on surface of the parasites. In an in vivo trial, formalin was applied prior to Mesanophrys sp. artificial infection as prophylaxis to P. trituberculatus. The results showed that formalin prophylactic treatment effectively prevented P. trituberculatus from Mesanophrys sp. infection, thus remarkably reducing the mortality of crabs compared with the non-formalin-exposed and infected crabs. Furthermore, the normal behavior and survival of P. trituberculatus were not impacted by the prophylactic treatment.


Assuntos
Antiparasitários/farmacologia , Braquiúros/parasitologia , Desinfetantes/farmacologia , Formaldeído/farmacologia , Oligoimenóforos/efeitos dos fármacos , Análise de Variância , Animais , Aquicultura , Braquiúros/crescimento & desenvolvimento , Cromatina/efeitos dos fármacos , Cílios/efeitos dos fármacos , Cílios/ultraestrutura , DNA de Protozoário/efeitos dos fármacos , DNA de Protozoário/isolamento & purificação , Relação Dose-Resposta a Droga , Eletroforese em Gel de Ágar , Hemolinfa/parasitologia , Microscopia Eletrônica de Varredura , Microscopia Eletrônica de Transmissão , Microscopia de Interferência , Mitocôndrias/efeitos dos fármacos , Mitocôndrias/ultraestrutura , Oligoimenóforos/genética , Oligoimenóforos/patogenicidade , Oligoimenóforos/ultraestrutura , Vacúolos/efeitos dos fármacos , Vacúolos/ultraestrutura
9.
Zoolog Sci ; 37(1): 7-13, 2020 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-32068369

RESUMO

The outer dynein arm-docking complex (ODA-DC), which was first identified in the green alga Chlamydomonas reinhardtii, is a protein complex that mediates the binding of axonemal dynein and doublet microtubules. To gain a better understanding of the evolutionary conservation and functional diversity of the ODA-DC, we knocked down a homolog of DC2, a major subunit of the ODA-DC, in the planarian Schmidtea mediterranea. Planaria are carnivorous flatworms that move by beating cilia on their ventral surface against a secreted mucus layer. These organisms have recently been used for cilia research because knockdown of flatworm genes by RNA interference (RNAi) is readily achieved through feeding with double-stranded RNA (dsRNA). Lack of DC2 in S. mediterranea caused several defects in cilia, including low beat frequency, decreased ciliary density, and a reduction in ciliary length. The loss of DC2 function C. reinhardtii mutant oda1 shows slow jerky swimming, but has two flagella of almost normal length. These data suggest that the function of a DC2 homolog in S. mediterranea cilia may be somewhat different from DC2 in C. reinhardtii flagella.


Assuntos
Dineínas do Axonema/metabolismo , Cílios/patologia , Planárias/metabolismo , Sequência de Aminoácidos , Animais , Dineínas do Axonema/genética , Cílios/genética , Cílios/metabolismo , Cílios/ultraestrutura , Flagelos , Microscopia Eletrônica de Transmissão , Movimento , Planárias/genética , Subunidades Proteicas/genética , Subunidades Proteicas/metabolismo , Interferência de RNA
10.
Cell Prolif ; 53(3): e12765, 2020 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-32034931

RESUMO

Cartilage is a connective tissue in the skeletal system and has limited regeneration ability and unique biomechanical reactivity. The growth and development of cartilage can be affected by different physical, chemical and biological factors, such as mechanical stress, inflammation, osmotic pressure, hypoxia and signalling transduction. Primary cilia are multifunctional sensory organelles that regulate diverse signalling transduction and cell activities. They are crucial for the regulation of cartilage development and act in a variety of ways, such as react to mechanical stress, mediate signalling transduction, regulate cartilage-related diseases progression and affect cartilage tumorigenesis. Therefore, research on primary cilia-mediated cartilage growth and development is currently extremely popular. This review outlines the role of primary cilia in cartilage development in recent years and elaborates on the potential regulatory mechanisms from different aspects.


Assuntos
Cartilagem/crescimento & desenvolvimento , Condrogênese , Cílios/metabolismo , Transdução de Sinais , Animais , Fenômenos Biomecânicos , Cartilagem/metabolismo , Cartilagem/ultraestrutura , Cílios/ultraestrutura , Humanos , Mecanotransdução Celular , Osteogênese
11.
Orthop Surg ; 12(2): 645-652, 2020 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-32064763

RESUMO

OBJECTIVE: To study the role of primary cilia formation disorder and osteoblasts autophagy in the pathogenesis of steroid-induced avascular necrosis of the femoral head (SANFH). METHODS: Osteoblasts were isolated from rabbit bones and treated with 1 µM Methylprednisolone for 0, 12, 24, 48, and 72 h. The Beclin1, MAP1LC3, Atg-5, Atg-12, IFT20 and OFD1 mRNAs and proteins were detected by PCR and Western blotting, and their correlation was statistically analyzed. The lengths of osteoblast cilia were measured under a laser confocal microscope, and the autophagy flux was tracked by transfecting the osteoblasts with GFP-RFP-LC3 lentivirus. RESULTS: Methylprednisolone significantly upregulated Beclin1, MAP1LC3, Atg-5, Atg-12 and OFD1 mRNAs and proteins in a time-dependent manner, and decreased that of IFT20 (P < 0.05). In addition, the autophagy flux in the osteoblasts also increased and the ciliary length decreased in a time-dependent manner after Methylprednisolone treatment. The length of the cilia were 5.46 ± 0.11 um at 0 h, 4.08 ± 0.09 um at 12 h, 3.07 ± 0.07 um at 24 h, 2.31 ± 0.10 um at 48 h, and finally 1.15 ± 0.04 um at 72 h. Methylprednisolone treatment also affects primary cilium numbers in cultures, for 0 to 72 h. The autophagy regulatory genes, Beclin1, MAP1LC3, Atg-5 and Atg-12, were found to be negatively correlated with IFT20, with an average correlation coefficient of -0.81. A negative correlation was also found between OFD1 and IFT20, with an average correlation coefficient of -0.53. CONCLUSION: Methylprednisolone inhibits primary cilia formation and promotes autophagy, which could be the pathological basis of SANFH. The exact regulatory mechanism needs to be further studied in vivo.


Assuntos
Autofagia/efeitos dos fármacos , Cílios/efeitos dos fármacos , Cílios/ultraestrutura , Metilprednisolona/farmacologia , Osteoblastos/efeitos dos fármacos , Osteoblastos/ultraestrutura , Animais , Necrose da Cabeça do Fêmur , Glucocorticoides/farmacologia , Coelhos
12.
Biol Cell ; 112(1): 22-37, 2020 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-31789463

RESUMO

BACKGROUND: The centrosome regulates cell spatial organisation by controlling the architecture of the microtubule (MT) cytoskeleton. Conversely, the position of the centrosome within the cell depends on cytoskeletal networks it helps organizing. In mammalian cells, centrosome positioning involves a population of MT stably anchored at centrioles, the core components of the centrosome. An MT-anchoring complex containing the proteins ninein and Cep170 is enriched at subdistal appendages (SAP) that decorate the older centriole (called mother centriole) and at centriole proximal ends. Here, we studied the role played at the centrosome by hVFL3/CCDC61, the human ortholog of proteins required for anchoring distinct sets of cytoskeletal fibres to centrioles in unicellular eukaryotes. RESULTS: We show that hVFL3 co-localises at SAP and at centriole proximal ends with components of the MT-anchoring complex, and physically interacts with Cep170. Depletion of hVFL3 increased the distance between mother and daughter centrioles without affecting the assembly of a filamentous linker that tethers the centrioles and contains the proteins rootletin and C-Nap1. When the linker was disrupted by inactivating C-Nap1, hVFL3-depletion exacerbated centriole splitting, a phenotype also observed following depletion of other SAP components. This supported that hVFL3 is required for SAP function, which we further established by showing that centrosome positioning is perturbed in hVFL3-depleted interphase cells. Finally, we found that hVFL3 is an MT-binding protein. CONCLUSIONS AND SIGNIFICANCE: Together, our results support that hVFL3 is required for anchoring MT at SAP during interphase and ensuring proper centrosome cohesion and positioning. The role of the VFL3 family of proteins thus appears to have been conserved in evolution despite the great variation in the shape of centriole appendages in different eukaryotic species.


Assuntos
Proteínas de Transporte/metabolismo , Centríolos , Centrossomo , Tubulina (Proteína)/metabolismo , Animais , Sistemas CRISPR-Cas , Proteínas de Transporte/genética , Linhagem Celular , Centríolos/metabolismo , Centríolos/ultraestrutura , Centrossomo/metabolismo , Centrossomo/ultraestrutura , Cílios/ultraestrutura , Proteínas do Citoesqueleto/metabolismo , Citoesqueleto/ultraestrutura , Humanos , Microscopia Eletrônica , Microtúbulos/metabolismo , Microtúbulos/ultraestrutura , RNA Interferente Pequeno
13.
Gastric Cancer ; 23(1): 64-72, 2020 01.
Artigo em Inglês | MEDLINE | ID: mdl-31267361

RESUMO

BACKGROUND: Gastrointestinal stromal tumour (GIST) is a mesenchymal cancer which derives from interstitial cells of Cajal. To determine whether a relationship between Hedgehog (Hh) signalling pathway and primary cilia exists in GIST tumours is intended here. METHODS: Immunohistochemical, immunofluorescence and ultrastructural techniques were performed in this study. RESULTS: We show that GIST cells present primary cilia (an antenna-like structure based on microtubules). But, moreover, we prove Hedgehog signalling pathway activation in these tumours (a pathway related with tumoural features such as proliferation, migration or stemness) and we show for the first time that this signalling pathway activation in GIST is mediated by primary cilia, likely in a paracrine way. CONCLUSION: Thus, primary cilia and Hedgehog signalling would be fundamental in tumoural microenvironment control of GIST cells for their maintenance, differentiation and proliferation.


Assuntos
Cílios/patologia , Neoplasias Gastrointestinais/metabolismo , Tumores do Estroma Gastrointestinal/metabolismo , Proteínas Hedgehog/metabolismo , Cílios/metabolismo , Cílios/ultraestrutura , Neoplasias Gastrointestinais/patologia , Tumores do Estroma Gastrointestinal/patologia , Humanos , Transdução de Sinais , Proteína GLI1 em Dedos de Zinco/metabolismo
14.
Cell Mol Life Sci ; 77(1): 195-212, 2020 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-31177295

RESUMO

During ciliogenesis, the mother centriole transforms into a basal body competent to nucleate a cilium. The mother centriole and basal body possess sub-distal appendages (SDAs) and basal feet (BF), respectively. SDAs and BF are thought to be equivalent structures. In contrast to SDA assembly, little is known about the players involved in BF assembly and its assembly order. Furthermore, the contribution of BF to ciliogenesis is not understood. Here, we found that SDAs are distinguishable from BF and that the protein NPHP5 is a novel SDA and BF component. Remarkably, NPHP5 is specifically required for BF assembly in cells able to form basal bodies but is dispensable for SDA assembly. Determination of the hierarchical assembly reveals that NPHP5 cooperates with a subset of SDA/BF proteins to organize BF. The assembly pathway of BF is similar but not identical to that of SDA. Loss of NPHP5 or a BF protein simultaneously inhibits BF assembly and primary ciliogenesis, and these phenotypes could be rescued by manipulating the expression of certain components in the BF assembly pathway. These findings define a novel role for NPHP5 in specifically regulating BF assembly, a process which is tightly coupled to primary ciliogenesis.


Assuntos
Corpos Basais/metabolismo , Proteínas de Ligação a Calmodulina/metabolismo , Cílios/metabolismo , Corpos Basais/ultraestrutura , Linhagem Celular , Centríolos/metabolismo , Centríolos/ultraestrutura , Cílios/ultraestrutura , Humanos , Mapas de Interação de Proteínas
15.
EMBO J ; 39(4): e102723, 2020 02 17.
Artigo em Inglês | MEDLINE | ID: mdl-31880004

RESUMO

Cilia serve as cellular antennae that translate sensory information into physiological responses. In the sperm flagellum, a single chemoattractant molecule can trigger a Ca2+ rise that controls motility. The mechanisms underlying such ultra-sensitivity are ill-defined. Here, we determine by mass spectrometry the copy number of nineteen chemosensory signaling proteins in sperm flagella from the sea urchin Arbacia punctulata. Proteins are up to 1,000-fold more abundant than the free cellular messengers cAMP, cGMP, H+ , and Ca2+ . Opto-chemical techniques show that high protein concentrations kinetically compartmentalize the flagellum: Within milliseconds, cGMP is relayed from the receptor guanylate cyclase to a cGMP-gated channel that serves as a perfect chemo-electrical transducer. cGMP is rapidly hydrolyzed, possibly via "substrate channeling" from the channel to the phosphodiesterase PDE5. The channel/PDE5 tandem encodes cGMP turnover rates rather than concentrations. The rate-detection mechanism allows continuous stimulus sampling over a wide dynamic range. The textbook notion of signal amplification-few enzyme molecules process many messenger molecules-does not hold for sperm flagella. Instead, high protein concentrations ascertain messenger detection. Similar mechanisms may occur in other small compartments like primary cilia or dendritic spines.


Assuntos
Arbacia/fisiologia , Quimiotaxia , Proteômica , Transdução de Sinais , Animais , Arbacia/ultraestrutura , Cálcio/metabolismo , Cílios/fisiologia , Cílios/ultraestrutura , GMP Cíclico/metabolismo , Tomografia com Microscopia Eletrônica , Flagelos/fisiologia , Flagelos/ultraestrutura , Guanilato Ciclase/metabolismo , Masculino , Espectrometria de Massas , Espermatozoides/fisiologia , Espermatozoides/ultraestrutura
16.
J Cell Biol ; 219(1)2020 01 06.
Artigo em Inglês | MEDLINE | ID: mdl-31874113

RESUMO

Cells use motile cilia to generate force in the extracellular space. The structure of a cilium can be classified into three subdomains: the intracellular basal body (BB) that templates cilium formation, the extracellular axoneme that generates force, and the transition zone (TZ) that bridges them. While the BB is composed of triplet microtubules (TMTs), the axoneme is composed of doublet microtubules (DMTs), meaning the cilium must convert between different microtubule geometries. Here, we performed electron cryotomography to define this conversion, and our reconstructions reveal identifying structural features of the BB, TZ, and axoneme. Each region is distinct in terms of microtubule number and geometry, microtubule inner proteins, and microtubule linkers. TMT to DMT conversion occurs within the BB, and microtubule geometry changes to axonemal by the end of the TZ, followed by the addition of axoneme-specific components essential for cilium motility. Our results provide the highest-resolution images of the motile cilium to date and reveal how BBs template axonemes.


Assuntos
Axonema/ultraestrutura , Corpos Basais/ultraestrutura , Cílios/ultraestrutura , Microscopia Crioeletrônica/métodos , Tomografia com Microscopia Eletrônica/métodos , Microtúbulos/ultraestrutura , Traqueia/ultraestrutura , Animais , Axonema/metabolismo , Corpos Basais/metabolismo , Bovinos , Cílios/metabolismo , Proteínas dos Microtúbulos/metabolismo , Microtúbulos/metabolismo , Traqueia/metabolismo
17.
Pediatr Pulmonol ; 55(1): 130-135, 2020 01.
Artigo em Inglês | MEDLINE | ID: mdl-31549486

RESUMO

BACKGROUND: The diagnosis of primary ciliary dyskinesia (PCD) is difficult and requires a combination of clinical features, nasal nitric oxide testing, cilia ultrastructural analysis by electron microscopy (EM), and genetics. A recently described cytoplasmic ultrastructural change termed "ciliary inclusions" was reported to be diagnostic of PCD; however, no supporting evidence of PCD was provided. In this study, we sought to confirm, or refute, the diagnosis of PCD in subjects with "ciliary inclusions" on EM. METHODS: Six subjects from five families with previous lab reports of "ciliary inclusions" on EMs of ciliated cells were identified and evaluated at a Genetic Disorders of Mucociliary Clearance Consortium site. We performed a detailed clinical history, nasal nitric oxide measurement, genetic testing including whole-exome sequencing (WES), and when possible, repeat ciliary EM study. RESULTS: Only one of six subjects had multiple and persistent clinical features congruent with PCD. No subject had situs inversus. Only one of six subjects had a very low nasal nitric oxide level. No "ciliary inclusions" were found in three subjects who had a repeat ciliary EM, and ciliary axonemal ultrastructures were normal. Genetic testing, including WES, was negative for PCD-causing genes, and for pathogenic variants in gene pathways that might cause "ciliary inclusions," such as ciliary biogenesis. CONCLUSION: "Ciliary Inclusions", in isolation, are not sufficient to diagnosis PCD. If seen, additional studies should be done to pursue an accurate diagnosis.


Assuntos
Cílios/ultraestrutura , Transtornos da Motilidade Ciliar/diagnóstico , Pré-Escolar , Transtornos da Motilidade Ciliar/genética , Transtornos da Motilidade Ciliar/metabolismo , Feminino , Testes Genéticos , Humanos , Lactente , Masculino , Mucosa Nasal/metabolismo , Óxido Nítrico/metabolismo , Sequenciamento Completo do Exoma
18.
Elife ; 82019 12 10.
Artigo em Inglês | MEDLINE | ID: mdl-31821146

RESUMO

Cilia and flagella are conserved eukaryotic organelles essential for cellular signaling and motility. Cilia dysfunctions cause life-threatening ciliopathies, many of which are due to defects in the transition zone (TZ), a complex structure of the ciliary base. Therefore, understanding TZ assembly, which relies on ordered interactions of multiprotein modules, is of critical importance. Here, we show that Drosophila Dzip1 and Fam92 form a functional module which constrains the conserved core TZ protein, Cep290, to the ciliary base. We identify cell type specific roles of this functional module in two different tissues. While it is required for TZ assembly in all Drosophila ciliated cells, it also regulates basal-body growth and docking to the plasma membrane during spermatogenesis. We therefore demonstrate a novel regulatory role for Dzip1 and Fam92 in mediating membrane/basal-body interactions and show that these interactions exhibit cell type specific functions in basal-body maturation and TZ organization.


Assuntos
Proteínas de Transporte de Cátions/metabolismo , Cílios/metabolismo , Proteínas de Drosophila/metabolismo , Drosophila/metabolismo , Alelos , Animais , Corpos Basais/metabolismo , Proteínas de Transporte/metabolismo , Proteínas de Transporte de Cátions/genética , Membrana Celular/metabolismo , Cílios/genética , Cílios/ultraestrutura , Drosophila/genética , Proteínas de Drosophila/genética , Flagelos/genética , Flagelos/metabolismo , Flagelos/ultraestrutura , Células Germinativas , Masculino , Proteínas Nucleares/metabolismo , Células Receptoras Sensoriais , Espermatogênese/fisiologia
19.
Cells ; 8(12)2019 12 11.
Artigo em Inglês | MEDLINE | ID: mdl-31835861

RESUMO

Primary ciliary dyskinesia (PCD) is a recessive heterogeneous disorder of motile cilia, affecting one per 15,000-30,000 individuals; however, the frequency of this disorder is likely underestimated. Even though more than 40 genes are currently associated with PCD, in the case of approximately 30% of patients, the genetic cause of the manifested PCD symptoms remains unknown. Because motile cilia are highly evolutionarily conserved organelles at both the proteomic and ultrastructural levels, analyses in the unicellular and multicellular model organisms can help not only to identify new proteins essential for cilia motility (and thus identify new putative PCD-causative genes), but also to elucidate the function of the proteins encoded by known PCD-causative genes. Consequently, studies involving model organisms can help us to understand the molecular mechanism(s) behind the phenotypic changes observed in the motile cilia of PCD affected patients. Here, we summarize the current state of the art in the genetics and biology of PCD and emphasize the impact of the studies conducted using model organisms on existing knowledge.


Assuntos
Transtornos da Motilidade Ciliar/genética , Modelos Animais de Doenças , Doenças Raras/metabolismo , Animais , Cílios/metabolismo , Cílios/ultraestrutura , Transtornos da Motilidade Ciliar/metabolismo , Redes Reguladoras de Genes , Predisposição Genética para Doença , Humanos
20.
Cell Rep ; 29(13): 4334-4348.e7, 2019 Dec 24.
Artigo em Inglês | MEDLINE | ID: mdl-31875544

RESUMO

In mammals, odorant receptors not only detect odors but also define the target in the olfactory bulb, where sensory neurons project to give rise to the sensory map. The odorant receptor is expressed at the cilia, where it binds odorants, and at the axon terminal. The mechanism of activation and function of the odorant receptor at the axon terminal is, however, still unknown. Here, we identify phosphatidylethanolamine-binding protein 1 as a putative ligand that activates the odorant receptor at the axon terminal and affects the turning behavior of sensory axons. Genetic ablation of phosphatidylethanolamine-binding protein 1 in mice results in a strongly disturbed olfactory sensory map. Our data suggest that the odorant receptor at the axon terminal of olfactory neurons acts as an axon guidance cue that responds to molecules originating in the olfactory bulb. The dual function of the odorant receptor links specificity of odor perception and axon targeting.


Assuntos
Axônios/metabolismo , Percepção Olfatória/fisiologia , Neurônios Receptores Olfatórios/metabolismo , Proteína de Ligação a Fosfatidiletanolamina/genética , Receptores Odorantes/genética , Animais , Axônios/ultraestrutura , Cálcio/metabolismo , Cílios/metabolismo , Cílios/ultraestrutura , Misturas Complexas/química , Embrião de Mamíferos , Regulação da Expressão Gênica , Células HEK293 , Humanos , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Odorantes/análise , Bulbo Olfatório/química , Bulbo Olfatório/metabolismo , Neurônios Receptores Olfatórios/ultraestrutura , Proteína de Ligação a Fosfatidiletanolamina/deficiência , Proteína de Ligação a Fosfatidiletanolamina/metabolismo , Ratos , Ratos Sprague-Dawley , Receptores Odorantes/metabolismo , Transdução de Sinais , Olfato/fisiologia
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