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1.
Plant Sci ; 303: 110753, 2021 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-33487341

RESUMO

Dynein light chain (DLC) proteins are an important component of dynein complexes, which are widely distributed in plants and animals and involved in a variety of cellular processes. The functions of DLC genes in plant chilling stress remain unclear. In this study, we isolated a DLC gene from tomato, designated SlLC6D. Promoter analysis revealed many cis-elements involved in abiotic stress in the SlLC6D promoter. Expression of SlLC6D was induced by heat and salt stress, and inhibited by polyethylene glycol and chilling stress. Knockdown of SlLC6D in tomato exhibited low relative electrolyte leakage, malondialdehyde content, and reactive oxygen species (ROS) accumulation under chilling stress. The content of proline and activities of superoxide dismutase and peroxidase in knockdown lines were higher than in the wild type and overexpression lines during chilling stress. The high transcript abundances of three cold-responsive genes were detected in knockdown lines in response to chilling stress. Seedling growth of knockdown lines was significantly higher than that of the wild type and overexpression lines under chilling stress. These results suggest that SlLC6D is a negative regulator of chilling stress tolerance, possibly by regulating ROS contents and the ICE1-CBF-COR pathway.


Assuntos
Dineínas/genética , Genes de Plantas/genética , Lycopersicon esculentum/genética , Proteínas de Plantas/genética , Resposta ao Choque Frio , Sequência Conservada/genética , Dineínas/metabolismo , Dineínas/fisiologia , Genes de Plantas/fisiologia , Lycopersicon esculentum/metabolismo , Lycopersicon esculentum/fisiologia , Malondialdeído/metabolismo , Proteínas de Plantas/metabolismo , Proteínas de Plantas/fisiologia , Prolina/metabolismo , Espécies Reativas de Oxigênio/metabolismo , Reação em Cadeia da Polimerase em Tempo Real
2.
PLoS Genet ; 16(8): e1008954, 2020 08.
Artigo em Inglês | MEDLINE | ID: mdl-32785227

RESUMO

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


Assuntos
Axonema/genética , Proteínas do Citoesqueleto/genética , Dineínas/genética , Infertilidade Masculina/genética , Animais , Chlamydomonas/genética , Cílios/genética , Cílios/patologia , Fertilização In Vitro , Humanos , Infertilidade Masculina/patologia , Masculino , Camundongos , Camundongos Knockout , Motilidade Espermática/genética , Cauda do Espermatozoide/metabolismo , Cauda do Espermatozoide/patologia , Espermatozoides/crescimento & desenvolvimento , Espermatozoides/patologia , Testículo/crescimento & desenvolvimento , Testículo/patologia
3.
Exp Cell Res ; 395(2): 112204, 2020 10 15.
Artigo em Inglês | MEDLINE | ID: mdl-32735892

RESUMO

BACKGROUND: SARS-CoV2, the agent responsible for the current pandemic, is also causing respiratory distress syndrome (RDS), hyperinflammation and high mortality. It is critical to dissect the pathogenetic mechanisms in order to reach a targeted therapeutic approach. METHODS: In the present investigation, we evaluated the effects of SARS-CoV2 on human bronchial epithelial cells (HBEC). We used RNA-seq datasets available online for identifying SARS-CoV2 potential genes target on human bronchial epithelial cells. RNA expression levels and potential cellular gene pathways have been analyzed. In order to identify possible common strategies among the main pandemic viruses, such as SARS-CoV2, SARS-CoV1, MERS-CoV, and H1N1, we carried out a hypergeometric test of the main genes transcribed in the cells of the respiratory tract exposed to these viruses. RESULTS: The analysis showed that two mechanisms are highly regulated in HBEC: the innate immunity recruitment and the disassembly of cilia and cytoskeletal structure. The granulocyte colony-stimulating factor (CSF3) and dynein heavy chain 7, axonemal (DNAH7) represented respectively the most upregulated and downregulated genes belonging to the two mechanisms highlighted above. Furthermore, the carcinoembryonic antigen-related cell adhesion molecule 7 (CEACAM7) that codifies for a surface protein is highly specific of SARS-CoV2 and not for SARS-CoV1, MERS-CoV, and H1N1, suggesting a potential role in viral entry. In order to identify potential new drugs, using a machine learning approach, we highlighted Flunisolide, Thalidomide, Lenalidomide, Desoximetasone, xylazine, and salmeterol as potential drugs against SARS-CoV2 infection. CONCLUSIONS: Overall, lung involvement and RDS could be generated by the activation and down regulation of diverse gene pathway involving respiratory cilia and muscle contraction, apoptotic phenomena, matrix destructuration, collagen deposition, neutrophil and macrophages recruitment.


Assuntos
Brônquios/metabolismo , Infecções por Coronavirus/genética , Redes Reguladoras de Genes , Pneumonia Viral/genética , Mucosa Respiratória/metabolismo , Transcriptoma , Brônquios/patologia , Antígeno Carcinoembrionário/genética , Antígeno Carcinoembrionário/metabolismo , Infecções por Coronavirus/metabolismo , Descoberta de Drogas/métodos , Dineínas/genética , Dineínas/metabolismo , Proteínas Ligadas por GPI/genética , Proteínas Ligadas por GPI/metabolismo , Fator Estimulador de Colônias de Granulócitos/genética , Fator Estimulador de Colônias de Granulócitos/metabolismo , Humanos , Imunidade Inata , Aprendizado de Máquina , Pandemias , Pneumonia Viral/metabolismo , Regulação para Cima
4.
Breast Cancer Res ; 22(1): 64, 2020 06 15.
Artigo em Inglês | MEDLINE | ID: mdl-32539762

RESUMO

BACKGROUND: Aging is a comorbidity of breast cancer suggesting that aging-associated transcriptome changes may promote breast cancer progression. However, the mechanism underlying the age effect on breast cancer remains poorly understood. METHOD: We analyzed transcriptomics of the matched normal breast tissues from the 82 breast cancer patients in The Cancer Genome Atlas (TCGA) dataset with linear regression for genes with age-associated expression that are not associated with menopause. We also analyzed differentially expressed genes between the paired tumor and non-tumor breast tissues in TCGA for the identification of age and breast cancer (ABC)-associated genes. A few of these genes were selected for further investigation of their malignancy-regulating activities with in vitro and in vivo assays. RESULTS: We identified 148 upregulated and 189 downregulated genes during aging. Overlapping of tumor-associated genes between normal and tumor tissues with age-dependent genes resulted in 14 upregulated and 24 downregulated genes that were both age and breast cancer associated. These genes are predictive in relapse-free survival, indicative of their potential tumor promoting or suppressive functions, respectively. Knockdown of two upregulated genes (DYNLT3 and P4HA3) or overexpression of the downregulated ALX4 significantly reduced breast cancer cell proliferation, migration, and clonogenicity. Moreover, knockdown of P4HA3 reduced growth and metastasis whereas overexpression of ALX4 inhibited the growth of xenografted breast cancer cells in mice. CONCLUSION: Our study suggests that transcriptome alterations during aging may contribute to breast tumorigenesis. DYNLT3, P4HA3, and ALX4 play significant roles in breast cancer progression.


Assuntos
Neoplasias da Mama/genética , Mama/fisiologia , Adulto , Fatores Etários , Idoso , Idoso de 80 Anos ou mais , Animais , Biomarcadores Tumorais/genética , Biomarcadores Tumorais/metabolismo , Mama/metabolismo , Mama/patologia , Neoplasias da Mama/metabolismo , Neoplasias da Mama/patologia , Progressão da Doença , Dineínas/genética , Dineínas/metabolismo , Feminino , Regulação Neoplásica da Expressão Gênica , Xenoenxertos , Humanos , Camundongos , Camundongos Endogâmicos NOD , Camundongos SCID , Pessoa de Meia-Idade , Pró-Colágeno-Prolina Dioxigenase/genética , Pró-Colágeno-Prolina Dioxigenase/metabolismo , Prognóstico , Taxa de Sobrevida , Células Tumorais Cultivadas
5.
BMC Med Genet ; 21(1): 87, 2020 05 01.
Artigo em Inglês | MEDLINE | ID: mdl-32357925

RESUMO

BACKGROUND: Developmental dyslexia (DD) is a neurodevelopmental learning disorder with high heritability. A number of candidate susceptibility genes have been identified, some of which are linked to the function of the cilium, an organelle regulating left-right asymmetry development in the embryo. Furthermore, it has been suggested that disrupted left-right asymmetry of the brain may play a role in neurodevelopmental disorders such as DD. However, it is unknown whether there is a common genetic cause to DD and laterality defects or ciliopathies. CASE PRESENTATION: Here, we studied two individuals with co-occurring situs inversus (SI) and DD using whole genome sequencing to identify genetic variants of importance for DD and SI. Individual 1 had primary ciliary dyskinesia (PCD), a rare, autosomal recessive disorder with oto-sino-pulmonary phenotype and SI. We identified two rare nonsynonymous variants in the dynein axonemal heavy chain 5 gene (DNAH5): a previously reported variant c.7502G > C; p.(R2501P), and a novel variant c.12043 T > G; p.(Y4015D). Both variants are predicted to be damaging. Ultrastructural analysis of the cilia revealed a lack of outer dynein arms and normal inner dynein arms. MRI of the brain revealed no significant abnormalities. Individual 2 had non-syndromic SI and DD. In individual 2, one rare variant (c.9110A > G;p.(H3037R)) in the dynein axonemal heavy chain 11 gene (DNAH11), coding for another component of the outer dynein arm, was identified. CONCLUSIONS: We identified the likely genetic cause of SI and PCD in one individual, and a possibly significant heterozygosity in the other, both involving dynein genes. Given the present evidence, it is unclear if the identified variants also predispose to DD and further studies into the association between laterality, ciliopathies and DD are needed.


Assuntos
Dineínas do Axonema/genética , Dislexia/genética , Situs Inversus/genética , Encéfalo/diagnóstico por imagem , Encéfalo/patologia , Criança , Transtornos da Motilidade Ciliar/genética , Transtornos da Motilidade Ciliar/patologia , Dineínas/genética , Dislexia/diagnóstico por imagem , Dislexia/patologia , Feminino , Predisposição Genética para Doença , Heterozigoto , Humanos , Masculino , Pessoa de Meia-Idade , Mutação/genética , Polimorfismo de Nucleotídeo Único/genética , Situs Inversus/diagnóstico por imagem , Situs Inversus/patologia
6.
Proc Natl Acad Sci U S A ; 117(14): 7799-7802, 2020 04 07.
Artigo em Inglês | MEDLINE | ID: mdl-32205434

RESUMO

Cytoskeletons are self-organized networks based on polymerized proteins: actin, tubulin, and driven by motor proteins, such as myosin, kinesin, and dynein. Their positive Darwinian evolution enables them to approach optimized functionality (self-organized criticality). Dynein has three distinct titled subunits, but how these units connect to function as a molecular motor is mysterious. Dynein binds to tubulin through two coiled coil stalks and a stalk head. The energy used to alter the head binding and propel cargo along tubulin is supplied by ATP at a ring 1,500 amino acids away. Here, we show how many details of this extremely distant interaction are explained by water waves quantified by thermodynamic scaling. Water waves have shaped all proteins throughout positive Darwinian evolution, and many aspects of long-range water-protein interactions are universal (described by self-organized criticality). Dynein water waves resembling tsunami produce nearly optimal energy transport over 1,500 amino acids along dynein's one-dimensional peptide backbone. More specifically, this paper identifies many similarities in the function and evolution of dynein compared to other cytoskeleton proteins such as actin, myosin, and tubulin.


Assuntos
Trifosfato de Adenosina/genética , Citoesqueleto/genética , Dineínas/genética , Evolução Molecular , Actinas , Sequência de Aminoácidos/genética , Animais , Fenômenos Biofísicos , Aptidão Genética/genética , Cinesina/genética , Microtúbulos/genética , Miosinas/genética , Conformação Proteica , Tubulina (Proteína)/genética
7.
Nat Commun ; 11(1): 1038, 2020 02 25.
Artigo em Inglês | MEDLINE | ID: mdl-32098965

RESUMO

The movements of cytoplasmic dynein on microtubule (MT) tracks is achieved by two-way communication between the microtubule-binding domain (MTBD) and the ATPase domain via a coiled-coil stalk, but the structural basis of this communication remains elusive. Here, we regulate MTBD either in high-affinity or low-affinity states by introducing a disulfide bond to the stalk and analyze the resulting structures by NMR and cryo-EM. In the MT-unbound state, the affinity changes of MTBD are achieved by sliding of the stalk α-helix by a half-turn, which suggests that structural changes propagate from the ATPase-domain to MTBD. In addition, MT binding induces further sliding of the stalk α-helix even without the disulfide bond, suggesting how the MT-induced conformational changes propagate toward the ATPase domain. Based on differences in the MT-binding surface between the high- and low-affinity states, we propose a potential mechanism for the directional bias of dynein movement on MT tracks.


Assuntos
Dineínas/química , Dineínas/metabolismo , Microtúbulos/metabolismo , Proteínas de Saccharomyces cerevisiae/química , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/química , Saccharomyces cerevisiae/metabolismo , Sítios de Ligação , Microscopia Crioeletrônica , Citoplasma/química , Citoplasma/genética , Citoplasma/metabolismo , Dissulfetos/química , Dineínas/genética , Modelos Moleculares , Mutação , Ressonância Magnética Nuclear Biomolecular , Domínios Proteicos , Estrutura Quaternária de Proteína , Estrutura Terciária de Proteína , Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/genética , Tubulina (Proteína)/química , Tubulina (Proteína)/metabolismo
8.
Andrologia ; 52(3): e13539, 2020 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-32030798

RESUMO

Asthenozoospermia (AZS), which characterised by reduced forward sperm motility, is a common cause of male infertility. Recently, mitochondrial dysfunction reported in AZS men came to attention for finding the molecular aetiology of AZS. Mitochondria-related microRNAs (miRNAs) are the most important regulators of mitochondrial function through post-transcriptionally modulation of gene expression. Therefore, this study aims to evaluate the expression of four recently reported mitochondrial-related miRNAs (miR-4485-3p/4484/4461 and 4463) in the sperm sample of asthenozoospermic men. RNA was extracted from spermatozoa of 74 volunteers (39 patients with idiopathic AZS and 35 controls with normal fertility), and relative gene expression analysis was performed by quantitative PCR. We used SNORD48 as a normaliser gene, and quantification was calculated by 2-ΔΔCt method. The expression of miR-4484 and miR-4461 was not detected in the spermatozoa of cases and controls. However, miR-4485-3p (p = .006) was significantly downregulated in the AZS men compared with the controls, but the miR-4463 expression was not significantly different between the two groups (p = .5). Bioinformatic analysis identified three target genes for miR-4485-3p (DNAH1, KIT and PARK7) that are related to male infertility. In conclusion, the downregulation of miR-4485-3p was associated with idiopathic AZS, which could be a molecular link between mitochondrial dysfunction and AZS.


Assuntos
Astenozoospermia/genética , MicroRNAs/metabolismo , RNA Mitocondrial/metabolismo , Espermatozoides/metabolismo , Adulto , Astenozoospermia/patologia , Estudos de Casos e Controles , Biologia Computacional , Regulação para Baixo , Dineínas/genética , Humanos , Masculino , MicroRNAs/isolamento & purificação , Mitocôndrias/metabolismo , Proteína Desglicase DJ-1/genética , Proteínas Proto-Oncogênicas c-kit/genética , Reação em Cadeia da Polimerase em Tempo Real , Motilidade Espermática/genética , Espermatozoides/citologia , Espermatozoides/patologia
9.
Nat Commun ; 11(1): 23, 2020 01 07.
Artigo em Inglês | MEDLINE | ID: mdl-31911620

RESUMO

Assembly of infectious influenza A viruses (IAV) is a complex process involving transport from the nucleus to the plasma membrane. Rab11A-containing recycling endosomes have been identified as a platform for intracellular transport of viral RNA (vRNA). Here, using high spatiotemporal resolution light-sheet microscopy (~1.4 volumes/second, 330 nm isotropic resolution), we quantify Rab11A and vRNA movement in live cells during IAV infection and report that IAV infection decreases speed and increases arrest of Rab11A. Unexpectedly, infection with respiratory syncytial virus alters Rab11A motion in a manner opposite to IAV, suggesting that Rab11A is a common host component that is differentially manipulated by respiratory RNA viruses. Using two-color imaging we demonstrate co-transport of Rab11A and IAV vRNA in infected cells and provide direct evidence that vRNA-associated Rab11A have altered transport. The mechanism of altered Rab11A movement is likely related to a decrease in dynein motors bound to Rab11A vesicles during IAV infection.


Assuntos
Dineínas/metabolismo , Vírus da Influenza A/fisiologia , Influenza Humana/metabolismo , Proteínas rab de Ligação ao GTP/metabolismo , Transporte Biológico , Dineínas/genética , Interações Hospedeiro-Patógeno , Humanos , Vírus da Influenza A/genética , Influenza Humana/genética , Influenza Humana/virologia , RNA Viral/genética , RNA Viral/metabolismo , Proteínas rab de Ligação ao GTP/genética
10.
Am J Hum Genet ; 106(2): 153-169, 2020 02 06.
Artigo em Inglês | MEDLINE | ID: mdl-31978331

RESUMO

Cilia and flagella are evolutionarily conserved organelles whose motility relies on the outer and inner dynein arm complexes (ODAs and IDAs). Defects in ODAs and IDAs result in primary ciliary dyskinesia (PCD), a disease characterized by recurrent airway infections and male infertility. PCD mutations in assembly factors have been shown to cause a combined ODA-IDA defect, affecting both cilia and flagella. We identified four loss-of-function mutations in TTC12, which encodes a cytoplasmic protein, in four independent families in which affected individuals displayed a peculiar PCD phenotype characterized by the absence of ODAs and IDAs in sperm flagella, contrasting with the absence of only IDAs in respiratory cilia. Analyses of both primary cells from individuals carrying TTC12 mutations and human differentiated airway cells invalidated for TTC12 by a CRISPR-Cas9 approach revealed an IDA defect restricted to a subset of single-headed IDAs that are different in flagella and cilia, whereas TTC12 depletion in the ciliate Paramecium tetraurelia recapitulated the sperm phenotype. Overall, our study, which identifies TTC12 as a gene involved in PCD, unveils distinct dynein assembly mechanisms in human motile cilia versus flagella.


Assuntos
Cílios/patologia , Transtornos da Motilidade Ciliar/etiologia , Dineínas/metabolismo , Flagelos/patologia , Mutação , Proteínas/genética , Cauda do Espermatozoide/patologia , Adulto , Axonema , Criança , Cílios/metabolismo , Transtornos da Motilidade Ciliar/patologia , Dineínas/genética , Feminino , Flagelos/metabolismo , Homozigoto , Humanos , Infertilidade Masculina/etiologia , Infertilidade Masculina/patologia , Masculino , Pessoa de Meia-Idade , Linhagem , Fenótipo , Motilidade Espermática , Cauda do Espermatozoide/metabolismo , Adulto Jovem
11.
Biol Cell ; 112(2): 53-72, 2020 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-31859373

RESUMO

BACKGROUND: Fibroblasts executing directional migration position their centrosome, and their Golgi apparatus, in front of the nucleus towards the cell leading edge. Centrosome positioning relative to the nucleus has been associated to mechanical forces exerted on the centrosome by the microtubule-dependent molecular motor cytoplasmic dynein 1, and to nuclear movements such as rearward displacement and rotation events. Dynein has been proposed to regulate the position of the centrosome by exerting pulling forces on microtubules from the cell leading edge, where the motor is enriched during migration. However, the mechanism explaining how dynein acts at the front of the cells has not been elucidated. RESULTS: We present here results showing that the protein Focal Adhesion Kinase (FAK) interacts with dynein and regulates the enrichment of the dynein/dynactin complex at focal adhesions at the cell the leading edge of migrating fibroblasts. This suggests that focal adhesions provide anchoring sites for dynein during the polarisation process. In support of this, we present evidence indicating that the interaction between FAK and dynein, which is regulated by the phosphorylation of FAK on its Ser732 residue, is required for proper centrosome positioning. Our results further show that the polarisation of the centrosome can occur independently of nuclear movements. Although FAK regulates both nuclear and centrosome motilities, downregulating the interaction between FAK and dynein affects only the nuclear independent polarisation of the centrosome. CONCLUSIONS: Our work highlights the role of FAK as a key player in the regulation of several aspects of cell polarity. We thus propose a model in which the transient localisation of dynein with focal adhesions provides a tuneable mechanism to bias dynein traction forces on microtubules allowing proper centrosome positioning in front of the nucleus. SIGNIFICANCE: We unravel here a new role for the cancer therapeutic target FAK in the regulation of cell morphogenesis.


Assuntos
Movimento Celular , Polaridade Celular , Dineínas/metabolismo , Quinase 1 de Adesão Focal/metabolismo , Animais , Dineínas/genética , Quinase 1 de Adesão Focal/genética , Camundongos , Células NIH 3T3 , Transporte Proteico
12.
Biochem Biophys Res Commun ; 523(1): 253-257, 2020 02 26.
Artigo em Inglês | MEDLINE | ID: mdl-31864710

RESUMO

Diverse inner arm dyneins cooperate with outer arm dyneins to produce ciliary beating. This study demonstrates an expression system for inner arm dyneins in Tetrahymena. The motor domain of inner arm dynein (Dyh8p or Dyh12p) was fused with the tail of outer arm dynein (Dyh3p) and expressed in viable DYH3-knockout (vKO-DYH3) cells. The chimeric dyneins were observed in the oral apparatus and cilia on the cell bodies, and did not change the swimming speed of vKO-DYH3 cells. In a gliding assay, the motor domains of Dyh8p and Dyh12p moved toward the minus ends of microtubules at 0.8 and 0.3 µm/s, respectively. The gliding velocities of Dyh8p and Dyh12p were decreased in 5 mM ATP but not increased in 0.1 or 0.5 mM ADP. This expression system will be useful for molecular studies on diverse inner arm dyneins.


Assuntos
Cílios/genética , Dineínas/genética , Tetrahymena/genética , Cílios/metabolismo , Dineínas/isolamento & purificação , Dineínas/metabolismo , Subunidades Proteicas/genética , Subunidades Proteicas/metabolismo , Tetrahymena/citologia , Tetrahymena/metabolismo
13.
J Cell Biol ; 219(2)2020 02 03.
Artigo em Inglês | MEDLINE | ID: mdl-31727776

RESUMO

Axon initial segments (AISs) initiate action potentials and regulate the trafficking of vesicles between somatodendritic and axonal compartments. However, the mechanisms controlling AIS assembly remain poorly defined. We performed differential proteomics and found nuclear mitotic apparatus protein 1 (NuMA1) is downregulated in AIS-deficient neonatal mouse brains and neurons. NuMA1 is transiently located at the AIS during development where it interacts with the scaffolding protein 4.1B and the dynein regulator lissencephaly 1 (Lis1). Silencing NuMA1 or protein 4.1B by shRNA disrupts AIS assembly, but not maintenance. Silencing Lis1 or overexpressing NuMA1 during AIS assembly increased the density of AIS proteins, including ankyrinG and neurofascin-186 (NF186). NuMA1 inhibits the endocytosis of AIS NF186 by impeding Lis1's interaction with doublecortin, a potent facilitator of NF186 endocytosis. Our results indicate the transient expression and AIS localization of NuMA1 stabilizes the developing AIS by inhibiting endocytosis and removal of AIS proteins.


Assuntos
Segmento Inicial do Axônio/metabolismo , Proteínas de Ciclo Celular/genética , Dineínas/genética , Endocitose/genética , Proteômica , 1-Alquil-2-acetilglicerofosfocolina Esterase/antagonistas & inibidores , 1-Alquil-2-acetilglicerofosfocolina Esterase/genética , Animais , Anquirinas/genética , Axônios/metabolismo , Moléculas de Adesão Celular/genética , Citoesqueleto/genética , Regulação da Expressão Gênica/genética , Humanos , Camundongos , Proteínas dos Microfilamentos/genética , Proteínas Associadas aos Microtúbulos/antagonistas & inibidores , Proteínas Associadas aos Microtúbulos/genética , Fatores de Crescimento Neural/genética , Neurônios/metabolismo , Transporte Proteico/genética , RNA Interferente Pequeno/farmacologia
14.
Dev Biol ; 459(2): 126-137, 2020 03 15.
Artigo em Inglês | MEDLINE | ID: mdl-31881198

RESUMO

Crumbs (Crb in Drosophila; CRB1-3 in mammals) is a transmembrane determinant of epithelial cell polarity and a regulator of Hippo signalling. Crb is normally localized to apical cell-cell contacts, just above adherens junctions, but how apical trafficking of Crb is regulated in epithelial cells remains unclear. We use the Drosophila follicular epithelium to demonstrate that polarized trafficking of Crb is mediated by transport along microtubules by the motor protein Dynein and along actin filaments by the motor protein Myosin-V (MyoV). Blocking transport of Crb-containing vesicles by Dynein or MyoV leads to accumulation of Crb within Rab11 endosomes, rather than apical delivery. The final steps of Crb delivery and stabilisation at the plasma membrane requires the exocyst complex and three apical FERM domain proteins - Merlin, Moesin and Expanded - whose simultaneous loss disrupts apical localization of Crb. Accordingly, a knock-in deletion of the Crb FERM-binding motif (FBM) also impairs apical localization. Finally, overexpression of Crb challenges this system, creating a sensitized background to identify components involved in cytoskeletal polarization, apical membrane trafficking and stabilisation of Crb at the apical domain.


Assuntos
Polaridade Celular/genética , Proteínas de Drosophila/metabolismo , Dineínas/metabolismo , Proteínas de Membrana/metabolismo , Miosina Tipo V/metabolismo , Junções Aderentes/metabolismo , Animais , Animais Geneticamente Modificados , Proteínas de Drosophila/genética , Drosophila melanogaster , Dineínas/genética , Células Epiteliais/metabolismo , Feminino , Técnicas de Introdução de Genes , Proteínas de Membrana/genética , Microtúbulos/metabolismo , Miosina Tipo V/genética , Neurofibromina 2/metabolismo , Folículo Ovariano/citologia , Transporte Proteico , Transdução de Sinais/genética
15.
Sci Rep ; 9(1): 15864, 2019 11 01.
Artigo em Inglês | MEDLINE | ID: mdl-31676830

RESUMO

Male infertility due to spermatogenesis defects affects millions of men worldwide. However, the genetic etiology of the vast majority remains unclear. Here we describe three men with primary infertility due to multiple morphological abnormalities of the sperm flagella (MMAF) from two unrelated Han Chinese families. We performed whole-exome sequencing (WES) and Sanger sequencing on the proband of family 1, and found that he carried novel compound heterozygous missense mutations in dynein axonemal heavy chain 6 (DNAH6) that resulted in the substitution of a conserved amino acid residue and co-segregated with the MMAF phenotype in this family. Papanicolaou staining and transmission electron microscopy analysis revealed morphological and ultrastructural abnormalities in the sperm flagella in carriers of these genetic variants. Immunostaining experiments showed that DNAH6 was localized in the sperm tail. This is the first report identifying novel recessive mutations in DNAH6 as a cause of MMAF. These findings expand the spectrum of known MMAF mutations and phenotypes and provide information that can be useful for genetic and reproductive counseling of MMAF patients.


Assuntos
Dineínas/genética , Infertilidade Masculina , Mutação de Sentido Incorreto , Cauda do Espermatozoide/ultraestrutura , Adulto , China , Humanos , Infertilidade Masculina/genética , Infertilidade Masculina/patologia , Masculino , Sequenciamento Completo do Exoma
16.
Sci Rep ; 9(1): 14902, 2019 10 17.
Artigo em Inglês | MEDLINE | ID: mdl-31624276

RESUMO

Ectopic lipid accumulation in lipid droplets (LD) has been linked to many metabolic diseases. In this study, DHS-3::GFP was used as a LD marker in C. elegans and a forward genetic screen was carried out to find novel LD regulators. There were 140 mutant alleles identified which were divided into four phenotypic categories: enlarged, aggregated, aggregated and small, and decreased. After genetic mapping, mutations in three known LD regulatory genes (maoc-1, dhs-28, daf-22) and a peroxisome-related gene (acox-3) were found to enlarge LDs, demonstrating the reliability of using DHS-3 as a living marker. In the screen, the cytoskeleton protein C27H5.2 was found to be involved in LD aggregation, as was the LD resident/structure-like protein, MDT-28/PLIN-1. Using yeast two-hybrid screening and pull-down assays, MDT-28/PLIN-1 was found to bind to DLC-1 (dynein light chain). Fluorescence imaging confirmed that MDT-28/PLIN-1 mediated the interaction between DHS-3 labeled LDs and DLC-1 labeled microtubules. Furthermore, MDT-28/PLIN-1 was directly bound to DLC-1 through its amino acids 1-210 and 275-415. Taken together, our results suggest that MDT-28/PLIN-1 is involved in the regulation of LD distribution through its interaction with microtubule-related proteins.


Assuntos
Proteínas de Caenorhabditis elegans/metabolismo , Caenorhabditis elegans/metabolismo , Dineínas/metabolismo , Gotículas Lipídicas/metabolismo , Complexo Mediador/metabolismo , Microtúbulos/metabolismo , Proteínas Nucleares/metabolismo , Animais , Animais Geneticamente Modificados , Caenorhabditis elegans/citologia , Caenorhabditis elegans/genética , Proteínas de Caenorhabditis elegans/genética , Dineínas/genética , Técnicas de Silenciamento de Genes , Complexo Mediador/genética , Mutação , Proteínas Nucleares/genética , Ligação Proteica/genética , Interferência de RNA
17.
Mol Biol Cell ; 30(21): 2659-2680, 2019 10 01.
Artigo em Inglês | MEDLINE | ID: mdl-31483737

RESUMO

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


Assuntos
Proteínas de Algas/metabolismo , Axonema/metabolismo , Cílios/metabolismo , Dineínas/metabolismo , Flagelos/metabolismo , Proteômica/métodos , Proteínas de Algas/genética , Sequência de Aminoácidos , Axonema/genética , Chlamydomonas reinhardtii/genética , Chlamydomonas reinhardtii/metabolismo , Cílios/genética , Cílios/ultraestrutura , Microscopia Crioeletrônica/métodos , Dineínas/genética , Tomografia com Microscopia Eletrônica , Flagelos/genética , Flagelos/ultraestrutura , Microscopia de Fluorescência/métodos , Microtúbulos/metabolismo , Microtúbulos/ultraestrutura , Mutação , Subunidades Proteicas/genética , Subunidades Proteicas/metabolismo , Gravação de Videoteipe/métodos
18.
Development ; 146(15)2019 08 09.
Artigo em Inglês | MEDLINE | ID: mdl-31391195

RESUMO

A conserved mechanism of polarity establishment is the localization of mRNA to specific cellular regions. Although it is clear that many mRNAs are transported along microtubules, much less is known about the mechanism by which these mRNAs are linked to microtubule motors. The RNA binding protein Egalitarian (Egl) is necessary for localization of several mRNAs in Drosophila oocytes and embryos. Egl also interacts with Dynein light chain (Dlc) and Bicaudal-D (BicD). The role of Dlc and BicD in mRNA localization has remained elusive. Both proteins are required for oocyte specification, as is Egl. Null alleles in these genes result in an oogenesis block. In this report, we used an shRNA-depletion strategy to overcome the oogenesis block. Our findings reveal that the primary function of Dlc is to promote Egl dimerization. Loss of dimerization compromises the ability of Egl to bind RNA. Consequently, Egl is not bound to cargo, and is not able to efficiently associate with BicD and the Dynein motor. Our results therefore identify the key molecular steps required for assembling a localization-competent mRNP.


Assuntos
Proteínas de Drosophila/metabolismo , Drosophila melanogaster/embriologia , Dineínas/metabolismo , Oócitos/citologia , Animais , Linhagem Celular , Proteínas de Drosophila/genética , Dineínas/genética , Microtúbulos/metabolismo , Oogênese/genética , Oogênese/fisiologia , Ligação Proteica/fisiologia , Interferência de RNA , RNA Mensageiro/genética , RNA Interferente Pequeno/genética
19.
Biophys J ; 117(4): 679-687, 2019 08 20.
Artigo em Inglês | MEDLINE | ID: mdl-31400919

RESUMO

Cilia and flagella are long, slender organelles found in many eukaryotic cells, where they have sensory, developmental, and motile functions. All cilia and flagella contain a microtubule-based structure called the axoneme. In motile cilia and flagella, which drive cell locomotion and fluid transport, the axoneme contains, along most of its length, motor proteins from the axonemal dynein family. These motor proteins drive motility by using energy derived from the hydrolysis of ATP to generate a bending wave, which travels down the axoneme. As a first step toward visualizing the ATPase activity of the axonemal dyneins during bending, we have investigated the kinetics of nucleotide binding to axonemes. Using a specially built ultraviolet total internal reflection fluorescence microscope, we found that the fluorescent ATP analog methylanthraniloyl ATP (mantATP), which has been shown to support axonemal motility, binds all along isolated, immobilized axonemes. By studying the recovery of fluorescence after photobleaching, we found that there are three mantATP binding sites: one that bleaches rapidly (time constant ≈ 1.7 s) and recovers slowly (time constant ≈ 44 s), one that bleaches with the same time constant but does not recover, and one that does not bleach. By reducing the dynein content in the axoneme using mutants and salt extraction, we provide evidence that the slow-recovering component, but not the other components, corresponds to axonemal dyneins. The recovery rate of this component, however, is too slow to be consistent with the activation of beating observed at higher mantATP concentrations; this indicates that the dyneins may be inhibited due to their immobilization at the surface. The development of this method is a first step toward direct observation of the traveling wave of dynein activity.


Assuntos
Trifosfato de Adenosina/metabolismo , Axonema/metabolismo , Dineínas/metabolismo , Proteínas de Plantas/metabolismo , Axonema/ultraestrutura , Sítios de Ligação , Chlamydomonas reinhardtii , Dineínas/química , Dineínas/genética , Recuperação de Fluorescência Após Fotodegradação , Cinética , Mutação , Proteínas de Plantas/química , Proteínas de Plantas/genética , Ligação Proteica
20.
Andrologia ; 51(10): e13380, 2019 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-31382319

RESUMO

This study aimed to evaluate dynein light chain type 1 (DYNLT1) mRNA expression in mature spermatozoa and to investigate its association with Beclin1 expression to help in understanding of pathogenesis of male infertility. It included 60 infertile men divided into idiopathic (n = 20), accessory gland inflammation (n = 20), and varicocele (n = 20) groups, and 20 healthy fertile men as a control group. Semen parameters were evaluated according to the 2010 World Health Organization criteria. Mature spermatozoa were isolated by Sil-select gradient. Relative quantification of DYNLT1 and Beclin1 mRNA expression in whole sperm pellet and mature spermatozoa was done using real-time PCR. Beclin1 protein was assessed in whole sperm pellet and mature spermatozoa by ELISA. Beclin1 mRNA and protein were significantly increased in spermatozoa from infertile patients of different aetiologies in comparison to healthy controls (p < .05). However, DYNLT1 mRNA expression was significantly decreased in infertile groups than controls (p < .05). Mature spermatozoa extracted from all studied subjects showed increased DYNLT1 mRNA and decreased Beclin1 mRNA and protein expression compared with the whole sample. It is concluded that decreased Beclin1 and increased DYNLT1 mRNA expression in mature spermatozoa may provide an insight into the biological processes that are activated or suppressed during sperm maturation.


Assuntos
Proteína Beclina-1/metabolismo , Dineínas/metabolismo , Fertilidade , Infertilidade Masculina/patologia , Espermatozoides/metabolismo , Adulto , Autofagia , Proteína Beclina-1/genética , Estudos de Casos e Controles , Dineínas/genética , Perfilação da Expressão Gênica , Voluntários Saudáveis , Humanos , Masculino , Pessoa de Meia-Idade , RNA Mensageiro/metabolismo , Análise do Sêmen , Adulto Jovem
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