Your browser doesn't support javascript.
loading
Mostrar: 20 | 50 | 100
Resultados 1 - 20 de 4.569
Filtrar
1.
Zool Res ; 42(2): 141-152, 2021 Mar 18.
Artigo em Inglês | MEDLINE | ID: mdl-33527802

RESUMO

Secretory pore-forming proteins (PFPs) have been identified in organisms from all kingdoms of life. Our studies with the toad species Bombina maxima found an interaction network among aerolysin family PFPs (af-PFPs) and trefoil factors (TFFs). As a toad af-PFP, BmALP1 can be reversibly regulated between active and inactive forms, with its paralog BmALP3 acting as a negative regulator. BmALP1 interacts with BmTFF3 to form a cellular active complex called ßγ-CAT. This PFP complex is characterized by acting on endocytic pathways and forming pores on endolysosomes, including stimulating cell macropinocytosis. In addition, cell exocytosis can be induced and/or modulated in the presence of ßγ-CAT. Depending on cell contexts and surroundings, these effects can facilitate the toad in material uptake and vesicular transport, while maintaining mucosal barrier function as well as immune defense. Based on experimental evidence, we hereby propose a secretory endolysosome channel (SELC) pathway conducted by a secreted PFP in cell endocytic and exocytic systems, with ßγ-CAT being the first example of a SELC protein. With essential roles in cell interactions and environmental adaptations, the proposed SELC protein pathway should be conserved in other living organisms.


Assuntos
Canais Iônicos/metabolismo , Animais , Anuros/metabolismo , Redes Reguladoras de Genes , Ácido Hialurônico , Canais Iônicos/genética , Família Multigênica , Fatores Trefoil/metabolismo
2.
Nature ; 590(7846): 509-514, 2021 02.
Artigo em Inglês | MEDLINE | ID: mdl-33568813

RESUMO

Mechanosensitive channels sense mechanical forces in cell membranes and underlie many biological sensing processes1-3. However, how exactly they sense mechanical force remains under investigation4. The bacterial mechanosensitive channel of small conductance, MscS, is one of the most extensively studied mechanosensitive channels4-8, but how it is regulated by membrane tension remains unclear, even though the structures are known for its open and closed states9-11. Here we used cryo-electron microscopy to determine the structure of MscS in different membrane environments, including one that mimics a membrane under tension. We present the structures of MscS in the subconducting and desensitized states, and demonstrate that the conformation of MscS in a lipid bilayer in the open state is dynamic. Several associated lipids have distinct roles in MscS mechanosensation. Pore lipids are necessary to prevent ion conduction in the closed state. Gatekeeper lipids stabilize the closed conformation and dissociate with membrane tension, allowing the channel to open. Pocket lipids in a solvent-exposed pocket between subunits are pulled out under sustained tension, allowing the channel to transition to the subconducting state and then to the desensitized state. Our results provide a mechanistic underpinning and expand on the 'force-from-lipids' model for MscS mechanosensation4,11.


Assuntos
Microscopia Crioeletrônica , Proteínas de Escherichia coli/metabolismo , Proteínas de Escherichia coli/ultraestrutura , Escherichia coli/química , Canais Iônicos/metabolismo , Canais Iônicos/ultraestrutura , Membranas Artificiais , Fosfatidilcolinas/metabolismo , Detergentes/farmacologia , Escherichia coli/ultraestrutura , Proteínas de Escherichia coli/química , Proteínas de Escherichia coli/genética , Interações Hidrofóbicas e Hidrofílicas , Canais Iônicos/química , Canais Iônicos/genética , Bicamadas Lipídicas/química , Bicamadas Lipídicas/metabolismo , Mecanotransdução Celular/efeitos dos fármacos , Modelos Moleculares , Mutação , Nanoestruturas/química , Nanoestruturas/ultraestrutura , Fosfatidilcolinas/química , Fosfatidilcolinas/farmacologia , Conformação Proteica/efeitos dos fármacos , beta-Ciclodextrinas/farmacologia
3.
Nat Commun ; 12(1): 657, 2021 01 28.
Artigo em Inglês | MEDLINE | ID: mdl-33510158

RESUMO

The sensation of pressure allows us to feel sustained compression and body strain. While our understanding of cutaneous touch has grown significantly in recent years, how deep tissue sensations are detected remains less clear. Here, we use quantitative sensory evaluations of patients with rare sensory disorders, as well as nerve blocks in typical individuals, to probe the neural and genetic mechanisms for detecting non-painful pressure. We show that the ability to perceive innocuous pressures is lost when myelinated fiber function is experimentally blocked in healthy volunteers and that two patients lacking Aß fibers are strikingly unable to feel innocuous pressures at all. We find that seven individuals with inherited mutations in the mechanoreceptor PIEZO2 gene, who have major deficits in touch and proprioception, are nearly as good at sensing pressure as healthy control subjects. Together, these data support a role for Aß afferents in pressure sensation and suggest the existence of an unknown molecular pathway for its detection.


Assuntos
Canais Iônicos/fisiologia , Mecanorreceptores/fisiologia , Sensação/fisiologia , Tato/fisiologia , Adulto , Idoso , Feminino , Humanos , Canais Iônicos/genética , Masculino , Mecanorreceptores/metabolismo , Pessoa de Meia-Idade , Mutação , Bloqueio Nervoso/métodos , Pressão , Propriocepção/genética , Propriocepção/fisiologia , Transtornos das Sensações/diagnóstico , Transtornos das Sensações/genética , Transtornos das Sensações/fisiopatologia , Pele/inervação , Pele/fisiopatologia , Adulto Jovem
4.
DNA Cell Biol ; 40(2): 239-246, 2021 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-33395371

RESUMO

Knockout (KO) or exogenous expression of a gene of interest in cultured cells is one of the most important ways to study the function of the gene. Compared with transient transfection, stable cell lines possess great advantages such as excellent cell homogeneity and feasibility for long-term use. However, technical challenges in generating stable cell lines still exist in many laboratories using conventional techniques like limiting dilution or cloning cylinders. In this study we describe an optimized method to efficiently create stable cell lines for functional studies. This method was successfully used to generate a PIEZO1 gene-KO cell line with the CRISPR/Cas9 technology, and TRPC5/GCaMP6f-mCherry-coexpressing cell lines without antibiotic selection. Monoclonal cell lines can be obtained in 2-4 weeks after transfection. This method does not require any special equipment or consumables and can be conducted in all laboratories with general cell-culture facility.


Assuntos
Técnicas de Inativação de Genes/métodos , Técnicas de Transferência de Genes , Sistemas CRISPR-Cas , Linhagem Celular , Humanos , Canais Iônicos/deficiência , Canais Iônicos/genética , Canais de Cátion TRPC/genética
5.
Arterioscler Thromb Vasc Biol ; 41(3): 1191-1204, 2021 03.
Artigo em Inglês | MEDLINE | ID: mdl-33406853

RESUMO

OBJECTIVE: Noncoding RNAs are emerging as important players in gene regulation and cardiovascular diseases. Their roles in the pathogenesis of atherosclerosis are not fully understood. The purpose of this study was to determine the role played by a previously uncharacterized long noncoding RNA, RP11-728F11.4, in the development of atherosclerosis and the mechanisms by which it acts. Approach and Results: Expression microarray analysis revealed that atherosclerotic plaques had increased expression of RP11-728F11.4 as well as the cognate gene FXYD6 (FXYD domain containing ion transport regulator 6), which encodes a modulator of Na+/K+-ATPase. In vitro experiments showed that RP11-728F11.4 interacted with the RNA-binding protein EWSR1 (Ewings sarcoma RNA binding protein-1) and upregulated FXYD6 expression. Lentivirus-induced overexpression of RP11-728F11.4 in cultured monocytes-derived macrophages resulted in higher Na+/K+-ATPase activity, intracellular cholesterol accumulation, and increased proinflammatory cytokine production. The effects of RP11-728F11.4 were enhanced by siRNA-mediated knockdown of EWSR1 and reduced by downregulation of FXYD domain containing ion transport regulator 6. In vivo experiments in apoE knockout mice fed a Western diet demonstrated that RP11-728F11.4 increased proinflammatory cytokine production and augmented atherosclerotic lesions. CONCLUSIONS: RP11-728F11.4 promotes atherosclerosis, with an influence on cholesterol homeostasis and proinflammatory molecule production, thus representing a potential therapeutic target. Graphic Abstract: A graphic abstract is available for this article.


Assuntos
Aterosclerose/genética , RNA Longo não Codificante/genética , Animais , Aterosclerose/etiologia , Aterosclerose/metabolismo , Células Cultivadas , Colesterol/metabolismo , Citocinas/metabolismo , Modelos Animais de Doenças , Células Endoteliais/metabolismo , Feminino , Técnicas de Silenciamento de Genes , Humanos , Canais Iônicos/genética , Canais Iônicos/metabolismo , Metabolismo dos Lipídeos , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout para ApoE , Pessoa de Meia-Idade , Placa Aterosclerótica/etiologia , Placa Aterosclerótica/genética , Placa Aterosclerótica/patologia , RNA Longo não Codificante/metabolismo , Proteína EWS de Ligação a RNA/antagonistas & inibidores , Proteína EWS de Ligação a RNA/genética , Proteína EWS de Ligação a RNA/metabolismo , ATPase Trocadora de Sódio-Potássio/metabolismo , Regulação para Cima
6.
Methods Mol Biol ; 2188: 51-65, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-33119846

RESUMO

Heterologous expression of recombinant ion channel subunits in mammalian cell lines allows for characterization of their functional properties and pharmacological regulation. In this chapter, we describe methods for thawing, refreezing, passaging, cell culture, and transfection of tsA201 cells suitable for electrophysiology and imaging experiments. Furthermore, we discuss the strengths and limitations of using these methods.


Assuntos
Canais Iônicos/genética , Transfecção/métodos , Contagem de Células/métodos , Técnicas de Cultura de Células/métodos , Linhagem Celular , Criopreservação/métodos , Expressão Gênica , Humanos , Canais de Potássio de Domínios Poros em Tandem/genética , Proteínas Recombinantes/genética , Técnicas de Cultura de Tecidos/métodos
7.
Methods Mol Biol ; 2188: 67-92, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-33119847

RESUMO

Artificial lipid bilayers have been used for several decades to study channel-forming pores and ion channels in membranes. Until recently, the classical two-chamber setups have been primarily used for studying the biophysical properties of pore forming proteins. Within the last 10 years, instruments for automated lipid bilayer measurements have been developed and are now commercially available. This chapter focuses on protein purification and reconstitution of channel-forming proteins into lipid bilayers using a classic setup and on the commercially available systems, the Orbit mini and Orbit 16.


Assuntos
Eletrofisiologia/instrumentação , Canais Iônicos/metabolismo , Bicamadas Lipídicas/metabolismo , Animais , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Fenômenos Eletrofisiológicos , Desenho de Equipamento , Escherichia coli/genética , Expressão Gênica , Humanos , Canais Iônicos/genética , Dispositivos Lab-On-A-Chip , Bicamadas Lipídicas/química , Mycobacterium smegmatis/genética , Mycobacterium smegmatis/metabolismo , Mutação Puntual , Porinas/genética , Porinas/metabolismo , Transformação Genética
8.
Methods Mol Biol ; 2188: 191-228, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-33119853

RESUMO

As key players in cell function, ion channels are important targets for drug discovery and therapeutic development against a wide range of health conditions. Thus, developing assays to reconstitute ion channel macromolecular complexes in physiological conditions and screen for chemical modifiers of protein-protein interactions within these complexes is timely in drug discovery campaigns. For most ion channels, expressing their pore-forming subunit in heterologous mammalian cells has now become a routine procedure. However, reconstituting protein-channel complexes in physiological environments is still challenging, limiting our ability to identify tools and probes based on allosteric mechanisms, which could lead to more targeted and precise modulation of the channel function. Here, we describe the assay development steps to stably reconstitute the interaction between voltage-gated Na+ (Nav) channel Nav1.6 and its accessory protein, fibroblast growth factor 14 (FGF14) using the split-luciferase complementation assay (LCA), followed by assay miniaturization and optimization in 384-well plates for in-cell high-throughput screening (HTS) against protein-channel interactions. This optimized LCA can subsequently be used for rapid estimation of hit potency and efficacy via dose-dependency studies, enabling ranking of hits prior to more labor-intensive validation studies. Lastly, we introduce the methodology for rapid functional hit validation studies using semi-automated planar patch-clamp electrophysiology. Our robust, in-cell HTS platform can be adapted to any suitable ion channel complex to explore regulatory pathways of cellular signaling using kinase inhibitors, as well as to screen small molecules for probe development and drug repurposing toward new targets/areas of medicine. Overall, the flexibility of this assay allows users to broadly explore therapeutic options for channelopathy-associated diseases at a fast pace, enabling rapid hypothesis generation in early phase drug discovery campaigns and narrowing down targets prior to more labor-intensive in vivo studies.


Assuntos
Ensaios de Triagem em Larga Escala/métodos , Canais Iônicos/metabolismo , Medições Luminescentes/métodos , Mapeamento de Interação de Proteínas/métodos , Animais , Técnicas de Cultura de Células/métodos , Descoberta de Drogas , Fatores de Crescimento de Fibroblastos/genética , Fatores de Crescimento de Fibroblastos/metabolismo , Células HEK293 , Humanos , Canais Iônicos/genética , Canal de Sódio Disparado por Voltagem NAV1.6/genética , Canal de Sódio Disparado por Voltagem NAV1.6/metabolismo , Mapas de Interação de Proteínas , Transfecção/métodos
9.
Methods Mol Biol ; 2188: 311-330, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-33119859

RESUMO

Automated patch clamping (APC) has been used for almost two decades to increase the throughput of electrophysiological measurements, especially in preclinical safety screening of drug compounds. Typically, cells are suctioned onto holes in planar surfaces and a stronger subsequent suction allows access to a whole cell configuration for electrical measurement of ion channel activity. The development of optogenetic tools over a wide range of wavelengths (UV to IR) provides powerful tools for improving spatiotemporal control of in vivo and in vitro experiments and is emerging as a powerful means of investigating cell networks (neuronal), single cell transduction, and subcellular pathways.Combining APC and optogenetic tools paves the way for improved investigation and control of cell kinetics and provides the opportunity for collecting robust data for new and exciting applications and therapeutic areas. Here, we present an APC optogenetics capability on the Qube Opto 384 system including experiments on light activated ion channels and photoactivated ligands.


Assuntos
Optogenética/métodos , Técnicas de Patch-Clamp/métodos , Técnicas de Cultura de Células/métodos , Channelrhodopsins/genética , Channelrhodopsins/metabolismo , Células HEK293 , Humanos , Canais Iônicos/genética , Canais Iônicos/metabolismo , Luz
10.
Methods Mol Biol ; 2258: 93-103, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-33340356

RESUMO

Embryogenesis, as well as regeneration, is increasingly recognized to be orchestrated by an interplay of transcriptional and bioelectric networks. Spatiotemporal patterns of resting potentials direct the size, shape, and locations of numerous organ primordia during patterning. These bioelectrical properties are established by the function of ion channels and pumps that set voltage potentials of individual cells, and gap junctions (electrical synapses) that enable physiological states to propagate across tissue networks. Functional experiments to probe the roles of bioelectrical states can be carried out by targeting endogenous ion channels during development. Here, we describe protocols, optimized for the highly tractable Xenopus laevis embryo, for molecular genetic targeting of ion channels and connexins based on CRISPR, and monitoring of resting potential states using voltage-sensing fluorescent dye. Similar strategies can be adapted to other model species.


Assuntos
Sistemas CRISPR-Cas , Conexinas/metabolismo , Sinapses Elétricas/metabolismo , Edição de Genes , Canais Iônicos/metabolismo , Xenopus laevis/metabolismo , Animais , Proteína 9 Associada à CRISPR/genética , Proteína 9 Associada à CRISPR/metabolismo , Repetições Palindrômicas Curtas Agrupadas e Regularmente Espaçadas , Conexinas/genética , Sinapses Elétricas/genética , Embrião não Mamífero/metabolismo , Desenvolvimento Embrionário , Regulação da Expressão Gênica no Desenvolvimento , Canais Iônicos/genética , Potenciais da Membrana , Microscopia de Fluorescência , RNA Guia/genética , RNA Guia/metabolismo , Fatores de Tempo , Xenopus laevis/embriologia , Xenopus laevis/genética
11.
Int J Mol Sci ; 22(1)2020 Dec 23.
Artigo em Inglês | MEDLINE | ID: mdl-33374679

RESUMO

Ion channels are membrane-spanning integral proteins expressed in multiple organs, including the eye. Here, ion channels play a role in several physiological processes, like signal transmission and visual processing. A wide range of mutations have been reported in the corresponding genes and their interacting subunit coding genes, which contribute significantly to a wide spectrum of ocular diseases collectively called channelopathies, a subgroup of inherited retinal dystrophies. Such mutations result in either a loss or gain-of channel functions affecting the structure, assembly, trafficking and localization of channel proteins. We investigated the probands of seven Italian and Egyptian families affected by not completely defined forms of inherited retinal dystrophies, by whole exome sequencing (WES) experiments, and found interesting variants in already known causative genes probably able to impair retinal functionalities. However, because such variants did not completely explain the phenotype manifested by each patient, we proceed to further investigate possible related genes carrying mutations that might complement previously found data, based on the common aspect linked to neurotransmission impairments. We found 10 mutated genes whose variants might alter important ligand binding sites differently distributed through all considered patients. Such genes encode for ion channels, or their regulatory proteins, and strictly interact with known causative genes, also sharing with them synaptic-related pathways. Taking into account several limitations that will be resolved by further experiments, we believe that our exploratory investigation will help scientists to provide a new promising paradigm for precise diagnosis of retinal dystrophies to facilitate the development of rational treatments.


Assuntos
Genes Modificadores , Canais Iônicos/genética , Fenótipo , Distrofias Retinianas/genética , Exoma , Feminino , Humanos , Masculino , Linhagem , Polimorfismo Genético , Sinapses/genética
12.
Neuron ; 108(1): 5-7, 2020 10 14.
Artigo em Inglês | MEDLINE | ID: mdl-33058766

RESUMO

Like axon guidance, the tuning of vascular tip cells during angiogenesis is an intriguing but puzzling developmental process. A new study in zebrafish (Liu et al., 2020) now demonstrates a critical role of the Piezo1 mechanosensitive ion channel in guiding vascular tip cells in pathfinding.


Assuntos
Cálcio , Mecanotransdução Celular , Animais , Encéfalo/metabolismo , Células Endoteliais/metabolismo , Canais Iônicos/genética , Canais Iônicos/metabolismo
13.
Nat Commun ; 11(1): 4070, 2020 08 13.
Artigo em Inglês | MEDLINE | ID: mdl-32792502

RESUMO

Human astroviruses are small non-enveloped viruses with positive-sense single-stranded RNA genomes. Astroviruses cause acute gastroenteritis in children worldwide and have been associated with encephalitis and meningitis in immunocompromised individuals. It is still unknown how astrovirus particles exit infected cells following replication. Through comparative genomic analysis and ribosome profiling we here identify and confirm the expression of a conserved alternative-frame ORF, encoding the protein XP. XP-knockout astroviruses are attenuated and pseudo-revert on passaging. Further investigation into the function of XP revealed plasma and trans Golgi network membrane-associated roles in virus assembly and/or release through a viroporin-like activity. XP-knockout replicons have only a minor replication defect, demonstrating the role of XP at late stages of infection. The discovery of XP advances our knowledge of these important human viruses and opens an additional direction of research into their life cycle and pathogenesis.


Assuntos
Canais Iônicos/metabolismo , Mamastrovirus/metabolismo , Proteínas não Estruturais Virais/metabolismo , Animais , Linhagem Celular , Cricetinae , Eletroforese em Gel de Poliacrilamida , Genômica/métodos , Células HeLa , Humanos , Immunoblotting , Imunoprecipitação , Canais Iônicos/genética , Mamastrovirus/genética , Microscopia de Fluorescência , Plasmídeos/genética , Ribossomos , Proteínas não Estruturais Virais/genética , Replicação Viral/genética , Replicação Viral/fisiologia
14.
Neuron ; 108(1): 180-192.e5, 2020 10 14.
Artigo em Inglês | MEDLINE | ID: mdl-32827455

RESUMO

During development, endothelial tip cells (ETCs) located at the leading edge of growing vascular plexus guide angiogenic sprouts to target vessels, and thus, ETC pathfinding is fundamental for vascular pattern formation in organs, including the brain. However, mechanisms of ETC pathfinding remain largely unknown. Here, we report that Piezo1-mediated Ca2+ activities at primary branches of ETCs regulate branch dynamics to accomplish ETC pathfinding during zebrafish brain vascular development. ETC branches display spontaneous local Ca2+ transients, and high- and low-frequency Ca2+ transients cause branch retraction through calpain and branch extension through nitric oxide synthase, respectively. These Ca2+ transients are mainly mediated by Ca2+-permeable Piezo1 channels, which can be activated by mechanical force, and mutating piezo1 largely impairs ETC pathfinding and brain vascular patterning. These findings reveal that Piezo1 and downstream Ca2+ signaling act as molecular bases for ETC pathfinding and highlight a novel function of Piezo1 and Ca2+ in vascular development.


Assuntos
Vasos Sanguíneos/crescimento & desenvolvimento , Encéfalo/irrigação sanguínea , Cálcio/metabolismo , Células Endoteliais/metabolismo , Canais Iônicos/genética , Neovascularização Fisiológica/genética , Proteínas de Peixe-Zebra/genética , Animais , Encéfalo/crescimento & desenvolvimento , Sinalização do Cálcio , Calpaína/metabolismo , Canais Iônicos/metabolismo , Mecanotransdução Celular , Mutação , Óxido Nítrico Sintase/metabolismo , Peixe-Zebra , Proteínas de Peixe-Zebra/metabolismo
15.
Nature ; 585(7823): 129-134, 2020 09.
Artigo em Inglês | MEDLINE | ID: mdl-32848250

RESUMO

Transmembrane channels and pores have key roles in fundamental biological processes1 and in biotechnological applications such as DNA nanopore sequencing2-4, resulting in considerable interest in the design of pore-containing proteins. Synthetic amphiphilic peptides have been found to form ion channels5,6, and there have been recent advances in de novo membrane protein design7,8 and in redesigning naturally occurring channel-containing proteins9,10. However, the de novo design of stable, well-defined transmembrane protein pores that are capable of conducting ions selectively or are large enough to enable the passage of small-molecule fluorophores remains an outstanding challenge11,12. Here we report the computational design of protein pores formed by two concentric rings of α-helices that are stable and monodisperse in both their water-soluble and their transmembrane forms. Crystal structures of the water-soluble forms of a 12-helical pore and a 16-helical pore closely match the computational design models. Patch-clamp electrophysiology experiments show that, when expressed in insect cells, the transmembrane form of the 12-helix pore enables the passage of ions across the membrane with high selectivity for potassium over sodium; ion passage is blocked by specific chemical modification at the pore entrance. When incorporated into liposomes using in vitro protein synthesis, the transmembrane form of the 16-helix pore-but not the 12-helix pore-enables the passage of biotinylated Alexa Fluor 488. A cryo-electron microscopy structure of the 16-helix transmembrane pore closely matches the design model. The ability to produce structurally and functionally well-defined transmembrane pores opens the door to the creation of designer channels and pores for a wide variety of applications.


Assuntos
Simulação por Computador , Genes Sintéticos/genética , Canais Iônicos/química , Canais Iônicos/genética , Modelos Moleculares , Biologia Sintética , Linhagem Celular , Microscopia Crioeletrônica , Cristalografia por Raios X , Condutividade Elétrica , Escherichia coli/genética , Escherichia coli/metabolismo , Hidrazinas , Canais Iônicos/metabolismo , Transporte de Íons , Lipossomos/metabolismo , Técnicas de Patch-Clamp , Porinas/química , Porinas/genética , Porinas/metabolismo , Engenharia de Proteínas , Estrutura Secundária de Proteína , Solubilidade , Água/química
16.
Nat Commun ; 11(1): 3351, 2020 07 03.
Artigo em Inglês | MEDLINE | ID: mdl-32620897

RESUMO

The sodium-leak channel NALCN forms a subthreshold sodium conductance that controls the resting membrane potentials of neurons. The auxiliary subunits of the channel and their functions in mammals are largely unknown. In this study, we demonstrate that two large proteins UNC80 and UNC79 are subunits of the NALCN complex. UNC80 knockout mice are neonatal lethal. The C-terminus of UNC80 contains a domain that interacts with UNC79 and overcomes a soma-retention signal to achieve dendritic localization. UNC80 lacking this domain, as found in human patients, still supports whole-cell NALCN currents but lacks dendritic localization. Our results establish the subunit composition of the NALCN complex, uncover the inter-subunit interaction domains, reveal the functional significance of regulation of dendritic membrane potential by the sodium-leak channel complex, and provide evidence supporting that genetic variations found in individuals with intellectual disability are the causes for the phenotype observed in patients.


Assuntos
Proteínas de Transporte/genética , Deficiência Intelectual/genética , Canais Iônicos/metabolismo , Proteínas de Membrana/genética , Proteínas de Membrana/metabolismo , Proteínas do Tecido Nervoso/genética , Animais , Proteínas de Transporte/metabolismo , Criança , Análise Mutacional de DNA , Conjuntos de Dados como Assunto , Dendritos/patologia , Modelos Animais de Doenças , Técnicas de Introdução de Genes , Células HEK293 , Hipocampo/citologia , Hipocampo/patologia , Humanos , Deficiência Intelectual/diagnóstico , Deficiência Intelectual/patologia , Canais Iônicos/genética , Masculino , Camundongos , Camundongos Knockout , Mutação , Proteínas do Tecido Nervoso/metabolismo , Cultura Primária de Células , Domínios Proteicos/genética , Índice de Gravidade de Doença , Sequenciamento Completo do Exoma
17.
Nat Commun ; 11(1): 3568, 2020 07 16.
Artigo em Inglês | MEDLINE | ID: mdl-32678085

RESUMO

Dissemination of transformed cells is a key process in metastasis. Despite its importance, how transformed cells disseminate from an intact tissue and enter the circulation is poorly understood. Here, we use a fully developed tissue, Drosophila midgut, and describe the morphologically distinct steps and the cellular events occurring over the course of RasV12-transformed cell dissemination. Notably, RasV12-transformed cells formed the Actin- and Cortactin-rich invasive protrusions that were important for breaching the extracellular matrix (ECM) and visceral muscle. Furthermore, we uncovered the essential roles of the mechanosensory channel Piezo in orchestrating dissemination of RasV12-transformed cells. Collectively, our study establishes an in vivo model for studying how transformed cells migrate out from a complex tissue and provides unique insights into the roles of Piezo in invasive cell behavior.


Assuntos
Proteínas de Drosophila/metabolismo , Canais Iônicos/metabolismo , Mecanotransdução Celular , Invasividade Neoplásica/patologia , Proteínas ras/metabolismo , Animais , Membrana Basal/metabolismo , Membrana Basal/patologia , Transformação Celular Neoplásica , Modelos Animais de Doenças , Drosophila , Proteínas de Drosophila/genética , Vesículas Extracelulares/metabolismo , Trato Gastrointestinal/patologia , Genes ras , Canais Iônicos/genética , Metástase Neoplásica/patologia , Podossomos/metabolismo , Proteínas ras/genética
18.
PLoS One ; 15(7): e0235922, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32673370

RESUMO

We have previously established that epigenetic regulator RING1 and YY1 binding protein (RYBP) is required for the contractility of embryonic stem (ES) cell derived cardiomyocytes (CMCs), suggesting its essential role in contractility. In order to investigate the underlying molecular events of this phenotype, we compared the transcriptomic profile of the wild type and Rybp null mutant ES cells and CMCs differentiated from these cell lines. We identified genes related to ion homeostasis, cell adhesion and sarcomeric organization affected in the Rybp null mutant CMCs, by using hierarchical gene clustering and Gene Ontology analysis. We have also demonstrated that the amount of RYBP is drastically reduced in the terminally differentiated wild type CMCs whilst it is broadly expressed in the early phase of differentiation when progenitors form. We also describe that RYBP is important for the proper expression of key cardiac transcription factors including Mesp1, Shh and Mef2c. These findings identify Rybp as a gene important for both early cardiac gene transcription and consequent sarcomere formation necessary for contractility. Since impairment of sarcomeric function and contractility plays a central role in reduced cardiac pump function leading to heart failures in human, current results might be relevant to the pathophysiology of cardiomyopathies.


Assuntos
Proteínas Repressoras/genética , Sarcômeros/fisiologia , Animais , Fatores de Transcrição Hélice-Alça-Hélice Básicos/genética , Fatores de Transcrição Hélice-Alça-Hélice Básicos/metabolismo , Moléculas de Adesão Celular/genética , Moléculas de Adesão Celular/metabolismo , Diferenciação Celular , Linhagem Celular , Regulação da Expressão Gênica no Desenvolvimento , Proteínas Hedgehog/genética , Proteínas Hedgehog/metabolismo , Canais Iônicos/genética , Canais Iônicos/metabolismo , Camundongos , Células-Tronco Embrionárias Murinas/citologia , Células-Tronco Embrionárias Murinas/metabolismo , Miócitos Cardíacos/citologia , Miócitos Cardíacos/metabolismo , RNA Mensageiro/metabolismo , Proteínas Repressoras/deficiência
19.
Nat Commun ; 11(1): 3168, 2020 06 23.
Artigo em Inglês | MEDLINE | ID: mdl-32576830

RESUMO

In humans, mutations in the PIEZO2 gene, which encodes for a mechanosensitive ion channel, were found to result in skeletal abnormalities including scoliosis and hip dysplasia. Here, we show in mice that loss of Piezo2 expression in the proprioceptive system recapitulates several human skeletal abnormalities. While loss of Piezo2 in chondrogenic or osteogenic lineages does not lead to human-like skeletal abnormalities, its loss in proprioceptive neurons leads to spine malalignment and hip dysplasia. To validate the non-autonomous role of proprioception in hip joint morphogenesis, we studied this process in mice mutant for proprioceptive system regulators Runx3 or Egr3. Loss of Runx3 in the peripheral nervous system, but not in skeletal lineages, leads to similar joint abnormalities, as does Egr3 loss of function. These findings expand the range of known regulatory roles of the proprioception system on the skeleton and provide a central component of the underlying molecular mechanism, namely Piezo2.


Assuntos
Canais Iônicos/metabolismo , Anormalidades Musculoesqueléticas/metabolismo , Sistema Musculoesquelético/metabolismo , Neurônios/metabolismo , Propriocepção/fisiologia , Anormalidades Múltiplas , Animais , Remodelação Óssea , Subunidade alfa 3 de Fator de Ligação ao Core/metabolismo , Modelos Animais de Doenças , Proteína 3 de Resposta de Crescimento Precoce/metabolismo , Predisposição Genética para Doença/genética , Luxação do Quadril/genética , Luxação do Quadril/metabolismo , Luxação do Quadril/patologia , Articulação do Quadril/anatomia & histologia , Articulação do Quadril/metabolismo , Articulação do Quadril/patologia , Canais Iônicos/genética , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Anormalidades Musculoesqueléticas/genética , Anormalidades Musculoesqueléticas/patologia , Sistema Musculoesquelético/patologia , Escoliose
20.
Am J Physiol Cell Physiol ; 319(2): C250-C257, 2020 08 01.
Artigo em Inglês | MEDLINE | ID: mdl-32579474

RESUMO

The classic view of the red blood cell (RBC) presents a biologically inert cell that upon maturation has limited capacity to alter its physical properties. This view developed largely because of the absence of translational machinery and inability to synthesize or repair proteins in circulating RBC. Recent developments have challenged this perspective, in light of observations supporting the importance of posttranslational modifications and greater understanding of ion movement in these cells, that each regulate a myriad of cellular properties. There is thus now sufficient evidence to induce a step change in understanding of RBC: rather than passively responding to the surrounding environment, these cells have the capacity to actively regulate their physical properties and thus alter flow behavior of blood. Specific evidence supports that the physical and rheological properties of RBC are subject to active modulation, primarily by the second-messenger molecules nitric oxide (NO) and calcium-ions (Ca2+). Furthermore, an isoform of nitric oxide synthase is expressed in RBC (RBC-NOS), which has been recently demonstrated to have an active role in regulating the physical properties of RBC. Mechanical stimulation of the cell membrane activates RBC-NOS, leading to NO generation, which has several intracellular effects, including the S-nitrosylation of integral membrane components. Intracellular concentration of Ca2+ is increased upon mechanical stimulation via the recently identified mechanosensitive cation channel piezo1. Increased intracellular Ca2+ modifies the physical properties of RBC by regulating cell volume and potentially altering several important intracellular proteins. A synthesis of recent advances in understanding of molecular processes within RBC thus challenges the classic view of these cells and rather indicates a highly active cell with self-regulated mechanical properties.


Assuntos
Eritrócitos/metabolismo , Canais Iônicos/genética , Mecanotransdução Celular/genética , Óxido Nítrico Sintase/genética , Cálcio/metabolismo , Membrana Celular/enzimologia , Membrana Celular/genética , Ativação Enzimática/genética , Eritrócitos/enzimologia , Regulação Enzimológica da Expressão Gênica/genética , Humanos , Canais Iônicos/sangue , Óxido Nítrico/metabolismo , Óxido Nítrico Sintase/metabolismo
SELEÇÃO DE REFERÊNCIAS
DETALHE DA PESQUISA
...