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1.
FASEB J ; 34(3): 4653-4669, 2020 03.
Artigo em Inglês | MEDLINE | ID: mdl-32017270

RESUMO

Transmissible gastroenteritis virus (TGEV) is a swine enteropathogenic coronavirus that causes significant economic losses in swine industry. Current studies on TGEV internalization mainly focus on viral receptors, but the internalization mechanism is still unclear. In this study, we used single-virus tracking to obtain the detailed insights into the dynamic events of the TGEV internalization and depict the whole sequential process. We observed that TGEVs could be internalized through clathrin- and caveolae-mediated endocytosis, and the internalization of TGEVs was almost completed within ~2 minutes after TGEVs attached to the cell membrane. Furthermore, the interactions of TGEVs with actin and dynamin 2 in real time during the TGEV internalization were visualized. To our knowledge, this is the first report that single-virus tracking technique is used to visualize the entire dynamic process of the TGEV internalization: before the TGEV internalization, with the assistance of actin, clathrin, and caveolin 1 would gather around the virus to form the vesicle containing the TGEV, and after ~60 seconds, dynamin 2 would be recruited to promote membrane fission. These results demonstrate that TGEVs enter ST cells via clathrin- and caveolae-mediated endocytic, actin-dependent, and dynamin 2-dependent pathways.


Assuntos
Gastroenterite Suína Transmissível/metabolismo , Gastroenterite Suína Transmissível/virologia , Vírus da Gastroenterite Transmissível/patogenicidade , Actinas/metabolismo , Animais , Cavéolas/metabolismo , Caveolina 1/metabolismo , Linhagem Celular , Membrana Celular/metabolismo , Membrana Celular/fisiologia , Membrana Celular/virologia , Clatrina/metabolismo , Dinamina II/metabolismo , Endocitose/fisiologia , Fusão de Membrana/fisiologia , Suínos , Internalização do Vírus
2.
J Mol Biol ; 432(4): 1235-1250, 2020 02 14.
Artigo em Inglês | MEDLINE | ID: mdl-31857086

RESUMO

The Bridging integrator 1 (BIN1)/Amphiphysin/Rvs (BAR) protein family is an essential part of the cell's machinery to bend membranes. BIN1 is a muscle-enriched BAR protein with an established role in muscle development and skeletal myopathies. Here, we demonstrate that BIN1, on its own, is able to form complex interconnected tubular systems in vitro, reminiscent of t-tubule system in muscle cells. We further describe how BIN1's electrostatic interactions regulate membrane bending: the ratio of negatively charged lipids in the bilayer altered membrane bending and binding properties of BIN1 and so did the manipulation of BIN1's surface charge. We show that the electrostatically mediated BIN1 membrane binding depended on the membrane curvature-it was less affected in liposomes with high curvature. Curiously, BIN1 membrane binding and bending was diminished in cells where the membrane's charge was experimentally reduced. Membrane bending was also reduced in BIN1 mutants where negative or positive charges in the BAR domain have been eliminated. This phenotype, characteristic of BIN1 mutants linked to myopathies, was rescued when the membrane charge was made more negative. The latter findings also show that cells can control tubulation at their membranes by simply altering the membrane charge and through it, the recruitment of BAR proteins and their interaction partners (e.g. dynamin).


Assuntos
Proteínas Adaptadoras de Transdução de Sinal/química , Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Membrana Celular/metabolismo , Lipossomos/química , Proteínas Nucleares/química , Proteínas Nucleares/metabolismo , Proteínas Supressoras de Tumor/química , Proteínas Supressoras de Tumor/metabolismo , Proteínas Adaptadoras de Transdução de Sinal/genética , Animais , Células COS , Chlorocebus aethiops , Dicroísmo Circular , Dinamina II/química , Dinamina II/metabolismo , Mutação , Proteínas do Tecido Nervoso/química , Proteínas do Tecido Nervoso/metabolismo , Proteínas Nucleares/genética , Eletricidade Estática , Proteínas Supressoras de Tumor/genética
3.
Cancer Res ; 79(11): 2878-2891, 2019 06 01.
Artigo em Inglês | MEDLINE | ID: mdl-30975647

RESUMO

The sialoglycoprotein podocalyxin is absent in normal pancreas but is overexpressed in pancreatic cancer and is associated with poor clinical outcome. Here, we investigate the role of podocalyxin in migration and metastasis of pancreatic adenocarcinomas using SW1990 and Pa03c as cell models. Although ezrin is regarded as a cytoplasmic binding partner of podocalyxin that regulates actin polymerization via Rac1 or RhoA, we did not detect podocalyxin-ezrin association in pancreatic cancer cells. Moreover, depletion of podocalyxin did not alter actin dynamics or modulate Rac1 and RhoA activities in pancreatic cancer cells. Using mass spectrometry, bioinformatics analysis, coimmunoprecipitation, and pull-down assays, we discovered a novel, direct binding interaction between the cytoplasmic tail of podocalyxin and the large GTPase dynamin-2 at its GTPase, middle, and pleckstrin homology domains. This podocalyxin-dynamin-2 interaction regulated microtubule growth rate, which in turn modulated focal adhesion dynamics and ultimately promoted efficient pancreatic cancer cell migration via microtubule- and Src-dependent pathways. Depletion of podocalyxin in a hemispleen mouse model of pancreatic cancer diminished liver metastasis without altering primary tumor size. Collectively, these findings reveal a novel mechanism by which podocalyxin facilitates pancreatic cancer cell migration and metastasis. SIGNIFICANCE: These findings reveal that a novel interaction between podocalyxin and dynamin-2 promotes migration and metastasis of pancreatic cancer cells by regulating microtubule and focal adhesion dynamics.


Assuntos
Dinamina II/metabolismo , Neoplasias Pancreáticas/patologia , Sialoglicoproteínas/metabolismo , Animais , Adesão Celular/genética , Linhagem Celular Tumoral , Movimento Celular , Citoesqueleto/metabolismo , Citoesqueleto/patologia , Dinamina II/genética , Feminino , Humanos , Neoplasias Hepáticas/secundário , Camundongos SCID , Microtúbulos/genética , Microtúbulos/metabolismo , Neoplasias Pancreáticas/metabolismo , Sialoglicoproteínas/genética , Proteínas rac1 de Ligação ao GTP/genética , Proteínas rac1 de Ligação ao GTP/metabolismo , Proteína rhoA de Ligação ao GTP/genética , Proteína rhoA de Ligação ao GTP/metabolismo , Quinases da Família src/metabolismo
4.
Cancer Invest ; 37(3): 144-155, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-30907150

RESUMO

Glioma stem cells (GSCs) play major roles in drug resistance, tumour maintenance and recurrence of glioblastoma. We investigated inhibition of the GTPase dynamin 2 as a therapy for glioblastoma. Glioma cell lines and patient-derived GSCs were treated with dynamin inhibitors, Dynole 34-2 and CyDyn 4-36. We studied about cell viability, and GSC neurosphere formation in vitro and orthotopic tumour growth in vivo. Dynamin inhibition reduced glioblastoma cell line viability and suppressed neurosphere formation and migration of GSCs. Tumour growth was reduced by CyDyn 4-36 treatment. Dynamin 2 inhibition therefore represents a novel approach for stem cell-directed Glioblastoma therapy.


Assuntos
Neoplasias Encefálicas/tratamento farmacológico , Cianoacrilatos/uso terapêutico , Dinamina II/antagonistas & inibidores , Glioma/tratamento farmacológico , Indóis/uso terapêutico , Células-Tronco Neoplásicas/efeitos dos fármacos , Neoplasias Encefálicas/metabolismo , Neoplasias Encefálicas/patologia , Linhagem Celular Tumoral , Movimento Celular/efeitos dos fármacos , Proliferação de Células/efeitos dos fármacos , Dinamina II/metabolismo , Glioma/metabolismo , Glioma/patologia , Humanos , Terapia de Alvo Molecular/métodos , Células-Tronco Neoplásicas/metabolismo , Carga Tumoral/efeitos dos fármacos , Ensaios Antitumorais Modelo de Xenoenxerto
5.
J Cell Biol ; 218(5): 1670-1685, 2019 05 06.
Artigo em Inglês | MEDLINE | ID: mdl-30894403

RESUMO

Skeletal muscle development requires the cell-cell fusion of differentiated myoblasts to form muscle fibers. The actin cytoskeleton is known to be the main driving force for myoblast fusion; however, how actin is organized to direct intercellular fusion remains unclear. Here we show that an actin- and dynamin-2-enriched protrusive structure, the invadosome, is required for the fusion process of myogenesis. Upon differentiation, myoblasts acquire the ability to form invadosomes through isoform switching of a critical invadosome scaffold protein, Tks5. Tks5 directly interacts with and recruits dynamin-2 to the invadosome and regulates its assembly around actin filaments to strengthen the stiffness of dynamin-actin bundles and invadosomes. These findings provide a mechanistic framework for the acquisition of myogenic fusion machinery during myogenesis and reveal a novel structural function for Tks5 and dynamin-2 in organizing actin filaments in the invadosome to drive membrane fusion.


Assuntos
Citoesqueleto de Actina/fisiologia , Fusão Celular , Dinamina II/metabolismo , Fusão de Membrana , Mioblastos/fisiologia , Proteínas de Ligação a Fosfato/metabolismo , Podossomos/metabolismo , Animais , Comunicação Celular , Diferenciação Celular , Movimento Celular , Células Cultivadas , Camundongos , Mioblastos/citologia
6.
Mol Biol Cell ; 30(5): 579-590, 2019 03 01.
Artigo em Inglês | MEDLINE | ID: mdl-30601711

RESUMO

Clathrin plaques are stable features of the plasma membrane observed in several cell types. They are abundant in muscle, where they localize at costameres that link the contractile apparatus to the sarcolemma and connect the sarcolemma to the basal lamina. Here, we show that clathrin plaques and surrounding branched actin filaments form microdomains that anchor a three-dimensional desmin intermediate filament (IF) web. Depletion of clathrin plaque and branched actin components causes accumulation of desmin tangles in the cytoplasm. We show that dynamin 2, whose mutations cause centronuclear myopathy (CNM), regulates both clathrin plaques and surrounding branched actin filaments, while CNM-causing mutations lead to desmin disorganization in a CNM mouse model and patient biopsies. Our results suggest a novel paradigm in cell biology, wherein clathrin plaques act as platforms capable of recruiting branched cortical actin, which in turn anchors IFs, both essential for striated muscle formation and function.


Assuntos
Actinas/metabolismo , Clatrina/metabolismo , Músculo Esquelético/metabolismo , Animais , Desmina/metabolismo , Dinamina II/metabolismo , Humanos , Filamentos Intermediários/metabolismo , Filamentos Intermediários/ultraestrutura , Camundongos Knockout , Fibras Musculares Esqueléticas/metabolismo , Fibras Musculares Esqueléticas/ultraestrutura , Mutação/genética , Miopatias Congênitas Estruturais/genética , Proteína da Síndrome de Wiskott-Aldrich/metabolismo
7.
Elife ; 82019 01 16.
Artigo em Inglês | MEDLINE | ID: mdl-30648534

RESUMO

Myelination requires extensive plasma membrane rearrangements, implying that molecules controlling membrane dynamics play prominent roles. The large GTPase dynamin 2 (DNM2) is a well-known regulator of membrane remodeling, membrane fission, and vesicular trafficking. Here, we genetically ablated Dnm2 in Schwann cells (SCs) and in oligodendrocytes of mice. Dnm2 deletion in developing SCs resulted in severely impaired axonal sorting and myelination onset. Induced Dnm2 deletion in adult SCs caused a rapidly-developing peripheral neuropathy with abundant demyelination. In both experimental settings, mutant SCs underwent prominent cell death, at least partially due to cytokinesis failure. Strikingly, when Dnm2 was deleted in adult SCs, non-recombined SCs still expressing DNM2 were able to remyelinate fast and efficiently, accompanied by neuropathy remission. These findings reveal a remarkable self-healing capability of peripheral nerves that are affected by SC loss. In the central nervous system, however, we found no major defects upon Dnm2 deletion in oligodendrocytes.


Assuntos
Dinamina II/metabolismo , Oligodendroglia/metabolismo , Células de Schwann/metabolismo , Animais , Axônios/metabolismo , Morte Celular , Diferenciação Celular , Sobrevivência Celular , Citocinese , Camundongos , Mitose , Bainha de Mielina/metabolismo , Nervos Periféricos/metabolismo , Transcriptoma/genética
8.
Virology ; 529: 43-56, 2019 03.
Artigo em Inglês | MEDLINE | ID: mdl-30660774

RESUMO

The large GTPase dynamin 2 controls both endosomal fission and microtubule acetylation. Here we report that dynamin 2 alters microtubules and regulates the trafficking of human adenovirus type 37. Dynamin 2 knockdown by siRNA in infected cells resulted in accumulation of acetylated tubulin, repositioning of microtubule organizing centers (MTOCs) closer to cell nuclei, increased virus in the cytosol (with a compensatory decrease in endosomal virus), reduced proinflammatory cytokine induction, and increased binding of virus to the nucleoporin, Nup358. These events led to increased viral DNA nuclear entry and viral replication. Overexpression of dynamin 2 generated opposite effects. Therefore, dynamin 2 inhibits adenovirus replication and promotes innate immune responses by the infected cell. MTOC transposition in dynamin 2 knockdown promotes a closer association with nuclear pore complexes to facilitate viral DNA delivery. Dynamin 2 plays a key role in adenoviral trafficking and influences host responses to infection.


Assuntos
Adenoviridae/fisiologia , Dinamina II/fisiologia , Internalização do Vírus , Citosol/fisiologia , Técnicas de Silenciamento de Genes , Humanos , Replicação Viral/fisiologia
9.
J Pathol ; 247(2): 177-185, 2019 02.
Artigo em Inglês | MEDLINE | ID: mdl-30350425

RESUMO

Dynamin plays an essential role in maintaining the structure and function of the glomerular filtration barrier. Specifically, dynamin regulates the actin cytoskeleton and the turnover of nephrin in podocytes, and knocking down dynamin expression causes proteinuria. Moreover, promoting dynamin oligomerization with Bis-T-23 restores podocyte function and reduces proteinuria in several animal models of chronic kidney disease. Thus, dynamin is a promising therapeutic target for treating chronic kidney disease. Here, we investigated the pathophysiological role of dynamin under proteinuric circumstances in a rat model and in humans. We found that glomerular Dnm2 and Dnm1 mRNA levels are increased prior to the onset of proteinuria in a rat model of spontaneous proteinuria. Also, in zebrafish embryos, we confirm that knocking down dynamin translation results in proteinuria. Finally, we show that the glomerular expression of dynamin and cathepsin L protein is increased in several human proteinuric kidney diseases. We propose that the increased expression of glomerular dynamin reflects an exhausted attempt to maintain and/or restore integrity of the glomerular filtration barrier. These results confirm that dynamin plays an important role in maintaining the glomerular filtration barrier, and they support the notion that dynamin is a promising therapeutic target in proteinuric kidney disease. © 2018 The Authors. The Journal of Pathology published by John Wiley & Sons Ltd on behalf of Pathological Society of Great Britain and Ireland.


Assuntos
Dinamina II/metabolismo , Dinamina I/metabolismo , Nefropatias/metabolismo , Glomérulos Renais/metabolismo , Proteinúria/metabolismo , Adulto , Idoso , Animais , Catepsina L/genética , Catepsina L/metabolismo , Modelos Animais de Doenças , Dinamina I/genética , Dinamina II/genética , Feminino , Taxa de Filtração Glomerular , Humanos , Nefropatias/genética , Nefropatias/fisiopatologia , Glomérulos Renais/fisiopatologia , Masculino , Pessoa de Meia-Idade , Proteinúria/genética , Proteinúria/fisiopatologia , Ratos Endogâmicos Dahl , Ratos Endogâmicos SHR , Fatores de Tempo , Regulação para Cima , Peixe-Zebra , Proteínas de Peixe-Zebra/genética , Proteínas de Peixe-Zebra/metabolismo
10.
Mol Biol Cell ; 30(1): 160-168, 2019 01 01.
Artigo em Inglês | MEDLINE | ID: mdl-30379601

RESUMO

The mitochondrial membrane undergoes extreme remodeling during fission. While a few membrane-squeezing proteins are recognized as the key drivers of fission, there is a growing body of evidence that strongly suggests that conical lipids play a critical role in regulating mitochondrial morphology and fission. However, the mechanisms by which proteins and lipids cooperate to execute fission have not been quantitatively investigated. Here, we computationally model the squeezing of the largely tubular mitochondrion and show that proteins and conical lipids can act synergistically to trigger buckling instability and achieve extreme constriction. More remarkably, the study reveals that the conical lipids can act with different fission proteins to induce hierarchical instabilities and create increasingly narrow and stable constrictions. We reason that this geometric plasticity imparts significant robustness to the fission reaction by arresting the elastic tendency of the membrane to rebound during protein polymerization and depolymerization cycles. Our in vitro study validates protein-lipid cooperativity in constricting membrane tubules. Overall, our work presents a general mechanism for achieving drastic topological remodeling in cellular membranes.


Assuntos
Dinâmica Mitocondrial , Actinas/metabolismo , Animais , Catálise , Linhagem Celular , Simulação por Computador , Dinamina II/metabolismo , Mamíferos , Lipídeos de Membrana/química , Mitocôndrias/metabolismo , Mitocôndrias/ultraestrutura , Membranas Mitocondriais/metabolismo , Membranas Mitocondriais/ultraestrutura , Modelos Biológicos , Reprodutibilidade dos Testes
11.
Blood Adv ; 2(23): 3540-3552, 2018 12 11.
Artigo em Inglês | MEDLINE | ID: mdl-30538113

RESUMO

Megakaryocyte (MK) migration from the bone marrow periosteal niche toward the vascular niche is a prerequisite for proplatelet extension and release into the circulation. The mechanism for this highly coordinated process is poorly understood. Here we show that dynasore (DNSR), a small-molecule inhibitor of dynamins (DNMs), or short hairpin RNA knockdown of DNM2 and DNM3 impairs directional migration in a human MK cell line or MKs derived from cultured CD34+ cells. Because cell migration requires actin cytoskeletal rearrangements, we measured actin polymerization and the activity of cytoskeleton regulator RhoA and found them to be decreased after inhibition of DNM2 and DNM3. Because SDF-1α is important for hematopoiesis, we studied the expression of its receptor CXCR4 in DNSR-treated cells. CXCR4 expression on the cell surface was increased, at least partially because of slower endocytosis and internalization after SDF-1α treatment. Combined inhibition of DNM2 and DNM3 or forced expression of dominant-negative Dnm2-K44A or GTPase-defective DNM3 diminished ß1 integrin (ITGB1) activity. DNSR-treated MKs showed an abnormally clustered staining pattern of Rab11, a marker of recycling endosomes. This suggests decreased recruitment of the recycling pathway in DNSR-treated cells. Altogether, we show that the GTPase activity of DNMs, which governs endocytosis and regulates cell receptor trafficking, exerts control on MK migration toward SDF-1α gradients, such as those originating from the vascular niche. DNMs play a critical role in MKs by triggering membrane-cytoskeleton rearrangements downstream of CXCR4 and integrins.


Assuntos
Dinamina III/metabolismo , Dinamina II/metabolismo , Integrina beta1/metabolismo , Receptores CXCR4/metabolismo , Citoesqueleto de Actina , Linhagem Celular , Membrana Celular/metabolismo , Movimento Celular , Dinamina II/antagonistas & inibidores , Dinamina II/genética , Dinamina III/antagonistas & inibidores , Dinamina III/genética , Humanos , Megacariócitos/citologia , Megacariócitos/metabolismo , Interferência de RNA , RNA Interferente Pequeno/metabolismo , Proteínas rab de Ligação ao GTP/metabolismo , Proteína rhoA de Ligação ao GTP/metabolismo
12.
Nat Commun ; 9(1): 5239, 2018 12 07.
Artigo em Inglês | MEDLINE | ID: mdl-30531964

RESUMO

Dynamin-related protein 1 (Drp1) is essential for mitochondrial and peroxisomal fission. Recent studies propose that Drp1 does not sever but rather constricts mitochondrial membranes allowing dynamin 2 (Dnm2) to execute final scission. Here, we report that unlike Drp1, Dnm2 is dispensable for peroxisomal and mitochondrial fission, as these events occurred in Dnm2 knockout cells. Fission events were also observed in mouse embryonic fibroblasts lacking Dnm1, 2 and 3. Using reconstitution experiments on preformed membrane tubes, we show that Drp1 alone both constricts and severs membrane tubes. Scission required the membrane binding, self-assembling and GTPase activities of Drp1 and occurred on tubes up to 250 nm in radius. In contrast, Dnm2 exhibited severely restricted fission capacity with occasional severing of tubes below 50 nm in radius. We conclude that Drp1 has both membrane constricting and severing abilities and is the dominant dynamin performing mitochondrial and peroxisomal fission.


Assuntos
Dinaminas/metabolismo , Fusão de Membrana , Mitocôndrias/metabolismo , Peroxissomos/metabolismo , Animais , Linhagem Celular , Dinamina II/genética , Dinamina II/metabolismo , Dinaminas/genética , Embrião de Mamíferos/citologia , Fibroblastos/metabolismo , Células HeLa , Humanos , Camundongos Knockout , Microscopia Confocal , Dinâmica Mitocondrial , Membranas Mitocondriais/metabolismo
13.
Nat Commun ; 9(1): 4849, 2018 11 19.
Artigo em Inglês | MEDLINE | ID: mdl-30451841

RESUMO

Myotubular myopathy (MTM) is a severe X-linked disease without existing therapies. Here, we show that tamoxifen ameliorates MTM-related histopathological and functional abnormalities in mice, and nearly doubles survival. The beneficial effects of tamoxifen are mediated primarily via estrogen receptor signaling, as demonstrated through in vitro studies and in vivo phenotypic rescue with estradiol. RNA sequencing and protein expression analyses revealed that rescue is mediated in part through post-transcriptional reduction of dynamin-2, a known MTM modifier. These findings demonstrate an unexpected ability of tamoxifen to improve the murine MTM phenotype, providing preclinical evidence to support clinical translation.


Assuntos
Dinamina II/genética , Músculo Esquelético/efeitos dos fármacos , Miopatias Congênitas Estruturais/tratamento farmacológico , Substâncias Protetoras/farmacologia , Proteínas Tirosina Fosfatases não Receptoras/genética , Receptores Estrogênicos/genética , Tamoxifeno/farmacologia , Animais , Modelos Animais de Doenças , Avaliação Pré-Clínica de Medicamentos , Dinamina II/metabolismo , Estradiol/metabolismo , Estradiol/farmacologia , Acoplamento Excitação-Contração/efeitos dos fármacos , Feminino , Expressão Gênica/efeitos dos fármacos , Sequenciamento de Nucleotídeos em Larga Escala , Humanos , Longevidade/efeitos dos fármacos , Masculino , Camundongos , Camundongos Knockout , Atividade Motora/efeitos dos fármacos , Músculo Esquelético/metabolismo , Músculo Esquelético/patologia , Miofibrilas/efeitos dos fármacos , Miofibrilas/metabolismo , Miofibrilas/ultraestrutura , Miopatias Congênitas Estruturais/genética , Miopatias Congênitas Estruturais/metabolismo , Miopatias Congênitas Estruturais/patologia , Proteínas Tirosina Fosfatases não Receptoras/deficiência , Receptores Estrogênicos/metabolismo
14.
Nat Commun ; 9(1): 4848, 2018 11 19.
Artigo em Inglês | MEDLINE | ID: mdl-30451843

RESUMO

X-linked myotubular myopathy (XLMTM, also known as XLCNM) is a severe congenital muscular disorder due to mutations in the myotubularin gene, MTM1. It is characterized by generalized hypotonia, leading to neonatal death of most patients. No specific treatment exists. Here, we show that tamoxifen, a well-known drug used against breast cancer, rescues the phenotype of Mtm1-deficient mice. Tamoxifen increases lifespan several-fold while improving overall motor function and preventing disease progression including lower limb paralysis. Tamoxifen corrects functional, histological and molecular hallmarks of XLMTM, with improved force output, myonuclei positioning, myofibrillar structure, triad number, and excitation-contraction coupling. Tamoxifen normalizes the expression level of the XLMTM disease modifiers DNM2 and PI3KC2B, likely contributing to the phenotypic rescue. Our findings demonstrate that tamoxifen is a promising candidate for clinical evaluation in XLMTM patients.


Assuntos
Atividade Motora/efeitos dos fármacos , Músculo Esquelético/efeitos dos fármacos , Miopatias Congênitas Estruturais/tratamento farmacológico , Substâncias Protetoras/farmacologia , Proteínas Tirosina Fosfatases não Receptoras/genética , Tamoxifeno/farmacologia , Animais , Classe II de Fosfatidilinositol 3-Quinases/genética , Classe II de Fosfatidilinositol 3-Quinases/metabolismo , Modelos Animais de Doenças , Progressão da Doença , Dinamina II/genética , Dinamina II/metabolismo , Estimulação Elétrica , Acoplamento Excitação-Contração/efeitos dos fármacos , Feminino , Expressão Gênica/efeitos dos fármacos , Genes Letais , Humanos , Longevidade/efeitos dos fármacos , Masculino , Camundongos , Camundongos Knockout , Músculo Esquelético/metabolismo , Músculo Esquelético/patologia , Miofibrilas/efeitos dos fármacos , Miofibrilas/metabolismo , Miofibrilas/ultraestrutura , Miopatias Congênitas Estruturais/genética , Miopatias Congênitas Estruturais/metabolismo , Miopatias Congênitas Estruturais/patologia , Proteínas Tirosina Fosfatases não Receptoras/deficiência
15.
J Struct Biol ; 204(3): 406-419, 2018 12.
Artigo em Inglês | MEDLINE | ID: mdl-30352275

RESUMO

Human cytomegalovirus (HCMV) entry into susceptible cells is a fast intricate process that is not fully understood. Although, previous studies explored different aspects of this process by means of biochemical and inhibitors assays, a clear morphological characterization of its steps at the ultrastructural level is still lacking. We attempted to characterize those intermediates involved during HCMV entry by developing a methodological approach that resulted in optimal ultrastructure preservation and allowed for 3D imaging. It involves rapid freezing and cryosubstitution which ensure a clear visibility of membranous leaflets as well as retained membranous continuity. Likewise, it delivered a reproducible optimization of the growth and infection conditions that are pivotal towards maintaining biologically active enriched input virus particles. Data acquisition was achieved through STEM tomography in a 3D context. Indeed, several intermediates that characterize HCMV entry-related events were observed both extra- and intracellularly. Some of the cell-membrane associated viral particles that we referred to as "Pinocchio particles" were morphologically altered in comparison to the cell-free virions. We were also able to characterize intracellular fusion intermediates taking place between the viral envelope and the vesicular membranes. Furthermore, inhibiting actin polymerization by Latrunculin-A enabled us to spot fusion-like intermediates of the viral envelope with the host cell plasma membrane that we did not observe in the untreated infected cells. Our data also suggests that Dyngo-4a; a dynamin-2 inhibitor, does not interfere with the internalization of the HCMV into the host cells as previously deduced.


Assuntos
Citomegalovirus/fisiologia , Tomografia com Microscopia Eletrônica/métodos , Vírion/fisiologia , Internalização do Vírus , Linhagem Celular , Membrana Celular/metabolismo , Membrana Celular/ultraestrutura , Membrana Celular/virologia , Células Cultivadas , Citomegalovirus/ultraestrutura , Dinamina II/antagonistas & inibidores , Dinamina II/metabolismo , Fibroblastos/metabolismo , Fibroblastos/ultraestrutura , Fibroblastos/virologia , Interações Hospedeiro-Patógeno/efeitos dos fármacos , Humanos , Hidrazonas/farmacologia , Imageamento Tridimensional/métodos , Naftóis/farmacologia , Reprodutibilidade dos Testes , Vírion/ultraestrutura
16.
Proc Natl Acad Sci U S A ; 115(43): 11066-11071, 2018 10 23.
Artigo em Inglês | MEDLINE | ID: mdl-30291191

RESUMO

Centronuclear myopathies (CNM) are a group of severe muscle diseases for which no effective therapy is currently available. We have previously shown that reduction of the large GTPase DNM2 in a mouse model of the X-linked form, due to loss of myotubularin phosphatase MTM1, prevents the development of the skeletal muscle pathophysiology. As DNM2 is mutated in autosomal dominant forms, here we tested whether DNM2 reduction can rescue DNM2-related CNM in a knock-in mouse harboring the p.R465W mutation (Dnm2 RW/+) and displaying a mild CNM phenotype similar to patients with the same mutation. A single intramuscular injection of adeno-associated virus-shRNA targeting Dnm2 resulted in reduction in protein levels 5 wk post injection, with a corresponding improvement in muscle mass and fiber size distribution, as well as an improvement in histopathological CNM features. To establish a systemic treatment, weekly i.p. injections of antisense oligonucleotides targeting Dnm2 were administered to Dnm2 RW/+mice for 5 wk. While muscle mass, histopathology, and muscle ultrastructure were perturbed in Dnm2 RW/+mice compared with wild-type mice, these features were indistinguishable from wild-type mice after reducing DNM2. Therefore, DNM2 knockdown via two different strategies can efficiently correct the myopathy due to DNM2 mutations, and it provides a common therapeutic strategy for several forms of centronuclear myopathy. Furthermore, we provide an example of treating a dominant disease by targeting both alleles, suggesting that this strategy may be applied to other dominant diseases.


Assuntos
Dinamina II/genética , Miopatias Congênitas Estruturais/genética , Animais , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Músculo Esquelético/metabolismo , Mutação/genética , Proteínas Tirosina Fosfatases não Receptoras/genética
17.
Mol Biol Cell ; 29(24): 2959-2968, 2018 11 26.
Artigo em Inglês | MEDLINE | ID: mdl-30188768

RESUMO

New methods in stem cell 3D organoid tissue culture, advanced imaging, and big data image analytics now allow tissue-scale 4D cell biology, but currently available analytical pipelines are inadequate for handing and analyzing the resulting gigabytes and terabytes of high-content imaging data. We expressed fluorescent protein fusions of clathrin and dynamin2 at endogenous levels in genome-edited human embryonic stem cells, which were differentiated into hESC-derived intestinal epithelial organoids. Lattice light-sheet imaging with adaptive optics (AO-LLSM) allowed us to image large volumes of these organoids (70 × 60 × 40 µm xyz) at 5.7 s/frame. We developed an open-source data analysis package termed pyLattice to process the resulting large (∼60 Gb) movie data sets and to track clathrin-mediated endocytosis (CME) events. CME tracks could be recorded from ∼35 cells at a time, resulting in ∼4000 processed tracks per movie. On the basis of their localization in the organoid, we classified CME tracks into apical, lateral, and basal events and found that CME dynamics is similar for all three classes, despite reported differences in membrane tension. pyLattice coupled with AO-LLSM makes possible quantitative high temporal and spatial resolution analysis of subcellular events within tissues.


Assuntos
Vesículas Revestidas por Clatrina/metabolismo , Clatrina/metabolismo , Células-Tronco Embrionárias Humanas/citologia , Processamento de Imagem Assistida por Computador/métodos , Intestinos/citologia , Animais , Big Data , Técnicas de Cultura de Células/métodos , Diferenciação Celular/fisiologia , Dinamina II/metabolismo , Endocitose/fisiologia , Células-Tronco Embrionárias Humanas/metabolismo , Humanos , Mucosa Intestinal/citologia , Mucosa Intestinal/metabolismo , Camundongos , Organoides/citologia , Organoides/diagnóstico por imagem , Organoides/metabolismo
18.
Neurol Sci ; 39(12): 2043-2051, 2018 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-30232666

RESUMO

Centronuclear myopathies (CNMs) are a group of clinically and genetically heterogeneous muscle disorders. Here, we report a cohort of seven CNM patients with their clinical, histological, and morphological features. In addition, using the next-generation sequencing (NGS) technique (5/7 patients), we identified small indels: intronic, exonic, and missense mutations in MTM1, DNM2, and RYR1 genes. Further genetic studies revealed skewed X-chromosome inactivation in two female patients carrying MTM1 mutations. Based on the results of genetic analysis, these seven patients were classified as (1) X-linked recessive myotubular myopathy (patients 1-3) with MTM1 mutations and mild phenotype, (2) the autosomal dominant CNM (patients 4-6) with DNM2 mutations, and (3) the autosomal recessive CNM (patient 7) with RYR1 mutations. In all patients, histological findings featured a high proportion of fibers with central nuclei. Radial arrangement of the sarcoplasmic strands was observed in DNM2-CNM and RYR1-CNM patients. Muscle magnetic resonance imaging (MRI) revealed a proximal pattern of involvement presented in both MTM1-CNM and RYR1-CNM patients. A distal pattern of involvement was present in DNM2-CNM patients. Our findings thereby identified a number of novel features that expand the reported clinicopathological phenotype of CNMs in China.


Assuntos
Dinamina II/genética , Mutação/genética , Miopatias Congênitas Estruturais/genética , Proteínas Tirosina Fosfatases não Receptoras/genética , Adulto , Grupo com Ancestrais do Continente Asiático , Pré-Escolar , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Músculo Esquelético/diagnóstico por imagem , Músculo Esquelético/patologia , Músculo Esquelético/ultraestrutura , Miopatias Congênitas Estruturais/diagnóstico por imagem , Canal de Liberação de Cálcio do Receptor de Rianodina/genética , Adulto Jovem
19.
Vet Res ; 49(1): 92, 2018 Sep 17.
Artigo em Inglês | MEDLINE | ID: mdl-30223898

RESUMO

Caliciviruses in the genus Sapovirus are a significant cause of viral gastroenteritis in humans and animals. However, the mechanism of their entry into cells is not well characterized. Here, we determined the entry mechanism of porcine sapovirus (PSaV) strain Cowden into permissive LLC-PK cells. The inhibition of clathrin-mediated endocytosis using chlorpromazine, siRNAs, and a dominant negative (DN) mutant blocked entry and infection of PSaV Cowden strain, confirming a role for clathrin-mediated internalization. Entry and infection were also inhibited by the cholesterol-sequestering drug methyl-ß-cyclodextrin and was restored by the addition of soluble cholesterol, indicating that cholesterol also contributes to entry and infection of this strain. Furthermore, the inhibition of dynamin GTPase activity by dynasore, siRNA depletion of dynamin II, or overexpression of a DN mutant of dynamin II reduced the entry and infection, suggesting that dynamin mediates the fission and detachment of clathrin- and cholesterol-pits for entry of this strain. In contrast, the inhibition of caveolae-mediated endocytosis using nystatin, siRNAs, or a DN mutant had no inhibitory effect on entry and infection of this strain. It was further determined that cell entry of PSaV Cowden strain required actin rearrangements for vesicle internalization, endosomal trafficking from early to late endosomes through microtubules, and late endosomal acidification for uncoating. We conclude that PSaV strain Cowden is internalized into LLC-PK cells by clathrin- and cholesterol-mediated endocytosis that requires dynamin II and actin rearrangement, and that the uncoating occurs in the acidified late endosomes after trafficking from the early endosomes through microtubules.


Assuntos
Infecções por Caliciviridae/veterinária , Colesterol/fisiologia , Clatrina/fisiologia , Dinamina II/fisiologia , Endocitose , Sapovirus/fisiologia , Doenças dos Suínos/virologia , Animais , Infecções por Caliciviridae/virologia , Gastroenterite/veterinária , Gastroenterite/virologia , Células HeLa , Humanos , Células LLC-PK1 , Suínos
20.
J Cell Physiol ; 233(10): 7047-7056, 2018 10.
Artigo em Inglês | MEDLINE | ID: mdl-29744878

RESUMO

Photochemical tissue bonding (PTB) has been found to promote the healing of Achilles tendon tissue injury and to reduce postoperative complications. However, the underlying cellular and molecular mechanisms are not clear. In this study, the cell proliferation, ROS generation, migration and the protein expression of DNM2, NF-κB p65, TGF-ß1 and VEGF in tenocytes after PTB treatment were measured by CCK-8, flow cytometry, Transwell and western blot assay, respectively. And those in tenocytes after DNM2 silencing or overexpressing or treatment with inhibitors of NF-κB, ROS and RhoA were also measured. Our results showed that 10 mW PTB treatment for 80 and 120 s significantly increased cell proliferation and increased ROS generation in tenocytes. 10 mW PTB treatment for 40 and 80 s significantly activated RhoA and increased the protein expression of DNM2, NF-κB p65, TGF-ß1 and VEGF, but 10 mW PTB treatment for 120 s decreased the protein expression of those. DNM2 silencing significantly suppressed cell migration and the expression of DNM2, TGF-ß1, and VEGF in tenocytes after PTB treatment (10 mW, 80 s), which was inhibited by DNM2 overexpression. Individual treatment with inhibitor of NF-κB, ROS, and RhoA in tenocytes showed decreased protein expression of DNM2, TGF-ß1, and VEGF. Moreover, in vivo experiment found that PTB treatment significantly inhibited cell apoptosis and the expression of DNM2, NF-κB p65, RhoA, TGF-ß1, and VEGF in a time-dependent manner. Taken together, our results suggest that PTB promotes the proliferation and migration of injured tenocytes through ROS/RhoA/NF-κB/DNM2 signaling pathway.


Assuntos
Proliferação de Células/efeitos dos fármacos , Dinamina II/metabolismo , Fármacos Fotossensibilizantes/farmacologia , Espécies Reativas de Oxigênio/metabolismo , Tenócitos/metabolismo , Movimento Celular/fisiologia , Proliferação de Células/fisiologia , Dinaminas/efeitos dos fármacos , Dinaminas/metabolismo , Humanos , NF-kappa B/metabolismo , Fosforilação/efeitos dos fármacos , Transdução de Sinais/efeitos dos fármacos , Tenócitos/efeitos dos fármacos , Proteína rhoA de Ligação ao GTP/metabolismo
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