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1.
Zhonghua Yi Xue Yi Chuan Xue Za Zhi ; 36(10): 965-969, 2019 Oct 10.
Artigo em Chinês | MEDLINE | ID: mdl-31598937

RESUMO

OBJECTIVE: To explore the genetic basis for a family with non-syndromic autosomal recessive deafness. METHODS: The proband and her parents were subjected to physical and audiological examinations. With genomic DNA extracted from peripheral blood samples, next-generation sequencing was carried out using a panel for deafness genes. Suspected mutation was validated by Sanger sequencing and qPCR analysis of her parents. RESULTS: The proband presented bilateral severe sensorineural hearing loss at three days after birth. Her auditory threshold was 110-120 dBnHL but with absence of vestibular and retinal symptoms. Her brother also had deafness but her parents were normal. No abnormality was found upon physical examination of her family members, while audiological examination showed no middle ear or retrocochlear diseases. Next-generation sequencing identified compound heterozygous mutations of the MYO7A gene, including a previously known c.462C>A (p. Cys154Ter) and a novel EX43_46 Del, which were respectively derived from her mother and father. CONCLUSION: The compound heterozygous mutations of the MYO7A gene probably underlie the disease in this family. Our findings has enriched the mutation spectrum for non-syndromic autosomal recessive deafness 2.


Assuntos
Perda Auditiva Neurossensorial/genética , Miosinas/genética , Feminino , Sequenciamento de Nucleotídeos em Larga Escala , Humanos , Masculino , Mutação , Linhagem
2.
PLoS Genet ; 15(10): e1008279, 2019 10.
Artigo em Inglês | MEDLINE | ID: mdl-31603892

RESUMO

Muscle development and lipid accumulation in muscle critically affect meat quality of livestock. However, the genetic factors underlying myofiber-type specification and intramuscular fat (IMF) accumulation remain to be elucidated. Using two independent intercrosses between Western commercial breeds and Korean native pigs (KNPs) and a joint linkage-linkage disequilibrium analysis, we identified a 488.1-kb region on porcine chromosome 12 that affects both reddish meat color (a*) and IMF. In this critical region, only the MYH3 gene, encoding myosin heavy chain 3, was found to be preferentially overexpressed in the skeletal muscle of KNPs. Subsequently, MYH3-transgenic mice demonstrated that this gene controls both myofiber-type specification and adipogenesis in skeletal muscle. We discovered a structural variant in the promotor/regulatory region of MYH3 for which Q allele carriers exhibited significantly higher values of a* and IMF than q allele carriers. Furthermore, chromatin immunoprecipitation and cotransfection assays showed that the structural variant in the 5'-flanking region of MYH3 abrogated the binding of the myogenic regulatory factors (MYF5, MYOD, MYOG, and MRF4). The allele distribution of MYH3 among pig populations worldwide indicated that the MYH3 Q allele is of Asian origin and likely predates domestication. In conclusion, we identified a functional regulatory sequence variant in porcine MYH3 that provides novel insights into the genetic basis of the regulation of myofiber type ratios and associated changes in IMF in pigs. The MYH3 variant can play an important role in improving pork quality in current breeding programs.


Assuntos
Adipogenia/genética , Proteínas do Citoesqueleto/genética , Fibras Musculares Esqueléticas/metabolismo , Músculo Esquelético/crescimento & desenvolvimento , Miosinas/genética , Tecido Adiposo/crescimento & desenvolvimento , Tecido Adiposo/metabolismo , Animais , Cruzamento , Regulação da Expressão Gênica , Estudo de Associação Genômica Ampla , Genótipo , Carne , Camundongos , Camundongos Transgênicos , Músculo Esquelético/metabolismo , Cadeias Pesadas de Miosina/genética , Motivos de Nucleotídeos , Sus scrofa/genética , Sus scrofa/metabolismo , Suínos
3.
Genes (Basel) ; 10(8)2019 07 31.
Artigo em Inglês | MEDLINE | ID: mdl-31370293

RESUMO

: Hearing loss (HL) is a common sensory disorder affecting over 5% of the global population. The etiology underlying HL includes congenital and acquired causes; genetic factors are the main cause in over 50% of congenital cases. Pathogenic variants in the GJB2 gene are a major cause of congenital non-syndromic hearing loss (NSHL), while their distribution is highly heterogeneous in different populations. To the best of our knowledge, there is no data regarding the genetic etiologies of HL in Peru. In this study, we screened 133 Peruvian families with NSHL living in Lima. We sequenced both exons of the GJB2 gene for all probands. Seven probands with familial NSHL that remained negative for GJB2 variants underwent whole genome sequencing (WGS). We identified biallelic pathogenic variants in GJB2 in 43 probands; seven were heterozygous for only one allele. The c.427C>T variant was the most common pathogenic variant followed by the c.35delG variant. WGS revealed three novel variants in MYO15A in two probands, one of them was predicted to affect splicing and the others produce a premature stop codon. The Peruvian population showed a complex profile for genetic variants in the GJB2 gene, this particular profile might be a consequence of the admixture history in Peru.


Assuntos
Surdez/genética , Polimorfismo de Nucleotídeo Único , Conexinas/genética , Humanos , Taxa de Mutação , Miosinas/genética , Linhagem , Peru
4.
BMC Med Genet ; 20(1): 130, 2019 07 25.
Artigo em Inglês | MEDLINE | ID: mdl-31345173

RESUMO

BACKGROUND: Oculocutaneous albinism (OCA) is a group of rare genetically heterogeneous disorders. The present study aimed to identify the genetic cause of a Chinese Han family with non-syndromic oculocutaneous albinism (OCA). CASE PRESENTATION: Here, we report an 11-month-old male proband from a Chinese Han non-consanguineous family, who presented with milky skin, yellow white hair, nystagmus, astigmatism, and hypermetropia. We performed the targeted next-generation sequencing (NGS) on the proband and identified two novel compound heterozygous variants (c.1865 T > C (p.Leu622Pro) and exons 17-21 deletion) in OCA2 gene associated with OCA type 2 (OCA2, OMIM 203200). Meanwhile, a previously reported heterozygous mutation (c.4805G > A) in MYO7 gene related with Usher syndrome type 1B was found. The online tools SIFT, PolyPhen-2, and Mutation Taster predicted variant c.1865 T > C was probably damaging. The residue p.Leu622 was in a highly conserved region among species by CLUSTALW. Three-dimensional homology model with I-TASSER indicated that p.Leu622Pro variant disturbed the formation of the α-helix, resulting in a random coil structure. The gross deletion (exons 17-21) in OCA2 gene has was not been reported previously. These two novel variants in OCA2 gene were inherited from each parent respectively, after verification by Sanger sequencing and quantitative PCR (qPCR) in the family. CONCLUSIONS: This study indicates the two novel compound heterozygous mutations in OCA2 gene may be responsible for clinical manifestations of OCA2. It expands the mutation spectrum of OCA2 gene and is helpful to screen for large deletions with targeted NGS protocol in monogenic disease. It also assists the genetic counselling, carrier screening and personalized healthcare of the disease.


Assuntos
Albinismo Oculocutâneo/genética , Grupo com Ancestrais do Continente Asiático/genética , Predisposição Genética para Doença/genética , Proteínas de Membrana Transportadoras/genética , Mutação , Albinismo Oculocutâneo/fisiopatologia , Éxons , Aconselhamento Genético , Heterozigoto , Sequenciamento de Nucleotídeos em Larga Escala , Humanos , Lactente , Masculino , Modelos Moleculares , Miosinas/genética , Conformação Proteica , Análise de Sequência de Proteína , Deleção de Sequência
5.
Int J Pediatr Otorhinolaryngol ; 125: 128-132, 2019 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-31301639

RESUMO

BACKGROUND: Millions of people around the world are plagued by hearing loss. More than 50% of congenital or pre-lingual deafness is associated with genetic factors and has highly genetic heterogeneity. To date, although hundreds of genes have been found to be implicated in non-syndromic deafness, there are still lots of genes or loci that we need to verify. METHODS: In this study, we performed target sequencing and Sanger sequencing in a Kazakh consanguineous family with autosomal recessive non-syndromic hearing loss. Following that, functional and structural studies predicted the pathogenic effect of novel mutations by use of the online tools. RESULTS: We identified a novel homozygous mutation p.R3191C in MYO15A gene causing deafness in this family. The mutation p.R3191C co-segregated with the disease phenotype in this family and was not present in any public databases. Automatic tools predict that the novel mutation makes a great impact on the function and structure of MYO15A protein. CONCLUSIONS: This is a novel mutation of MYO15A causing deafness and also the first report of MYO15A mutations causing deafness in the Kazakh families. This finding expanded the spectrum of MYO15A mutations, making it more precise for future genetic diagnosis in patients with deafness.


Assuntos
Grupo com Ancestrais do Continente Asiático/genética , Perda Auditiva/etnologia , Perda Auditiva/genética , Mutação/genética , Miosinas/genética , China , Feminino , Genes Recessivos , Homozigoto , Humanos , Masculino , Linhagem , Fenótipo
6.
Nat Cell Biol ; 21(8): 933-939, 2019 08.
Artigo em Inglês | MEDLINE | ID: mdl-31358965

RESUMO

Actomyosin networks, the cell's major force production machineries, remodel cellular membranes during myriad dynamic processes1,2 by assembling into various architectures with distinct force generation properties3,4. While linear and branched actomyosin architectures are well characterized in cell-culture and cell-free systems3, it is not known how actin and myosin networks form and function to remodel membranes in complex three-dimensional mammalian tissues. Here, we use four-dimensional spinning-disc confocal microscopy with image deconvolution to acquire macromolecular-scale detail of dynamic actomyosin networks in exocrine glands of live mice. We address how actin and myosin organize around large membrane-bound secretory vesicles and generate the forces required to complete exocytosis5-7. We find that actin and non-muscle myosin II (NMII) assemble into previously undescribed polyhedral-like lattices around the vesicle membrane. The NMII lattice comprises bipolar minifilaments8-10 as well as non-canonical three-legged configurations. Using photobleaching and pharmacological perturbations in vivo, we show that actomyosin contractility and actin polymerization together push on the underlying vesicle membrane to overcome the energy barrier and complete exocytosis7. Our imaging approach thus unveils a force-generating actomyosin lattice that regulates secretion in the exocrine organs of live animals.


Assuntos
Actomiosina/metabolismo , Exocitose/fisiologia , Contração Muscular/fisiologia , Miosinas/metabolismo , Citoesqueleto de Actina/metabolismo , Actomiosina/genética , Animais , Membrana Celular/metabolismo , Exocitose/genética , Camundongos Transgênicos , Microscopia Confocal/métodos , Miosinas/genética , Vesículas Secretórias/metabolismo
7.
Eur J Med Genet ; 62(8): 103701, 2019 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-31195167

RESUMO

Klippel-Feil syndrome (KFS) is an exceedingly rare constitutional disorder in which a paucity of knowledge exists about the disease and its associated morbidity and mortality. We present a 4-year-old male with KFS, who notably was also diagnosed with large-cell anaplastic medulloblastoma. We evaluated the genetic basis of co-occurring KFS and medulloblastoma and the role of MYO18B as related to medulloblastoma. Constitutional and somatic variant and copy number analyses were performed from DNA-based exome studies, along with RNA-sequencing of tumor tissue, to elucidate the genetic etiology of the co-existing disease states. We identified novel constitutional compound heterozygous frameshift variants (NM_032608.5: p.Leu2257SerfsTer16 and p.Arg2220SerfsTer74) each encoding a premature stop of translation in MYO18B, consistent with a diagnosis of KFS. We did not identify any somatic variants of known relevance or disease-relevant therapeutic targets in the tumor. The somatic copy number profile was suggestive of Group 3γ medulloblastoma. Relative to pediatric brain tumors, medulloblastoma, particularly, Group 3, had increased gene expression of MYO18B. In summary, coexisting constitutional and somatic diagnoses in this patient enabled the elucidation of the genetic etiology of KFS and provided support for the role of MYO18B in tumor suppression.


Assuntos
Síndrome de Klippel-Feil/genética , Meduloblastoma/genética , Miosinas/genética , Proteínas Supressoras de Tumor/genética , Sequenciamento Completo do Exoma , Pré-Escolar , Exoma/genética , Mutação da Fase de Leitura/genética , Heterozigoto , Humanos , Síndrome de Klippel-Feil/complicações , Síndrome de Klippel-Feil/diagnóstico , Síndrome de Klippel-Feil/patologia , Masculino , Meduloblastoma/complicações , Meduloblastoma/diagnóstico , Meduloblastoma/patologia
8.
BMC Genomics ; 20(1): 432, 2019 May 28.
Artigo em Inglês | MEDLINE | ID: mdl-31138127

RESUMO

BACKGROUND: Accompanied with rapid growth and high density aquaculture, gibel carp has been seriously threatened by Carassius auratus herpesvirus (CaHV) since 2012. In previous study, distinct CaHV resistances and immune responses were revealed in the diseased individuals of three gibel carp gynogenetic clones (A+, F and H). However, little is known about the gene expression changes in the survivors after CaHV challenge, particularly their differences of innate and adaptive immune system between susceptible clone and resistant clone. RESULTS: We firstly confirmed the CaHV carrier state in the survivors of three gibel carp clones after CaHV challenge by evaluating the abundances of five CaHV genes. The assay of viral loads indicated the resistant clone H possessed not only stronger resistance but also higher tolerance to CaHV. Then, 2818, 4047 and 3323 differentially expressed unigenes (DEUs) were screened from the head-kidney transcriptome profiles of survivors compared with controls from clone A+, F and H. GO and KEGG analysis suggested that a persistent immune response might sustain in resistant clone H and F, while susceptible clone A+ had a long-term impact on the circulatory system which was consistent with the major symptoms of bleeding caused by CaHV. Among the top 30 enriched pathways of specifically up-regulated DEUs in respective clones, 26, 7 and 15 pathways in clone H, F and A+ were associated with infections, diseases, or immune-related pathways respectively. In addition, 20 pathways in clone F belonged to "metabolism" or "biogenesis", and 7 pathways involved in "circulatory system" were enriched in clone A+. Significantly, we revealed the differential expression changes of IFN system genes and immunoglobulin (Ig) genes among the survivors of three clones. Finally, myosins and Igs were identified as co-expression modules which were positively or negatively correlated to CaHV viral loads respectively. CONCLUSIONS: Our results revealed the common and distinct gene expression changes in immune and circulatory system in the survivors of three gibel carp gynogenetic clones with different CaHV resistances. The current study represents a paradigm of differential innate and adaptive immune reactions in teleost, and will be beneficial to the disease-resistance breeding of gibel carp.


Assuntos
Carpas/genética , Carpas/imunologia , Doenças dos Peixes/imunologia , Doenças dos Peixes/virologia , Infecções por Herpesviridae/veterinária , Imunidade Adaptativa/genética , Animais , Carpas/metabolismo , Carpas/virologia , Doenças dos Peixes/genética , Doenças dos Peixes/metabolismo , Genes de Imunoglobulinas , Herpesviridae , Infecções por Herpesviridae/genética , Infecções por Herpesviridae/imunologia , Infecções por Herpesviridae/metabolismo , Imunidade Inata/genética , Interferons/metabolismo , Miosinas/genética , Transdução de Sinais
9.
Can J Vet Res ; 83(2): 142-148, 2019 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-31097876

RESUMO

Bilateral deafness with concurrent vestibular dysfunction was first reported in the Doberman pinscher in 1980. Here, we identify a coding mutation in the MYO7A gene that is perfectly associated with the disorder. The lack of visual deficits in affected dogs suggests that, like rodents but unlike humans, MYO7A is not required for retinal function. DNA testing of the mutation will enable dog breeders to manage the incidence of this genetic defect.


Assuntos
Surdez/veterinária , Doenças do Cão/genética , Predisposição Genética para Doença , Mutação de Sentido Incorreto , Miosinas/genética , Doenças Vestibulares/veterinária , Animais , Estudos de Casos e Controles , DNA/genética , Surdez/genética , Cães , Regulação da Expressão Gênica , Estudo de Associação Genômica Ampla , Genótipo , Miosinas/metabolismo , Doenças Vestibulares/genética , Sequenciamento Completo do Genoma
10.
PLoS Genet ; 15(5): e1008083, 2019 05.
Artigo em Inglês | MEDLINE | ID: mdl-31116733

RESUMO

How biochemical and mechanical information are integrated during tissue development is a central question in morphogenesis. In many biological systems, the PIX-GIT complex localises to focal adhesions and integrates both physical and chemical information. We used Drosophila melanogaster egg chamber formation to study the function of PIX and GIT orthologues (dPix and Git, respectively), and discovered a central role for this complex in controlling myosin activity and epithelial monolayering. We found that Git's focal adhesion targeting domain mediates basal localisation of this complex to filament structures and the leading edge of migrating cells. In the absence of dpix and git, tissue disruption is driven by contractile forces, as reduction of myosin activators restores egg production and morphology. Further, dpix and git mutant eggs closely phenocopy defects previously reported in pak mutant epithelia. Together, these results indicate that the dPix-Git complex controls egg chamber morphogenesis by controlling myosin contractility and Pak kinase downstream of focal adhesions.


Assuntos
Transportadores de Cassetes de Ligação de ATP/genética , Proteínas de Drosophila/genética , Drosophila melanogaster/genética , Proteínas Ativadoras de GTPase/genética , Morfogênese/genética , Miosinas/genética , Transportadores de Cassetes de Ligação de ATP/metabolismo , Animais , Movimento Celular , Proteínas de Drosophila/metabolismo , Drosophila melanogaster/citologia , Drosophila melanogaster/crescimento & desenvolvimento , Drosophila melanogaster/metabolismo , Células Epiteliais/citologia , Células Epiteliais/metabolismo , Feminino , Adesões Focais/metabolismo , Adesões Focais/ultraestrutura , Proteínas Ativadoras de GTPase/metabolismo , Perfilação da Expressão Gênica , Regulação da Expressão Gênica no Desenvolvimento , Mecanotransdução Celular , Miosinas/metabolismo , Zigoto/citologia , Zigoto/crescimento & desenvolvimento , Zigoto/metabolismo , Quinases Ativadas por p21/genética , Quinases Ativadas por p21/metabolismo
11.
BMC Med Genet ; 20(1): 60, 2019 04 05.
Artigo em Inglês | MEDLINE | ID: mdl-30953472

RESUMO

BACKGROUND: MYO15A variants are responsible for human non-syndromic autosomal recessive deafness (DFNB3). The majority of MYO15A variants are associated with a congenital severe-to-profound hearing loss phenotype, except for MYO15A variants in exon 2, which cause a milder auditory phenotype, suggesting a genotype-phenotype correlation of MYO15A. However, MYO15A variants not in exon 2 related to a milder phenotype have also been reported, indicating that the genotype-phenotype correlation of MYO15A is complicated. This study aimed to provide more cases of MYO15A variation with diverse phenotypes to analyse this complex correlation. METHODS: Fifteen Chinese autosomal recessive non-syndromic hearing loss (ARNSHL) individuals with MYO15A variants (8 males and 7 females) from 14 unrelated families, identified by targeted gene capture of 127 known candidate deafness genes, were recruited. Additionally, we conducted a review of the literature to further analyses all reported MYO15A genotype-phenotype relationships worldwide. RESULTS: We identified 16 novel variants and 12 reported pathogenic MYO15A variants in 15 patients, two of which presented with a milder phenotype. Interestingly, one of these cases carried two reported pathogenic variants in exon 2, while the other carried two novel variants not in exon 2. Based on our literature review, MYO15A genotype-phenotype correlation analysis showed that almost all domains were reported to be correlated with a milder phenotype. However, variants in the N-terminal domain were more likely to cause a milder phenotype. Using next-generation sequencing (NGS), we also found that the number of known MYO15A variants with milder phenotypes in Southeast Asia has increased in recent years. CONCLUSION: Our work extended the MYO15A variant spectrum, enriched our knowledge of auditory phenotypes, and tried to explore the genotype-phenotype correlation in different populations in order to investigate the cause of the complex MYO15A genotype-phenotype correlation.


Assuntos
Genes Recessivos , Genótipo , Perda Auditiva/genética , Miosinas/genética , Fenótipo , Adolescente , Adulto , Pré-Escolar , China , Grupos Étnicos , Feminino , Humanos , Lactente , Masculino , Pessoa de Meia-Idade , Adulto Jovem
12.
Res Vet Sci ; 124: 270-279, 2019 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-31003009

RESUMO

In ungulates the stability of the fetlock joint is dependent on several muscles, which are exposed to high stress and strain. Among those muscles, the proximal sesamoidean ligament or PSL (also known as the suspensory ligament or Ruini's elasto-tendinous organ) is organized at birth in layers of muscle fibres alternated with abundant tendinous tissue that, during the postnatal development, becomes the predominant tissue. In this study we analysed the PSL of the sheep at the age of 1, 30 and 180 days and determined the expression of several genes which either (a) are markers of muscle fibre growth and maturation, or (b) play a role as signal molecules. We observed an accelerated maturation, as indicated by the transition of MyHC isoform expression towards the slow isoforms and a reduced regenerative potential indicated by the low Pax7 expression and the altered Wnt signalling. We also found a specific myogenic expression pattern of MyoD, Myf5 and Myogenin in the developing PSL and high mRNA levels of specific fibrogenic factors, as TGF-ß1, that, undoubtedly, stimulate the growth of connective tissue. Our observations confirmed, at molecular level, the peculiarity of the fast involution observed in PSL a muscle that undergoes a very specific active differentiation process during early development, which implies myofibres involution and their replacement with connective tissue.


Assuntos
Ligamentos/crescimento & desenvolvimento , Desenvolvimento Muscular/genética , Miosinas/genética , Carneiro Doméstico/genética , Fatores Etários , Animais , Diferenciação Celular , Fatores de Regulação Miogênica , Miosinas/metabolismo , Isoformas de Proteínas/genética , Isoformas de Proteínas/metabolismo , Ossos Sesamoides , Carneiro Doméstico/crescimento & desenvolvimento
13.
J Neuroinflammation ; 16(1): 77, 2019 Apr 10.
Artigo em Inglês | MEDLINE | ID: mdl-30971272

RESUMO

BACKGROUND: During the acute stroke phase, neutrophils from the peripheral blood are first to arrive in the ischemic brain, which then attracts other immune cells that exacerbate neuroinflammation in the ischemic tissue. Myosin1f was reported to specifically mediate neutrophil migration in the peripheral tissues, but whether it plays a critical role in the neuroinflammatory response after ischemic stroke remains unknown. In this study, we aim to test the hypothesis that myosin1f-mediated neutrophil migration is critical in acute neuroinflammation induced by ischemic stroke. METHODS: Myosin1f -/- and wild type (WT) mice were subjected to transient middle cerebral artery occlusion (MCAO). To determine which cells determine myosin1f's transmigration ability, bone marrow transplantation, neutrophil depletion, and adoptive neutrophil transfer were performed. The myosin1f RNA level was assessed in peripheral neutrophils by reverse transcription polymerase chain reaction (RT-PCR) at 1 day and 3 days after stroke. The infiltrating neutrophils were quantified by immunofluorescence staining and FACS at 72 h after reperfusion. RESULTS: The myosin1f -/- mice had significantly smaller infarctions than the myosin1f +/+ mice. Bone marrow transplantation from myosin1f -/- mice to recipient mice also had smaller infarctions compared to animals receiving bone marrow from myosin1f +/+ mice. By performing neutrophil depletion and adoptive transfer, we confirmed that myosin1f acts mainly in circulating neutrophils. RT-PCR showed that myosin1f gene expression was increased in the circulating blood neutrophils at 3 days after ischemia. The confocal immunostaining and FACS results confirmed that fewer neutrophils infiltrated into the ischemic brain in myosin1f -/- mice compared to WT mice. CONCLUSIONS: Myosin1f determines neutrophil migration into the ischemic hemisphere, which directly affects stroke outcome.


Assuntos
Lesões Encefálicas/etiologia , Movimento Celular/genética , Encefalite/etiologia , Infarto da Artéria Cerebral Média/complicações , Miosinas/metabolismo , Neutrófilos/fisiologia , Animais , Transplante de Medula Óssea/métodos , Lesões Encefálicas/patologia , Lesões Encefálicas/cirurgia , Antígeno CD11b/metabolismo , Modelos Animais de Doenças , Encefalite/cirurgia , Citometria de Fluxo , Regulação da Expressão Gênica/genética , Antígenos Comuns de Leucócito/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Miosinas/genética , Peroxidase/metabolismo , RNA Mensageiro/metabolismo , Índice de Gravidade de Doença , Fatores de Tempo
14.
Nat Commun ; 10(1): 1249, 2019 03 19.
Artigo em Inglês | MEDLINE | ID: mdl-30890704

RESUMO

Phagocytosis of invading pathogens or cellular debris requires a dramatic change in cell shape driven by actin polymerization. For antibody-covered targets, phagocytosis is thought to proceed through the sequential engagement of Fc-receptors on the phagocyte with antibodies on the target surface, leading to the extension and closure of the phagocytic cup around the target. We find that two actin-dependent molecular motors, class 1 myosins myosin 1e and myosin 1f, are specifically localized to Fc-receptor adhesions and required for efficient phagocytosis of antibody-opsonized targets. Using primary macrophages lacking both myosin 1e and myosin 1f, we find that without the actin-membrane linkage mediated by these myosins, the organization of individual adhesions is compromised, leading to excessive actin polymerization, slower adhesion turnover, and deficient phagocytic internalization. This work identifies a role for class 1 myosins in coordinated adhesion turnover during phagocytosis and supports a mechanism involving membrane-cytoskeletal crosstalk for phagocytic cup closure.


Assuntos
Actinas/metabolismo , Adesão Celular/fisiologia , Miosina Tipo I/metabolismo , Miosinas/metabolismo , Fagocitose/fisiologia , Actinas/ultraestrutura , Animais , Células da Medula Óssea , Membrana Celular/metabolismo , Membrana Celular/ultraestrutura , Citoesqueleto/metabolismo , Citoesqueleto/ultraestrutura , Feminino , Microscopia Intravital , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Microscopia Eletrônica , Microscopia de Fluorescência , Miosina Tipo I/genética , Miosinas/genética , Cultura Primária de Células , Células RAW 264.7 , Receptores Fc/metabolismo , Receptores Fc/ultraestrutura , Imagem com Lapso de Tempo
15.
Int J Pediatr Otorhinolaryngol ; 120: 166-172, 2019 May.
Artigo em Inglês | MEDLINE | ID: mdl-30826590

RESUMO

INTRODUCTION: MYO7A gene has been shown to be associated with Usher syndrome 1B and nonsyndromic deafness. Although a lot of mutations have been reported in MYO7A gene, novel MYO7A mutations are continuously to be identified. METHODS: Targeted next generation sequencing was performed on the two unrelated patients with Usher syndrome 1B and nonsyndromic deafness respectively. The identified mutations from targeted next generation sequencing were further validated by Sanger sequencing, and analyzed by bioinformatics tools, like SIFT, Polyphen-2, PyMOL, I-Mutant 2.0 and so on. RESULTS: By analyzing the sequencing data of these two patients, four novel MYO7A mutations were revealed: (i) MYO7A p.Tyr560Ser and p.Ala2039Pro were associated with Usher syndrome 1B. (ii) MYO7A c.2187 + 2_+8 delTGAGCAC and p.Leu728Pro were related to nonsyndromic hearing loss. These mutations were further proved to be possibly disease-causing by segregation analysis, conservation analysis and bioinformatics tools. CONCLUSIONS: Four novel MYO7A mutations were identified in the present study. These findings provided new evidence for the genetic counseling of Usher syndrome 1B and nonsyndromic deafness.


Assuntos
Surdez/genética , Mutação , Miosinas/genética , Síndromes de Usher/genética , Biologia Computacional , Feminino , Sequenciamento de Nucleotídeos em Larga Escala , Humanos
16.
J Biol Chem ; 294(18): 7202-7218, 2019 05 03.
Artigo em Inglês | MEDLINE | ID: mdl-30737279

RESUMO

Whereas myosin 18B (Myo18B) is known to be a critical sarcomeric protein, the function of myosin 18A (Myo18A) is unclear, although it has been implicated in cell motility and Golgi shape. Here, we show that homozygous deletion (homozygous tm1a, tm1b, or tm1d alleles) of Myo18a in mouse is embryonic lethal. Reminiscent of Myo18b, Myo18a was highly expressed in the embryo heart, and cardiac-restricted Myo18a deletion in mice was embryonic lethal. Surprisingly, using Western blot analysis, we were unable to detect the known isoforms of Myo18A, Myo18Aα and Myo18Aß, in mouse heart using a custom C-terminal antibody. However, alternative anti-Myo18A antibodies detected a larger than expected protein, and RNA-Seq analysis indicated that a novel Myo18A transcript is expressed in mouse ventricular myocytes (and human heart). Cloning and sequencing revealed that this cardiac isoform, denoted Myo18Aγ, lacks the PDZ-containing N terminus of Myo18Aα but includes an alternative N-terminal extension and a long serine-rich C terminus. EGFP-tagged Myo18Aγ expressed in ventricular myocytes localized to the level of A-bands in sarcomeres, and Myo18a knockout embryos at day 10.5 exhibited disorganized sarcomeres with wavy thick filaments. We additionally generated myeloid-restricted Myo18a knockout mice to investigate the role of Myo18A in nonmuscle cells, exemplified by macrophages, which express more Myo18Aß than Myo18Aα, but no defects in cell shape, motility, or Golgi shape were detected. In summary, we have identified a previously unrecognized sarcomere component, a large novel isoform (denoted Myo18Aγ) of Myo18A. Thus, both members of class XVIII myosins are critical components of cardiac sarcomeres.


Assuntos
Miocárdio/metabolismo , Miosinas/metabolismo , Sarcômeros/metabolismo , Animais , Deleção de Genes , Genes Letais , Proteínas de Fluorescência Verde/metabolismo , Humanos , Camundongos , Camundongos Knockout , Miosinas/genética , Isoformas de Proteínas/genética , Isoformas de Proteínas/metabolismo
17.
Plant Physiol ; 179(4): 1537-1555, 2019 04.
Artigo em Inglês | MEDLINE | ID: mdl-30705068

RESUMO

In plants, cellulose is synthesized at the cell surface by plasma membrane (PM)-localized cellulose synthase (CESA) complexes (CSCs). The molecular and cellular mechanisms that underpin delivery of CSCs to the PM, however, are poorly understood. Cortical microtubules have been shown to interact with CESA-containing compartments and mark the site for CSC delivery, but are not required for the delivery itself. Here, we demonstrate that myosin XI and the actin cytoskeleton mediate CSC delivery to the PM by coordinating the exocytosis of CESA-containing compartments. Measurement of cellulose content indicated that cellulose biosynthesis was significantly reduced in a myosin xik xi1 xi2 triple-knockout mutant. By combining genetic and pharmacological disruption of myosin activity with quantitative live-cell imaging, we observed decreased abundance of PM-localized CSCs and reduced delivery rate of CSCs in myosin-deficient cells. These phenotypes correlated with a significant increase in failed vesicle secretion events at the PM as well as an abnormal accumulation of CESA-containing compartments at the cell cortex. Through high-resolution spatiotemporal assays of cortical vesicle behavior, we identified defects in CSC vesicle tethering and fusion at the PM. Furthermore, disruption of myosin activity reduced the delivery of several other secretory markers to the PM and reduced constitutive and receptor-mediated endocytosis. These findings reveal a previously undescribed role for myosin in vesicle secretion and cellulose production at the cytoskeleton-PM-cell wall nexus.


Assuntos
Proteínas de Arabidopsis/fisiologia , Arabidopsis/metabolismo , Exocitose , Glucosiltransferases/metabolismo , Miosinas/fisiologia , Arabidopsis/citologia , Arabidopsis/genética , Proteínas de Arabidopsis/genética , Proteínas de Arabidopsis/metabolismo , Membrana Celular , Celulose/metabolismo , Citoplasma/metabolismo , Técnicas de Inativação de Genes , Modelos Moleculares , Miosinas/genética , Miosinas/metabolismo
18.
Nat Commun ; 10(1): 427, 2019 01 25.
Artigo em Inglês | MEDLINE | ID: mdl-30683875

RESUMO

Adeno-associated virus (AAV) has been successfully used to deliver gene therapy to improve auditory function in mouse models of hereditary hearing loss. Many forms of hereditary hearing loss have mutations which affect the cochlear hair cells, the mechanosensory cells which allow for sound detection and processing. While most conventional AAVs infect inner hair cells (IHCs) with various efficiencies, they infect outer hair cells (OHCs) and supporting cells at lower levels in the cochlea. Here we examine the infection patterns of two synthetic AAVs (AAV2.7m8 and AAV8BP2) in the mouse inner ear. AAV2.7m8 infects both IHCs and OHCs with high efficiency. In addition, AAV2.7m8 infects inner pillar cells and inner phalangeal cells with high efficiency. Our results suggest that AAV2.7m8 is an excellent viral vector for inner ear gene therapy targeting cochlear hair cells and supporting cells, and it will likely greatly expand the potential applications for inner ear gene therapy.


Assuntos
Dependovirus/genética , Terapia Genética/métodos , Vetores Genéticos/metabolismo , Células Ciliadas Auditivas Internas/metabolismo , Perda Auditiva Neurossensorial/terapia , Miosinas/genética , Animais , Animais Recém-Nascidos , Dependovirus/metabolismo , Modelos Animais de Doenças , Expressão Gênica , Genes Reporter , Vetores Genéticos/química , Proteínas de Fluorescência Verde/genética , Proteínas de Fluorescência Verde/metabolismo , Células Ciliadas Auditivas Internas/patologia , Células Ciliadas Auditivas Externas/metabolismo , Células Ciliadas Auditivas Externas/patologia , Audição/genética , Perda Auditiva Neurossensorial/genética , Perda Auditiva Neurossensorial/metabolismo , Perda Auditiva Neurossensorial/patologia , Camundongos , Miosinas/metabolismo
19.
PLoS Genet ; 15(1): e1007955, 2019 01.
Artigo em Inglês | MEDLINE | ID: mdl-30699121

RESUMO

The Drosophila protocadherins Dachsous and Fat regulate growth and tissue polarity by modulating the levels, membrane localization and polarity of the atypical myosin Dachs. Localization to the apical junctional membrane is critical for Dachs function, and the adapter protein Vamana/Dlish and palmitoyl transferase Approximated are required for Dachs membrane localization. However, how Dachs levels are regulated is poorly understood. Here we identify the early girl gene as playing an essential role in Fat signaling by limiting the levels of Dachs protein. early girl mutants display overgrowth of the wings and reduced cross vein spacing, hallmark features of mutations affecting Fat signaling. Genetic experiments reveal that it functions in parallel with Fat to regulate Dachs. early girl encodes an E3 ubiquitin ligase, physically interacts with Dachs, and regulates its protein stability. Concomitant loss of early girl and approximated results in accumulation of Dachs and Vamana in cytoplasmic punctae, suggesting that it also regulates their trafficking to the apical membrane. Our findings establish a crucial role for early girl in Fat signaling, involving regulation of Dachs and Vamana, two key downstream effectors of this pathway.


Assuntos
Aciltransferases/genética , Moléculas de Adesão Celular/genética , Proteínas de Drosophila/genética , Ubiquitina-Proteína Ligases/genética , Animais , Polaridade Celular/genética , Drosophila melanogaster/genética , Humanos , Proteínas de Membrana/genética , Mutação , Miosinas/genética , Transporte Proteico/genética , Transdução de Sinais , Asas de Animais/crescimento & desenvolvimento
20.
Gene ; 691: 45-55, 2019 Apr 05.
Artigo em Inglês | MEDLINE | ID: mdl-30611842

RESUMO

Myosins are a large family of actin filament-based motor proteins with a broad range of functions such as intracellular membrane trafficking, endocytosis, exocytosis, organellar transport, growth cone motility, cytokinesis, and cell locomotion. They are found in many organisms from fungi to humans. The myosin gene family in Bombyx mori is poorly studied, even though the molecular functions of these genes in vertebrates and insects, such as Drosophila, are well known. We identified 16 myosin genes from B. mori and identified the myosin genes in 12 vertebrates, eight insects, three nematodes, and seven protozoa. The number of myosin genes in vertebrates is double the number in invertebrates. The number of myosin isoforms in classes I and II is larger in vertebrates compared to invertebrates. B. mori myosin genes can be classified into 11 classes. Compared to B. mori, some myosin classes are not present in other insects. Classes I, II, XVIII, and XXI appear to be important for insect survival because they are conserved among nine insects. The relatively large sizes of B. mori myosin genes are due to their longer introns. Reverse transcription PCR (RT-PCR) and quantitative real-time PCR (qRT-PCR) analysis demonstrated that many B. mori myosin genes have tissue-specific expression and exhibit temporal-specific activity during metamorphosis. These data provide insights into evolutionary and functional aspects of B. mori myosin genes that could be useful for the study of homologous myosins in other Lepidoptera species.


Assuntos
Bombyx/crescimento & desenvolvimento , Bombyx/genética , Miosinas/genética , Sequenciamento Completo do Genoma/métodos , Animais , Mapeamento Cromossômico , Sequência Conservada , Evolução Molecular , Perfilação da Expressão Gênica , Regulação da Expressão Gênica no Desenvolvimento , Proteínas de Insetos/classificação , Proteínas de Insetos/genética , Família Multigênica , Miosinas/classificação , Especificidade de Órgãos , Filogenia , Vertebrados/genética
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