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1.
PLoS Biol ; 17(11): e3000531, 2019 11.
Artigo em Inglês | MEDLINE | ID: mdl-31682603

RESUMO

Recycling endosomes regulate plasma membrane recycling. Recently, recycling endosome-associated proteins have been implicated in the positioning and orientation of the mitotic spindle and cytokinesis. Loss of MYO5B, encoding the recycling endosome-associated myosin Vb, is associated with tumor development and tissue architecture defects in the gastrointestinal tract. Whether loss of MYO5B expression affects mitosis is not known. Here, we demonstrate that loss of MYO5B expression delayed cytokinesis, perturbed mitotic spindle orientation, led to the misorientation of the plane of cell division during the course of mitosis, and resulted in the delamination of epithelial cells. Remarkably, the effects on spindle orientation, but not cytokinesis, were a direct consequence of physical hindrance by giant late endosomes, which were formed in a chloride channel-sensitive manner concomitant with a redistribution of chloride channels from the cell periphery to late endosomes upon loss of MYO5B. Rab7 availability was identified as a limiting factor for the development of giant late endosomes. In accordance, increasing rab7 availability corrected mitotic spindle misorientation and cell delamination in cells lacking MYO5B expression. In conclusion, we identified a novel role for MYO5B in the regulation of late endosome size control and identify the inability to control late endosome size as an unexpected novel mechanism underlying defects in cell division orientation and epithelial architecture.


Assuntos
Endossomos/metabolismo , Cadeias Pesadas de Miosina/metabolismo , Miosina Tipo V/metabolismo , Fuso Acromático/metabolismo , Animais , Células CACO-2 , Adesão Celular/fisiologia , Divisão Celular/fisiologia , Linhagem Celular , Membrana Celular/metabolismo , Citocinese/genética , Citocinese/fisiologia , Endossomos/genética , Células Epiteliais/metabolismo , Feminino , Células HEK293 , Humanos , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Mitose/fisiologia , Cadeias Pesadas de Miosina/genética , Miosina Tipo V/genética , Proteínas rab de Ligação ao GTP/metabolismo
2.
Int J Biochem Cell Biol ; 116: 105620, 2019 11.
Artigo em Inglês | MEDLINE | ID: mdl-31561018

RESUMO

Diazepam is a medicament of the benzodiazepine family and it typically produces a sedative effect. Researchers have revealed that diazepam can induce melanogenesis and produce dendrite-like structures in B16 melanoma cells. However, the associated mechanisms of melanogenesis and phenotypic alterations have mostly remained unknown. In this study, we determined the effects of diazepam on melanogenesis, cellular phenotypic alterations, the location of melanosomes and the expression of relevant proteins in melanocytes using Masson-Fontana ammoniacal silver staining, scanning electron microscopy, immunocytochemistry and western blot analysis. Our results collectively indicated that diazepam had a pivotal role in melanocytes by enhancing melanin synthesis, melanocyte dendricity, melanosome trafficking, and capture at the dendrite tips. These functions might be attributed to the fact that diazepam activated the peripheral benzodiazepine receptor (PBR). This increased intracellular levels of cAMP, which stimulated the phosphorylation of cAMP response element-binding (CREB). As a result, this increased the tyrosinase, microphthalmia-associated transcription factor (MITF), Rab27a, Myosin Va, Rab17 and Cdc42 expression. This caused melanogenesis and melanosome transport. Therefore, our findings may provide a potential strategy for treating anti-hypopigmentation disorders.


Assuntos
Proteínas Quinases Dependentes de AMP Cíclico/genética , AMP Cíclico/metabolismo , Diazepam/farmacologia , Hipnóticos e Sedativos/farmacologia , Melanócitos/efeitos dos fármacos , Melanossomas/efeitos dos fármacos , Receptores de GABA-A/genética , Animais , Transporte Biológico/efeitos dos fármacos , Proteína de Ligação a CREB/genética , Proteína de Ligação a CREB/metabolismo , Linhagem Celular , Linhagem Celular Tumoral , Proteínas Quinases Dependentes de AMP Cíclico/metabolismo , Regulação da Expressão Gênica , Humanos , Melanócitos/citologia , Melanócitos/metabolismo , Melanossomas/metabolismo , Camundongos , Fator de Transcrição Associado à Microftalmia/genética , Fator de Transcrição Associado à Microftalmia/metabolismo , Monofenol Mono-Oxigenase/genética , Monofenol Mono-Oxigenase/metabolismo , Cadeias Pesadas de Miosina/genética , Cadeias Pesadas de Miosina/metabolismo , Miosina Tipo V/genética , Miosina Tipo V/metabolismo , Receptores de GABA-A/metabolismo , Transdução de Sinais , Proteína cdc42 de Ligação ao GTP/genética , Proteína cdc42 de Ligação ao GTP/metabolismo , Proteínas rab de Ligação ao GTP/genética , Proteínas rab de Ligação ao GTP/metabolismo , Proteínas rab27 de Ligação ao GTP/genética , Proteínas rab27 de Ligação ao GTP/metabolismo
3.
Neurourol Urodyn ; 38(5): 1266-1277, 2019 06.
Artigo em Inglês | MEDLINE | ID: mdl-31006139

RESUMO

AIM: Diabetic bladder dysfunction (DBD) is one of the most common and bothersome complications of diabetes mellitus (DM). This study aimed to investigate the functional, structural, and molecular changes of the bladder at 0, 3, 6, 9, and 12 weeks after DM induction by streptozotocin (STZ) in male C57BL/6 mice. METHODS: Male C57BL/6J mice were injected with STZ (130 mg/kg). Then, diabetic general characteristics, cystometry test, histomorphometry, and contractile responses to α, ß-methylene ATP, KCl, electrical-field stimulation, carbachol were performed at 0, 3, 6, 9, and 12 weeks after induction. Finally, protein and messenger RNA (mRNA) expressions of myosin Va and SLC17A9 were quantified. RESULTS: DM mice exhibited lower body weight, voiding efficiency and higher water intake, urine production, fasting blood glucose, oral glucose tolerance test, bladder wall thickness, maximum bladder capacity, residual volume, bladder compliance. In particular, nonvoiding contractions has increased more than five times at 6 weeks. And the amplitudes of spontaneous activity, contractile responses to all stimulus was about two times higher at 6 weeks but cut almost in half at 12 weeks. The protein and mRNA expressions of myosin Va and SLC17A9 were about two times higher at 6 weeks, but myosin Va was reverted nearly 40% while SLC17A9 is still higher at 12 weeks. CONCLUSIONS: DBD transitioned from a compensated state to a decompensated state in STZ-induced DM mice at 9 to 12 weeks after DM induction. Our molecular data suggest that the transition may be closely related to the alterations of myosin Va and SLC17A9 expression levels in the bladder with time.


Assuntos
Diabetes Mellitus Experimental/patologia , Doenças da Bexiga Urinária/patologia , Animais , Peso Corporal , Diabetes Mellitus Experimental/complicações , Diabetes Mellitus Experimental/genética , Ingestão de Líquidos , Estimulação Elétrica , Teste de Tolerância a Glucose , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Contração Muscular/efeitos dos fármacos , Cadeias Pesadas de Miosina/biossíntese , Cadeias Pesadas de Miosina/genética , Miosina Tipo V/biossíntese , Miosina Tipo V/genética , Proteínas de Transporte de Nucleotídeos/biossíntese , Proteínas de Transporte de Nucleotídeos/genética , RNA Mensageiro/biossíntese , RNA Mensageiro/genética , Estimulação Química , Doenças da Bexiga Urinária/etiologia , Doenças da Bexiga Urinária/genética , Urodinâmica
4.
Dev Dyn ; 248(4): 284-295, 2019 04.
Artigo em Inglês | MEDLINE | ID: mdl-30801852

RESUMO

BACKGROUND: During heart morphogenesis, the cardiac chambers undergo ballooning: a process involving regionalized elongation of cardiomyocytes. Cardiomyocyte shape changes require reorganization of the actin cytoskeleton; however, the genetic regulation of this process is not well understood. RESULTS: From a forward genetic screen, we identified the zebrafish uq 23ks mutant which manifests chamber ballooning defects. Whole-genome sequencing-mapping identified a truncating mutation in the gene, myo5b. myo5b encodes an atypical myosin required for endosome recycling and, consistent with this, increased vesicles were observed in myo5b mutant cardiomyocytes. Expression of RFP-Rab11a (a recycling endosome marker) confirmed increased recycling endosomes in cardiomyocytes of myo5b mutants. To investigate potential cargo of MyoVb-associated vesicles, we examined the adherens junction protein, N-cadherin. N-cadherin appeared mispatterned at cell junctions, and an increase in the number of intracellular particles was also apparent. Co-localization with RFP-Rab11a confirmed increased N-cadherin-positive recycling endosomes, demonstrating N-cadherin trafficking is perturbed in myo5b mutants. Finally, phalloidin staining showed disorganized F-actin in myo5b cardiomyocytes, suggesting the cytoskeleton fails to remodel, obstructing chamber ballooning. CONCLUSIONS: MyoVb is required for cardiomyocyte endosomal recycling and appropriate N-cadherin localization during the onset of chamber ballooning. Cardiomyocytes lacking MyoVb are unable to reorganize their actin cytoskeleton, resulting in failed chamber ballooning. Developmental Dynamics 248:284-295, 2019. © 2019 Wiley Periodicals, Inc.


Assuntos
Caderinas/metabolismo , Citoesqueleto/ultraestrutura , Coração/crescimento & desenvolvimento , Miócitos Cardíacos/metabolismo , Miosina Tipo V/fisiologia , Animais , Forma Celular , Citoesqueleto/metabolismo , Endossomos/metabolismo , Humanos , Miocárdio/citologia , Miócitos Cardíacos/citologia , Miócitos Cardíacos/ultraestrutura , Miosina Tipo V/genética , Miosinas/genética , Miosinas/fisiologia , Peixe-Zebra , Proteínas de Peixe-Zebra/genética , Proteínas de Peixe-Zebra/fisiologia
5.
Curr Genet ; 65(4): 919-940, 2019 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-30715564

RESUMO

Cells have elaborated a complex strategy to maintain protein homeostasis under physiological as well as stress conditions with the aim to ensure the smooth functioning of vital processes and producing healthy offspring. Impairment of one of the most important processes in living cells, translation, might have serious consequences including various brain disorders in humans. Here, we describe a variant of the translation initiation factor eIF3a, Rpg1-3, mutated in its PCI domain that displays an attenuated translation efficiency and formation of reversible assemblies at physiological growth conditions. Rpg1-3-GFP assemblies are not sequestered within mother cells only as usual for misfolded-protein aggregates and are freely transmitted from the mother cell into the bud although they are of non-amyloid nature. Their bud-directed transmission and the active movement within the cell area depend on the intact actin cytoskeleton and the related molecular motor Myo2. Mutations in the Rpg1-3 protein render not only eIF3a but, more importantly, also the eIF3 core complex prone to aggregation that is potentiated by the limited availability of Hsp70 and Hsp40 chaperones. Our results open the way to understand mechanisms yeast cells employ to cope with malfunction and aggregation of essential proteins and their complexes.


Assuntos
Fator de Iniciação 3 em Eucariotos/genética , Cadeias Pesadas de Miosina/genética , Miosina Tipo V/genética , Agregados Proteicos/genética , Proteínas de Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/genética , Citoesqueleto de Actina/genética , Proteínas de Choque Térmico HSP40/genética , Proteínas de Choque Térmico HSP70/genética , Humanos , Mitocôndrias , Mutação , Saccharomyces cerevisiae/crescimento & desenvolvimento
6.
J Biol Chem ; 294(15): 5896-5906, 2019 04 12.
Artigo em Inglês | MEDLINE | ID: mdl-30804213

RESUMO

Class V myosins are actin-dependent motors, which recognize numerous cellular cargos mainly via the C-terminal globular tail domain (GTD). Myo2, a yeast class V myosin, can transport a broad range of organelles. However, little is known about the capacity of Myo2-GTD to recognize such a diverse array of cargos specifically at the molecular level. Here, we solved crystal structures of Myo2-GTD (at 1.9-3.1 Å resolutions) in complex with three cargo adaptor proteins: Smy1 (for polarization of secretory vesicles), Inp2 (for peroxisome transport), and Mmr1 (for mitochondria transport). The structures of Smy1- and Inp2-bound Myo2-GTD, along with site-directed mutagenesis experiments, revealed a binding site in subdomain-I having a hydrophobic groove with high flexibility enabling Myo2-GTD to accommodate different protein sequences. The Myo2-GTD-Mmr1 complex structure confirmed and complemented a previously identified mitochondrion/vacuole-specific binding region. Moreover, differences between the conformations and locations of cargo-binding sites identified here for Myo2 and those reported for mammalian MyoVA (MyoVA) suggest that class V myosins potentially have co-evolved with their specific cargos. Our structural and biochemical analysis not only uncovers a molecular mechanism that explains the diverse cargo recognition by Myo2-GTD, but also provides structural information useful for future functional studies of class V myosins in cargo transport.


Assuntos
Cadeias Pesadas de Miosina/química , Cadeias Pesadas de Miosina/metabolismo , Miosina Tipo V/química , Miosina Tipo V/metabolismo , Proteínas de Saccharomyces cerevisiae/química , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/enzimologia , Sítios de Ligação , Evolução Molecular , Proteínas Associadas aos Microtúbulos/química , Proteínas Associadas aos Microtúbulos/genética , Proteínas Associadas aos Microtúbulos/metabolismo , Proteínas Mitocondriais/química , Proteínas Mitocondriais/genética , Proteínas Mitocondriais/metabolismo , Complexos Multiproteicos/química , Complexos Multiproteicos/genética , Complexos Multiproteicos/metabolismo , Cadeias Pesadas de Miosina/genética , Miosina Tipo V/genética , Domínios Proteicos , Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/genética
7.
Biosci Rep ; 39(3)2019 03 29.
Artigo em Inglês | MEDLINE | ID: mdl-30733278

RESUMO

Myosin Va (MyoVa) is an actin-based molecular motor that plays key roles in the final stages of secretory pathways, including neurotransmitter release. Several studies have addressed how MyoVa coordinates the trafficking of secretory vesicles, but why this molecular motor is found in exosomes is still unclear. In this work, using a yeast two-hybrid screening system, we identified the direct interaction between the globular tail domain (GTD) of MyoVa and four protein components of exosomes: the WD repeat-containing protein 48 (WDR48), the cold shock domain-containing protein E1 (CSDE1), the tandem C2 domain-containing protein 1 (TC2N), and the enzyme spermine synthase (SMS). The interaction between the GTD of MyoVa and SMS was further validated in vitro and displayed a K d in the low micromolar range (3.5 ± 0.5 µM). SMS localized together with MyoVa in cytoplasmic vesicles of breast cancer MCF-7 and neuroblastoma SH-SY5Y cell lines, known to produce exosomes. Moreover, MYO5A knockdown decreased the expression of SMS gene and rendered the distribution of SMS protein diffuse, supporting a role for MyoVa in SMS expression and targeting.


Assuntos
Vesículas Citoplasmáticas/metabolismo , Exossomos/metabolismo , Cadeias Pesadas de Miosina/metabolismo , Miosina Tipo V/metabolismo , Espermina Sintase/metabolismo , Sítios de Ligação , Linhagem Celular Tumoral , Células Cultivadas , Exossomos/genética , Fibroblastos/citologia , Fibroblastos/metabolismo , Regulação da Expressão Gênica , Humanos , Células MCF-7 , Cadeias Pesadas de Miosina/genética , Miosina Tipo V/genética , Ligação Proteica , Transporte Proteico , Interferência de RNA , Espermina Sintase/genética , Técnicas do Sistema de Duplo-Híbrido
8.
Fungal Genet Biol ; 125: 13-27, 2019 04.
Artigo em Inglês | MEDLINE | ID: mdl-30615944

RESUMO

In filamentous fungi, polarized growth is the result of vesicle secretion at the hyphal apex. Motor proteins mediate vesicle transport to target destinations on the plasma membrane via actin and microtubule cytoskeletons. Myosins are motor proteins associated with actin filaments. Specifically, class V myosins are responsible for cargo transport in eukaryotes. We studied the dynamics and localization of myosin V in wild type hyphae of Neurospora crassa and in hyphae that lacked MYO-5. In wild type hyphae, MYO-5-GFP was localized concentrated in the hyphal apex and colocalized with Spitzenkörper. Photobleaching studies showed that MYO-5-GFP was transported to the apex from subapical hyphal regions. The deletion of the class V myosin resulted in a reduced rate of hyphal growth, apical hyperbranching, and intermittent loss of hyphal polarity. MYO-5 did not participate in breaking the symmetrical growth during germination but contributed in the apical organization upon establishment of polarized growth. In the Δmyo-5 mutant, actin was organized into thick cables in the apical and subapical hyphal regions, and the number of endocytic patches was reduced. The microvesicles-chitosomes observed with CHS-1-GFP were distributed as a cloud occupying the apical dome and not in the Spitzenkörper as the WT strain. The mitochondrial movement was not associated with MYO-5, but tubular vacuole position is MYO-5-dependent. These results suggest that MYO-5 plays a role in maintaining apical organization and the integrity of the Spitzenkörper and is required for normal hyphal growth, polarity, septation, conidiation, and proper conidial germination.


Assuntos
Citoesqueleto de Actina/genética , Hifas/genética , Miosina Tipo V/genética , Neurospora crassa/genética , Membrana Celular/genética , Polaridade Celular/genética , Proteínas Fúngicas/genética , Regulação Fúngica da Expressão Gênica/genética , Proteínas de Fluorescência Verde/genética , Hifas/crescimento & desenvolvimento , Neurospora crassa/crescimento & desenvolvimento
9.
Genome Biol Evol ; 11(2): 415-430, 2019 02 01.
Artigo em Inglês | MEDLINE | ID: mdl-30496538

RESUMO

We analyzed evolutionary rates of conserved, duplicated myosin V (myo5) genes in nine teleost species to examine the outcomes of duplication events. Syntenic analysis and ancestral chromosome mapping suggest one tandem gene duplication event leading to the appearance of myo5a and myo5c, two rounds of whole genome duplication for vertebrates, and an additional round of whole genome duplication for teleosts account for the presence and location of the myo5 genes and their duplicates in teleosts and other vertebrates and the timing of the duplication events. Phylogenetic analyses reveal a previously unidentified myo5 clade that we refer to now as myo5bb. Analysis using dN/dS rate comparisons revealed large regions within duplicated myo5 genes that are highly conserved. Codons identified in other studies as encoding functionally important portions of the Myo5a and Myo5b proteins are shown to be highly conserved within the newly identified myo5bb clade and in other myo5 duplicates. As much as 30% of 319 codons encoding the cargo-binding domain in the myo5aa genes are conserved in all three codon positions in nine teleost species. For the myo5bb cargo-binding domain, 6.6% of 336 codons have zero substitutions in all nine teleost species. Using molecular evolution assays, we identify the myo5bb branch as being subject to evolutionary rate variation with the cargo-binding domain, having 20% of the sites under positive selection and the motor domain having 8% of its sites under positive selection. The high number of invariant codons coupled with relatively high dN/dS values in the region of the myo5 genes encoding the ATP-binding domain suggests the encoded proteins retain function and may have acquired novel functions associated with changes to the cargo-binding domain.


Assuntos
Evolução Molecular , Peixes/genética , Modelos Genéticos , Miosina Tipo V/genética , Seleção Genética , Animais , Duplicação Gênica , Filogenia , Sintenia
10.
EMBO Rep ; 20(1)2019 01.
Artigo em Inglês | MEDLINE | ID: mdl-30467237

RESUMO

CDC14A codes for a conserved proline-directed phosphatase, and mutations in the gene are associated with autosomal-recessive severe to profound deafness, due to defective kinocilia. A role of CDC14A in cilia formation has also been described in other organisms. However, how human CDC14A impacts on cilia formation remains unclear. Here, we show that human RPE1 hCDC14APD cells, encoding a phosphatase dead version of hCDC14A, have longer cilia than wild-type cells, while hCDC14A overexpression reduces cilia formation. Phospho-proteome analysis of ciliated RPE1 cells identified actin-associated and microtubule binding proteins regulating cilia length as hCDC14A substrates, including the actin-binding protein drebrin. Indeed, we find that hCDC14A counteracts the CDK5-dependent phosphorylation of drebrin at S142 during ciliogenesis. Further, we show that drebrin and hCDC14A regulate the recruitment of the actin organizer Arp2 to centrosomes. In addition, during ciliogenesis hCDC14A also regulates endocytosis and targeting of myosin Va vesicles to the basal body in a drebrin-independent manner, indicating that it impacts primary cilia formation in a multilayered manner.


Assuntos
Proteína 2 Relacionada a Actina/genética , Cílios/genética , Neuropeptídeos/genética , Monoéster Fosfórico Hidrolases/genética , Actinas/genética , Linhagem Celular , Movimento Celular/genética , Centrossomo/metabolismo , Cílios/metabolismo , Quinase 5 Dependente de Ciclina/genética , Endocitose/genética , Regulação da Expressão Gênica no Desenvolvimento/genética , Humanos , Microtúbulos/genética , Mutação , Cadeias Pesadas de Miosina/genética , Miosina Tipo V/genética , Fosforilação , Ligação Proteica , Proteoma/genética
11.
J Biol Chem ; 294(5): 1554-1567, 2019 02 01.
Artigo em Inglês | MEDLINE | ID: mdl-30518549

RESUMO

Myosins are molecular motors that use a conserved ATPase cycle to generate force. We investigated two mutations in the converter domain of myosin V (R712G and F750L) to examine how altering specific structural transitions in the motor ATPase cycle can impair myosin mechanochemistry. The corresponding mutations in the human ß-cardiac myosin gene are associated with hypertrophic and dilated cardiomyopathy, respectively. Despite similar steady-state actin-activated ATPase and unloaded in vitro motility-sliding velocities, both R712G and F750L were less able to overcome frictional loads measured in the loaded motility assay. Transient kinetic analysis and stopped-flow FRET demonstrated that the R712G mutation slowed the maximum ATP hydrolysis and recovery-stroke rate constants, whereas the F750L mutation enhanced these steps. In both mutants, the fast and slow power-stroke as well as actin-activated phosphate release rate constants were not significantly different from WT. Time-resolved FRET experiments revealed that R712G and F750L populate the pre- and post-power-stroke states with similar FRET distance and distance distribution profiles. The R712G mutant increased the mole fraction in the post-power-stroke conformation in the strong actin-binding states, whereas the F750L decreased this population in the actomyosin ADP state. We conclude that mutations in key allosteric pathways can shift the equilibrium and/or alter the activation energy associated with key structural transitions without altering the overall conformation of the pre- and post-power-stroke states. Thus, therapies designed to alter the transition between structural states may be able to rescue the impaired motor function induced by disease mutations.


Assuntos
Mecanotransdução Celular , Atividade Motora , Mutação , Miosina Tipo V/química , Miosina Tipo V/metabolismo , Difosfato de Adenosina/metabolismo , Adenosina Trifosfatases/metabolismo , Trifosfato de Adenosina/metabolismo , Sequência de Aminoácidos , Animais , Galinhas , Modelos Moleculares , Miosina Tipo V/genética , Ligação Proteica , Conformação Proteica , Domínios Proteicos , Homologia de Sequência
12.
J Biosci ; 43(4): 605-619, 2018 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-30207308

RESUMO

In vertebrates, early developing epidermis is a bilayered epithelium consisting of an outer periderm and the underlying basal epidermis. It eventually develops into a multi-layered epithelium. The mechanisms that control the architecture and homeostasis of early developing bilayered epidermis have remained poorly understood. Recently, we have shown that the function of Myosin Vb, an actin based molecular motor, is essential in peridermal cells for maintenance of plasma membrane homeostasis. Furthermore, our analyses of the goosepimples/myosin Vb mutant unravelled a direct link between plasma membrane homeostasis, cell size maintenance and tissue homeostasis in the developing epidermis. However, it remained unclear whether this link is specific to myosin Vb mutant or this is a general principle. Here we have identified two more genetic conditions, romeharsha mutant and clint1 knockdown, in which membrane homeostasis is perturbed, as evident by increased endocytosis and accumulation of lysosomes. As a consequence, peridermal cells exhibit smaller size and increased proliferation. We further show that decreasing endocytosis in romeharsha mutant and clint1 morphants rescues or mitigates the effect on cell size, cell proliferation and morphological phenotype. Our data confirms generality of the principle by reaffirming the causal link between plasma membrane homeostasis, cell size maintenance and tissue homeostasis.


Assuntos
Desenvolvimento Embrionário/genética , Endocitose/genética , Proteínas de Peixe-Zebra/genética , Peixe-Zebra/genética , Animais , Membrana Celular/genética , Tamanho Celular , Epiderme/crescimento & desenvolvimento , Homeostase/genética , Proteínas Mutantes/genética , Miosina Tipo V/genética , Fenótipo , Peixe-Zebra/crescimento & desenvolvimento
13.
Pestic Biochem Physiol ; 150: 1-9, 2018 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-30195381

RESUMO

Fungal resistance to fungicides is a serious challenge in crop protection. Although strategies have been found to prevent the development of fungicide resistance, rare strategy has been found to quickly reduce such resistance once it has occurred. We demonstrate that the application of dsRNAs, which inhibit the expression of the phenamacril (fungicide JS399-19) target gene-Myosin 5 (Myo5) in Fusarium, decreased F. asiaticum resistance to phenamacril and infection. RNAi molecules derived from different regions of Myo5 gene had different effects on phenamacril-resistance. Myo5-8 (one of Myo5 segments) exhibited great and stable effect on phenamacril-resistant reduction both in vivo and in vitro. Myo5 mRNA and protein were both reduced when mycelium was treated with Myo5-8 dsRNA. After a mixture of Myo5-8 dsRNA and phenamacril treatment, plants can highly control the infection of phenamacril-resistant strain. The antifungal activity of Myo5-8 dsRNA plus phenamacril effected longer than a single Myo5-8 dsRNA. In addition, no off-target sequences were found in wheat and/or other plant and animal species for Myo5-8 dsRNA sequence. Our findings suggest a new strategy for fungicide resistant reduction and for designing new fungicides to control pathogens which easily develop fungicide resistance.


Assuntos
Resistência Microbiana a Medicamentos/genética , Fungicidas Industriais/farmacologia , Fusarium/efeitos dos fármacos , Fusarium/genética , Miosina Tipo V/genética , RNA de Cadeia Dupla/genética , Fusarium/patogenicidade , Inativação Gênica , Testes de Sensibilidade Microbiana , Interferência de RNA , Virulência/genética
14.
Clin Liver Dis ; 22(4): 657-669, 2018 11.
Artigo em Inglês | MEDLINE | ID: mdl-30266155
15.
Gastroenterology ; 155(6): 1883-1897.e10, 2018 12.
Artigo em Inglês | MEDLINE | ID: mdl-30144427

RESUMO

BACKGROUND & AIMS: Inactivating mutations in MYO5B cause microvillus inclusion disease (MVID), but the physiological cause of the diarrhea associated with this disease is unclear. We investigated whether loss of MYO5B results in aberrant expression of apical enterocyte transporters. METHODS: We studied alterations in apical membrane transporters in MYO5B-knockout mice, as well as mice with tamoxifen-inducible, intestine-specific disruption of Myo5b (VilCreERT2;Myo5bflox/flox mice) or those not given tamoxifen (controls). Intestinal tissues were collected from mice and analyzed by immunostaining, immunoelectron microscopy, or cultured enteroids were derived. Functions of brush border transporters in intestinal mucosa were measured in Ussing chambers. We obtained duodenal biopsy specimens from individuals with MVID and individuals without MVID (controls) and compared transporter distribution by immunocytochemistry. RESULTS: Compared to intestinal tissues from littermate controls, intestinal tissues from MYO5B-knockout mice had decreased apical localization of SLC9A3 (also called NHE3), SLC5A1 (also called SGLT1), aquaporin (AQP) 7, and sucrase isomaltase, and subapical localization of intestinal alkaline phosphatase and CDC42. However, CFTR was present on apical membranes of enterocytes from MYO5B knockout and control mice. Intestinal biopsies from patients with MVID had subapical localization of NHE3, SGLT1, and AQP7, but maintained apical CFTR. After tamoxifen administration, VilCreERT2;Myo5bflox/flox mice lost apical NHE3, SGLT1, DRA, and AQP7, similar to germline MYO5B knockout mice. Intestinal tissues from VilCreERT2;Myo5bflox/flox mice had increased CFTR in crypts and CFTR localized to the apical membranes of enterocytes. Intestinal mucosa from VilCreERT2;Myo5bflox/flox mice given tamoxifen did not have an intestinal barrier defect, based on Ussing chamber analysis, but did have decreased SGLT1 activity and increased CFTR activity. CONCLUSIONS: Although trafficking of many apical transporters is regulated by MYO5B, trafficking of CFTR is largely independent of MYO5B. Decreased apical localization of NHE3, SGLT1, DRA, and AQP7 might be responsible for dysfunctional water absorption in enterocytes of patients with MVID. Maintenance of apical CFTR might exacerbate water loss by active secretion of chloride into the intestinal lumen.


Assuntos
Cloretos/metabolismo , Regulador de Condutância Transmembrana em Fibrose Cística/metabolismo , Enterócitos/metabolismo , Síndromes de Malabsorção/genética , Microvilosidades/patologia , Mucolipidoses/genética , Miosina Tipo V/genética , Trocadores de Sódio-Hidrogênio/metabolismo , Animais , Aquaporinas/metabolismo , Duodeno/metabolismo , Duodeno/patologia , Inativação Gênica , Humanos , Mucosa Intestinal , Intestinos/citologia , Intestinos/patologia , Síndromes de Malabsorção/patologia , Camundongos , Camundongos Knockout , Microvilosidades/genética , Mucolipidoses/patologia , Transporte Proteico , Transportador 1 de Glucose-Sódio/metabolismo , Trocador 3 de Sódio-Hidrogênio/metabolismo , Complexo Sacarase-Isomaltase/metabolismo , Tamoxifeno/administração & dosagem
16.
Artigo em Inglês | MEDLINE | ID: mdl-30138681

RESUMO

Myosin Va, a member of the myosin superfamily, has been widely identified associated with processes of cellular motility, which include neurotransmitter release and synaptic plasticity during neurodevelopment. However, the function of myosin Va in the growth and development of crustaceans has not yet been reported. In this study, a full-length cDNA of myosin Va (named as EsMyoVa) was cloned from the Chinese mitten crab, Eriocheir sinensis, and the expression patterns were detected in different tissues and larval developmental stages. The full-length cDNA of EsMyoVa was 6037 bp in length. Real time quantitative reverse transcription PCR (qRT-PCR) analysis showed that EsMyoVa transcript has a wide tissue distribution pattern and is expressed in zoeae, megalopa, juvenile crab stages and adults. In order to further study the function of this gene, we used RNAi technology in the muscle, hepatopancreas, gill, and gonad. After double-stranded RNA (dsRNA) injection, the expression level of EsMyoVa was significantly decreased in all tissues in both sexes and the gene knockdown effects of dsRNA persisted for at least 6 days. Subsequently, the role of EsMyoVa was revealed by silencing the transcript through one month injections of Myosin Va dsRNA. Crabs with reduced levels of EsMyoVa transcripts displayed a dramatic slowing in growth rate and considerably higher mortality compared to control groups, which indicated that this gene had important role of regulating growth and development.


Assuntos
Braquiúros/fisiologia , Regulação da Expressão Gênica no Desenvolvimento , Hepatopâncreas/metabolismo , Larva/fisiologia , Cadeias Pesadas de Miosina/metabolismo , Miosina Tipo V/metabolismo , Sequência de Aminoácidos , Animais , Sequência de Bases , Braquiúros/crescimento & desenvolvimento , Biologia Computacional , DNA Complementar/química , DNA Complementar/metabolismo , Feminino , Hepatopâncreas/crescimento & desenvolvimento , Larva/crescimento & desenvolvimento , Masculino , Músculo Esquelético/crescimento & desenvolvimento , Músculo Esquelético/metabolismo , Cadeias Pesadas de Miosina/antagonistas & inibidores , Cadeias Pesadas de Miosina/química , Cadeias Pesadas de Miosina/genética , Miosina Tipo V/antagonistas & inibidores , Miosina Tipo V/química , Miosina Tipo V/genética , Especificidade de Órgãos , Ovário/crescimento & desenvolvimento , Ovário/metabolismo , Filogenia , Interferência de RNA , Distribuição Aleatória , Proteínas Recombinantes/química , Proteínas Recombinantes/metabolismo , Homologia de Sequência de Aminoácidos , Testículo/crescimento & desenvolvimento , Testículo/metabolismo
17.
Exp Dermatol ; 27(10): 1120-1125, 2018 10.
Artigo em Inglês | MEDLINE | ID: mdl-30044001

RESUMO

The long noncoding RNA H19 was reported to associate with melanogenesis. However, it remains unknown whether H19 expression will be changed by UVB irradiation and whether H19 will regulate melanocytes melanogenesis by paracrine effects. Here, we analysed the expression changes of H19 irradiated by UVB in keratinocytes and explored the mechanism of melanogenesis stimulated by H19 through paracrine effects. First, after keratinocytes were exposed to UVB irradiation, expression of H19 and pro-opiomelanocortin (POMC) was measured by qRT-PCR. Also, α-melanocyte-stimulating hormone (α-MSH) contents in cells supernatant were measured by ELISA. Then, H19 siRNAs were designed and transfected into keratinocytes by liposome. The expression changes of H19, POMC and α-MSH were detected. Besides, expression of p53 was detected by Western blot. After that, supernatant of keratinocytes with H19 siRNAs or negative control siRNA was cocultured with immortalized melanocyte line PIG1. Expression levels of MiTF, TYR, Rab27A, TYRP2, FSCN1 and MYO5A in PIG1 cells were detected by Western blot and qRT-PCR. We found that H19 expression of keratinocytes cells decreased after UVB irradiation. However, the levels of POMC, α-MSH and p53 were upregulated in UVB-irradiated cells. Compared with the negative control, H19 siRNAs could significantly increase the expression of POMC, α-MSH and p53. After supernatant of keratinocytes transfected with H19 siRNAs was cocultured with PIG1 cells, the levels of MiTF, TYR and Rab27A were upregulated in PIG1 cells. In conclusion, UVB-inhibited H19 may promote α-MSH secretion by p53 in keratinocytes and then regulate melanocytes melanogenesis through paracrine effects.


Assuntos
Melaninas/biossíntese , Comunicação Parácrina/efeitos da radiação , Pró-Opiomelanocortina/genética , RNA Longo não Codificante/genética , RNA Longo não Codificante/metabolismo , Raios Ultravioleta , Proteínas de Transporte/genética , Proteínas de Transporte/metabolismo , Linhagem Celular , Sobrevivência Celular/efeitos da radiação , Técnicas de Cocultura , Ciclo-Oxigenase 2/genética , Relação Dose-Resposta à Radiação , Regulação para Baixo/efeitos da radiação , Fator 2 de Crescimento de Fibroblastos/genética , Humanos , Oxirredutases Intramoleculares/genética , Oxirredutases Intramoleculares/metabolismo , Queratinócitos/fisiologia , Queratinócitos/efeitos da radiação , Melanócitos/fisiologia , Melanócitos/efeitos da radiação , Proteínas dos Microfilamentos/genética , Proteínas dos Microfilamentos/metabolismo , Fator de Transcrição Associado à Microftalmia/genética , Fator de Transcrição Associado à Microftalmia/metabolismo , Cadeias Pesadas de Miosina/genética , Cadeias Pesadas de Miosina/metabolismo , Miosina Tipo V/genética , Miosina Tipo V/metabolismo , RNA Interferente Pequeno/genética , Proteína Supressora de Tumor p53/metabolismo , Tirosina/genética , Tirosina/metabolismo , Regulação para Cima/efeitos da radiação , alfa-MSH/metabolismo , Proteínas rab27 de Ligação ao GTP/genética , Proteínas rab27 de Ligação ao GTP/metabolismo
18.
Pestic Biochem Physiol ; 147: 127-132, 2018 May.
Artigo em Inglês | MEDLINE | ID: mdl-29933982

RESUMO

Fusarium wilt caused by Fusarium oxysporum f. sp. melonis (FOM) is one of the most notorious seed-borne diseases worldwide. Phenamacril is a cyanoacrylate fungicide with novel chemical structure and strong inhibitive activity against FOM. To evaluate the risk of FOM developing phenamacril resistance, five phenamacril-resistant mutants with >800µgml-1 minimum inhibitory concentration were obtained by repeated exposure to the fungicide in the laboratory. Compared with the parental isolate, four of the five phenamacril-resistant mutants showed enhanced biological fitness in sporulation and virulence, but not in sensitivity to various stresses (oxidative and osmotic pressure, cell membrane and wall inhibitor). No positive cross-resistance was observed among phenamacril and the other five fungicides, including azoxystrobin, carbendazim, boscalid, fluazinam and tebuconazole. Sequencing alignment results of the myosin 5 from the five resistant mutants and the parental strain indicated that the three resistant mutants fo-2, fo-3 and fo-4 had a single point mutation (S175L), which may confer the resistance of FOM against phenamacril. Interestingly, the resistant mutant fo-4 harbored not only one mutation (S175L) at myosin 5, but also the other mutation (A52G) at ß2-tublin. Our data supported that resistance risk of Fusarium oxysporum f. sp. melonis against phenamacril was between the moderate to high level.


Assuntos
Farmacorresistência Fúngica/genética , Fungicidas Industriais/farmacologia , Fusarium/efeitos dos fármacos , Miosina Tipo V/antagonistas & inibidores , Cucurbitaceae/microbiologia , Fusarium/genética , Fusarium/patogenicidade , Fusarium/fisiologia , Genes Fúngicos , Testes de Sensibilidade Microbiana , Micélio/efeitos dos fármacos , Micélio/crescimento & desenvolvimento , Miosina Tipo V/genética , Doenças das Plantas/microbiologia , Doenças das Plantas/prevenção & controle , Mutação Puntual , Medição de Risco , Alinhamento de Sequência , Esporos Bacterianos/efeitos dos fármacos , Virulência
19.
J Hum Genet ; 63(7): 821-829, 2018 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-29670293

RESUMO

MODY 5 and 6 have been shown to be low-penetrant MODYs. As the genetic background of unknown MODY is assumed to be similar, a new analytical strategy is applied here to elucidate genetic predispositions to unknown MODY. We examined to find whether there are major MODY gene loci remaining to be identified using SNP linkage analysis in Japanese. Whole-exome sequencing was performed with seven families with typical MODY. Candidates for novel MODY genes were examined combined with in silico network analysis. Some peaks were found only in either parametric or non-parametric analysis; however, none of these peaks showed a LOD score greater than 3.7, which is approved to be the significance threshold of evidence for linkage. Exome sequencing revealed that three mutated genes were common among 3 families and 42 mutated genes were common in two families. Only one of these genes, MYO5A, having rare amino acid mutations p.R849Q and p.V1601G, was involved in the biological network of known MODY genes through the intermediary of the INS. Although only one promising candidate gene, MYO5A, was identified, no novel, high penetrant MODY genes might remain to be found in Japanese MODY.


Assuntos
Diabetes Mellitus Tipo 2/epidemiologia , Diabetes Mellitus Tipo 2/genética , Predisposição Genética para Doença , Mutação , Cadeias Pesadas de Miosina/genética , Miosina Tipo V/genética , Adolescente , Adulto , Sequência de Bases , Criança , Mapeamento Cromossômico , Diabetes Mellitus Tipo 2/diagnóstico , Exoma , Feminino , Expressão Gênica , Redes Reguladoras de Genes , Ligação Genética , Humanos , Japão/epidemiologia , Escore Lod , Masculino , Linhagem , Penetrância
20.
Environ Microbiol ; 20(4): 1607-1621, 2018 04.
Artigo em Inglês | MEDLINE | ID: mdl-29575486

RESUMO

The vascular wilt fungus Verticillium dahliae is one of the most destructive pathogens of cotton (Gossypium hirsutum) and many other economically important dicot plants. Fungal pathogens require Myosin-mediated actomyosin motility system for colonization of their host plants; however, the mechanisms underlying this process have not been fully characterized for V. dahliae. Here, in a knock-out experiment, we characterized the role of VdMyo5, a member of the Myosin V family, before and during infection of cotton and Arabidopsis thaliana. The VdMyo5 deletion mutant (ΔVdmyo5) fungi showed obvious defects in the development of conidia and the polarized elongation of vegetative hyphae, but no inhibition of host root penetration. Overall, the ΔVdmyo5 fungi exhibited dramatically reduced virulence in cotton and Arabidopsis, with almost no colonization in sections of host vascular tissue. We found labelled Myosin5-GFP to be specifically enriched at the hyphal tip, co-localized with FM4-64 labelled Spitzenkörper, which is the vesicle supply centre in filamentous fungi. Comparative secretome analysis revealed that proteins associated with cell wall modification and degradation of reactive oxygen species were significantly altered in mutant strains. Our results indicate that Myosin5 is required for vegetative growth and full virulence, possibly by regulating vesicle transport. The findings provide important insight into the cellular mechanisms of Verticillium pathogenesis.


Assuntos
Actomiosina/metabolismo , Arabidopsis/microbiologia , Gossypium/microbiologia , Miosina Tipo V/metabolismo , Doenças das Plantas/microbiologia , Verticillium/patogenicidade , Técnicas de Inativação de Genes , Hifas/crescimento & desenvolvimento , Miosina Tipo V/genética , Raízes de Plantas/microbiologia , Esporos Fúngicos/crescimento & desenvolvimento , Verticillium/genética , Verticillium/metabolismo , Virulência/genética
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