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1.
Nat Commun ; 10(1): 2635, 2019 06 14.
Artigo em Inglês | MEDLINE | ID: mdl-31201302

RESUMO

Multidrug efflux pumps actively expel a wide range of toxic substrates from the cell and play a major role in intrinsic and acquired drug resistance. In Gram-negative bacteria, these pumps form tripartite assemblies that span the cell envelope. However, the in situ structure and assembly mechanism of multidrug efflux pumps remain unknown. Here we report the in situ structure of the Escherichia coli AcrAB-TolC multidrug efflux pump obtained by electron cryo-tomography and subtomogram averaging. The fully assembled efflux pump is observed in a closed state under conditions of antibiotic challenge and in an open state in the presence of AcrB inhibitor. We also observe intermediate AcrAB complexes without TolC and discover that AcrA contacts the peptidoglycan layer of the periplasm. Our data point to a sequential assembly process in living bacteria, beginning with formation of the AcrAB subcomplex and suggest domains to target with efflux pump inhibitors.


Assuntos
Proteínas da Membrana Bacteriana Externa/metabolismo , Proteínas de Transporte/fisiologia , Proteínas de Escherichia coli/metabolismo , Proteínas de Escherichia coli/fisiologia , Escherichia coli/fisiologia , Lipoproteínas/metabolismo , Proteínas de Membrana Transportadoras/metabolismo , Proteínas Associadas à Resistência a Múltiplos Medicamentos/metabolismo , Antibacterianos/farmacologia , Proteínas de Transporte/efeitos dos fármacos , Proteínas de Transporte/ultraestrutura , Microscopia Crioeletrônica/métodos , Farmacorresistência Bacteriana Múltipla/efeitos dos fármacos , Tomografia com Microscopia Eletrônica/métodos , Escherichia coli/efeitos dos fármacos , Escherichia coli/ultraestrutura , Proteínas de Escherichia coli/antagonistas & inibidores , Proteínas de Escherichia coli/efeitos dos fármacos , Proteínas de Escherichia coli/ultraestrutura , Microscopia Intravital/métodos , Proteínas Associadas à Resistência a Múltiplos Medicamentos/antagonistas & inibidores , Peptidoglicano/metabolismo , Periplasma/metabolismo , Ligação Proteica/efeitos dos fármacos , Estrutura Quaternária de Proteína/efeitos dos fármacos
2.
Nat Commun ; 10(1): 2637, 2019 06 14.
Artigo em Inglês | MEDLINE | ID: mdl-31201332

RESUMO

The brain stores and recalls memories through a set of neurons, termed engram cells. However, it is unclear how these cells are organized to constitute a corresponding memory trace. We established a unique imaging system that combines Ca2+ imaging and engram identification to extract the characteristics of engram activity by visualizing and discriminating between engram and non-engram cells. Here, we show that engram cells detected in the hippocampus display higher repetitive activity than non-engram cells during novel context learning. The total activity pattern of the engram cells during learning is stable across post-learning memory processing. Within a single engram population, we detected several sub-ensembles composed of neurons collectively activated during learning. Some sub-ensembles preferentially reappear during post-learning sleep, and these replayed sub-ensembles are more likely to be reactivated during retrieval. These results indicate that sub-ensembles represent distinct pieces of information, which are then orchestrated to constitute an entire memory.


Assuntos
Hipocampo/fisiologia , Memória/fisiologia , Neurônios/fisiologia , Animais , Mapeamento Encefálico/métodos , Feminino , Hipocampo/citologia , Microscopia Intravital/métodos , Proteínas Luminescentes/química , Masculino , Camundongos Endogâmicos C57BL , Camundongos Endogâmicos ICR , Camundongos Transgênicos , Microscopia de Fluorescência/métodos , Modelos Animais , Imagem Óptica/métodos , Optogenética/métodos , Sono/fisiologia
3.
Nat Commun ; 10(1): 2693, 2019 06 19.
Artigo em Inglês | MEDLINE | ID: mdl-31217419

RESUMO

The kinesin-3 KIF1C is a fast organelle transporter implicated in the transport of dense core vesicles in neurons and the delivery of integrins to cell adhesions. Here we report the mechanisms of autoinhibition and release that control the activity of KIF1C. We show that the microtubule binding surface of KIF1C motor domain interacts with its stalk and that these autoinhibitory interactions are released upon binding of protein tyrosine phosphatase PTPN21. The FERM domain of PTPN21 stimulates dense core vesicle transport in primary hippocampal neurons and rescues integrin trafficking in KIF1C-depleted cells. In vitro, human full-length KIF1C is a processive, plus-end directed motor. Its landing rate onto microtubules increases in the presence of either PTPN21 FERM domain or the cargo adapter Hook3 that binds the same region of KIF1C tail. This autoinhibition release mechanism allows cargo-activated transport and might enable motors to participate in bidirectional cargo transport without undertaking a tug-of-war.


Assuntos
Cinesina/metabolismo , Proteínas Associadas aos Microtúbulos/metabolismo , Proteínas Tirosina Fosfatases não Receptoras/metabolismo , Animais , Transporte Biológico , Linhagem Celular , Vesículas Citoplasmáticas/metabolismo , Hipocampo/citologia , Humanos , Integrinas/metabolismo , Microscopia Intravital/métodos , Cinesina/genética , Cinesina/isolamento & purificação , Camundongos , Proteínas Associadas aos Microtúbulos/isolamento & purificação , Microtúbulos/metabolismo , Neurônios/citologia , Cultura Primária de Células , Ligação Proteica , Domínios Proteicos , Proteínas Tirosina Fosfatases não Receptoras/genética , Proteínas Tirosina Fosfatases não Receptoras/isolamento & purificação , RNA Interferente Pequeno/metabolismo , Ratos , Proteínas Recombinantes/genética , Proteínas Recombinantes/isolamento & purificação , Proteínas Recombinantes/metabolismo , Imagem Individual de Molécula/métodos
4.
Nat Commun ; 10(1): 2745, 2019 06 21.
Artigo em Inglês | MEDLINE | ID: mdl-31227705

RESUMO

Small molecule probes are indispensable tools to explore diverse cellular events. However, finding a specific probe of a target remains a high challenge. Here we report the discovery of Fast-TRFS, a specific and superfast fluorogenic probe of mammalian thioredoxin reductase, a ubiquitous enzyme involved in regulation of diverse cellular redox signaling pathways. By systematically examining the processes of fluorophore release and reduction of cyclic disulfides/diselenides by the enzyme, structural factors that determine the response rate and specificity of the probe are disclosed. Mechanistic studies reveal that the fluorescence signal is switched on by a simple reduction of the disulfide bond within the probe, which is in stark contrast to the sensing mechanism of published probes. The favorable properties of Fast-TRFS enable development of a high-throughput screening assay to discover inhibitors of thioredoxin reductase by using crude tissue extracts as a source of the enzyme.


Assuntos
Descoberta de Drogas/métodos , Corantes Fluorescentes/química , Imagem Molecular/métodos , Sondas Moleculares/química , Tiorredoxina Redutase 1/metabolismo , Animais , Produtos Biológicos/farmacologia , Misturas Complexas , Dissulfetos/química , Corantes Fluorescentes/metabolismo , Células HeLa , Ensaios de Triagem em Larga Escala/métodos , Humanos , Microscopia Intravital/métodos , Microscopia de Fluorescência/métodos , Sondas Moleculares/metabolismo , Oxirredução , RNA Interferente Pequeno/metabolismo , Proteínas Recombinantes/metabolismo , Especificidade por Substrato , Tiorredoxina Redutase 1/antagonistas & inibidores , Tiorredoxina Redutase 1/genética
5.
Methods Mol Biol ; 1966: 27-38, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31041737

RESUMO

Immunohistochemistry using formalin-fixed, paraffin-embedded tissue, chromogen label, and light microscopy has traditionally been used to semiquantify estrogen receptor (ER) to guide diagnosis and management of breast cancer. Quantitation of ER for this purpose currently only assesses levels of the ER-alpha subtype. Considerable variability in results reported has been due to protocol and fixation variability, intraobserver and interobserver variability, and different scoring systems and thresholds for scoring ER positivity. Results can also vary with low expression levels of ER. ER-beta expression is reduced in breast and ovarian cancers and requires quantitation.Herein we describe a novel approach to quantifying ERß using older mouse ovarian surface epithelium, where ERß is expressed at lower levels than ERα and is therefore harder to detect. We use an antibody highly specific to the ERß1 isoform, together with immunofluorescence, confocal microscopy, and imaging and statistical software to achieve clear, reproducible, and unbiased quantitation of ERß.


Assuntos
Receptor beta de Estrogênio/análise , Regulação da Expressão Gênica , Imuno-Histoquímica/métodos , Microscopia Confocal/métodos , Ovário/metabolismo , Epitélio/metabolismo , Receptor beta de Estrogênio/genética , Feminino , Regulação Neoplásica da Expressão Gênica , Humanos , Microscopia Intravital/métodos , Neoplasias Ovarianas/genética , Neoplasias Ovarianas/metabolismo
6.
Nat Commun ; 10(1): 2194, 2019 05 16.
Artigo em Inglês | MEDLINE | ID: mdl-31097704

RESUMO

Although the physical properties of chromosomes, including their morphology, mechanics, and dynamics are crucial for their biological function, many basic questions remain unresolved. Here we directly image the circular chromosome in live E. coli with a broadened cell shape. We find that it exhibits a torus topology with, on average, a lower-density origin of replication and an ultrathin flexible string of DNA at the terminus of replication. At the single-cell level, the torus is strikingly heterogeneous, with blob-like Mbp-size domains that undergo major dynamic rearrangements, splitting and merging at a minute timescale. Our data show a domain organization underlying the chromosome structure of E. coli, where MatP proteins induce site-specific persistent domain boundaries at Ori/Ter, while transcription regulators HU and Fis induce weaker transient domain boundaries throughout the genome. These findings provide an architectural basis for the understanding of the dynamic spatial organization of bacterial genomes in live cells.


Assuntos
Cromossomos Bacterianos/química , DNA Bacteriano/química , DNA Circular/química , Escherichia coli/genética , Genoma Bacteriano , Proteínas Cromossômicas não Histona/metabolismo , Cromossomos Bacterianos/metabolismo , Replicação do DNA , DNA Bacteriano/metabolismo , DNA Circular/metabolismo , Escherichia coli/metabolismo , Proteínas de Escherichia coli/metabolismo , Microscopia Intravital/instrumentação , Microscopia Intravital/métodos , Microscopia de Fluorescência/instrumentação , Microscopia de Fluorescência/métodos , Conformação de Ácido Nucleico , Análise de Célula Única/instrumentação , Análise de Célula Única/métodos
7.
Nat Commun ; 10(1): 2181, 2019 05 16.
Artigo em Inglês | MEDLINE | ID: mdl-31097714

RESUMO

During the blood stage of human malaria, Plasmodium falciparum parasites divide by schizogony-a process wherein components for several daughter cells are produced within a common cytoplasm and then segmentation, a synchronized cytokinesis, produces individual invasive daughters. The basal complex is hypothesized to be required for segmentation, acting as a contractile ring to establish daughter cell boundaries. Here we identify an essential component of the basal complex which we name PfCINCH. Using three-dimensional reconstructions of parasites at electron microscopy resolution, we show that while parasite organelles form and divide normally, PfCINCH-deficient parasites develop inviable conjoined daughters that contain components for multiple cells. Through biochemical evaluation of the PfCINCH-containing complex, we discover multiple previously undescribed basal complex proteins. Therefore, this work provides genetic evidence that the basal complex is required for precise segmentation and lays the groundwork for a mechanistic understanding of how the parasite contractile ring drives cell division.


Assuntos
Divisão Celular/fisiologia , Proteínas Contráteis/fisiologia , Plasmodium falciparum/fisiologia , Proteínas de Protozoários/fisiologia , Animais , Eritrócitos/parasitologia , Microscopia Intravital/métodos , Proteínas Luminescentes/química , Proteínas Luminescentes/genética , Microscopia Eletrônica de Transmissão , Plasmodium falciparum/ultraestrutura , Esquizontes/fisiologia , Imagem com Lapso de Tempo
8.
Nat Commun ; 10(1): 2013, 2019 05 01.
Artigo em Inglês | MEDLINE | ID: mdl-31043592

RESUMO

Tight control over protein degradation is a fundamental requirement for cells to respond rapidly to various stimuli and adapt to a fluctuating environment. Here we develop a versatile, easy-to-handle library of destabilizing tags (degrons) for the precise regulation of protein expression profiles in mammalian cells by modulating target protein half-lives in a predictable manner. Using the well-established tetracycline gene-regulation system as a model, we show that the dynamics of protein expression can be tuned by fusing appropriate degron tags to gene regulators. Next, we apply this degron library to tune a synthetic pulse-generating circuit in mammalian cells. With this toolbox we establish a set of pulse generators with tailored pulse lengths and magnitudes of protein expression. This methodology will prove useful in the functional roles of essential proteins, fine-tuning of gene-expression systems, and enabling a higher complexity in the design of synthetic biological systems in mammalian cells.


Assuntos
Sequência de Aminoácidos/genética , Regulação da Expressão Gênica , Engenharia de Proteínas/métodos , Proteólise , Biotecnologia/métodos , Células HEK293 , Meia-Vida , Células HeLa , Humanos , Microscopia Intravital/métodos , Células-Tronco Mesenquimais , Microscopia de Fluorescência , Biologia Sintética/métodos
10.
Nat Commun ; 10(1): 2220, 2019 05 17.
Artigo em Inglês | MEDLINE | ID: mdl-31101805

RESUMO

Both medullary thymic epithelial cells (mTEC) and dendritic cells (DC) present tissue-restricted antigens (TRA) to thymocytes to induce central tolerance, but the relative contributions of these antigen-presenting cell (APC) subsets remain unresolved. Here we developed a two-photon microscopy approach to observe thymocytes interacting with intact APCs presenting TRAs. We find that mTECs and DCs cooperate extensively to induce tolerance, with their relative contributions regulated by the cellular form of the TRA and the class of major histocompatibility complex (MHC) on which antigen is presented. Even when TRA expression is restricted to mTECs, DCs still present self-antigens at least as frequently as mTECs. Notably, the DC subset cDC2 efficiently acquires secreted mTEC-derived TRAs for cross-presentation on MHC-I. By directly imaging interactions between thymocytes and APCs, while monitoring intracellular signaling, this study reveals that distinct DC subsets and AIRE+ mTECs contribute substantially to presentation of diverse self-antigens for establishing central tolerance.


Assuntos
Tolerância Central/imunologia , Células Dendríticas/imunologia , Timócitos/imunologia , Timo/imunologia , Animais , Apresentação do Antígeno/imunologia , Autoantígenos/imunologia , Autoantígenos/metabolismo , Transplante de Medula Óssea , Separação Celular/métodos , Células Dendríticas/metabolismo , Células Epiteliais/imunologia , Feminino , Citometria de Fluxo/métodos , Microscopia Intravital/métodos , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Microscopia de Fluorescência por Excitação Multifotônica/métodos , Linfócitos T Reguladores/imunologia , Timócitos/metabolismo , Timo/citologia , Fatores de Transcrição/imunologia , Fatores de Transcrição/metabolismo , Quimeras de Transplante/imunologia
11.
Nat Commun ; 10(1): 2064, 2019 05 02.
Artigo em Inglês | MEDLINE | ID: mdl-31048701

RESUMO

Afterglow imaging with long-lasting luminescence after cessation of light excitation provides opportunities for ultrasensitive molecular imaging; however, the lack of biologically compatible afterglow agents has impeded exploitation in clinical settings. This study presents a generic approach to transforming ordinary optical agents (including fluorescent polymers, dyes, and inorganic semiconductors) into afterglow luminescent nanoparticles (ALNPs). This approach integrates a cascade photoreaction into a single-particle entity, enabling ALNPs to chemically store photoenergy and spontaneously decay it in an energy-relay process. Not only can the afterglow profiles of ALNPs be finetuned to afford emission from visible to near-infrared (NIR) region, but also their intensities can be predicted by a mathematical model. The representative NIR ALNPs permit rapid detection of tumors in living mice with a signal-to-background ratio that is more than three orders of magnitude higher than that of NIR fluorescence. The biodegradability of the ALNPs further heightens their potential for ultrasensitive in vivo imaging.


Assuntos
Engenharia Química/métodos , Microscopia Intravital/métodos , Imagem Molecular/métodos , Nanopartículas/química , Neoplasias/diagnóstico por imagem , Animais , Linhagem Celular Tumoral/transplante , Estudos de Viabilidade , Feminino , Corantes Fluorescentes/química , Luminescência , Camundongos , Camundongos Nus , Microscopia de Fluorescência/métodos , Nanotecnologia/métodos , Sensibilidade e Especificidade
12.
Nat Commun ; 10(1): 2167, 2019 05 15.
Artigo em Inglês | MEDLINE | ID: mdl-31092821

RESUMO

Ribbon synapses transmit information in sensory systems, but their development is not well understood. To test the hypothesis that ribbon assembly stabilizes nascent synapses, we performed simultaneous time-lapse imaging of fluorescently-tagged ribbons in retinal cone bipolar cells (BCs) and postsynaptic densities (PSD95-FP) of retinal ganglion cells (RGCs). Ribbons and PSD95-FP clusters were more stable when these components colocalized at synapses. However, synapse density on ON-alpha RGCs was unchanged in mice lacking ribbons (ribeye knockout). Wildtype BCs make both ribbon-containing and ribbon-free synapses with these GCs even at maturity. Ribbon assembly and cone BC-RGC synapse maintenance are thus regulated independently. Despite the absence of synaptic ribbons, RGCs continued to respond robustly to light stimuli, although quantitative examination of the responses revealed reduced frequency and contrast sensitivity.


Assuntos
Células Fotorreceptoras Retinianas Cones/fisiologia , Sinapses/metabolismo , Transmissão Sináptica/fisiologia , Animais , Células Cultivadas , Proteína 4 Homóloga a Disks-Large/genética , Proteína 4 Homóloga a Disks-Large/metabolismo , Microscopia Intravital/métodos , Luz , Proteínas Luminescentes/química , Proteínas Luminescentes/genética , Camundongos , Camundongos Transgênicos , Microscopia de Fluorescência/métodos , Estimulação Luminosa , Cultura Primária de Células , Células Bipolares da Retina/fisiologia , Células Ganglionares da Retina/fisiologia , Imagem com Lapso de Tempo/métodos
13.
Nat Commun ; 10(1): 2285, 2019 05 23.
Artigo em Inglês | MEDLINE | ID: mdl-31123251

RESUMO

Growing tissue and bacterial colonies are active matter systems where cell divisions and cellular motion generate active stress. Although they operate in the non-equilibrium regime, these biological systems can form large-scale ordered structures. How mechanical instabilities drive the dynamics of active matter systems and form ordered structures are not well understood. Here, we use chaining Bacillus subtilis, also known as a biofilm, to study the relation between mechanical instabilities and nematic ordering. We find that bacterial biofilms have intrinsic length scales above which a series of mechanical instabilities occur. Localized stress and friction drive buckling and edge instabilities which further create nematically aligned structures and topological defects. We also observe that topological defects control stress distribution and initiate the formation of sporulation sites by creating three-dimensional structures. In this study we propose an alternative active matter platform to study the essential roles of mechanics in growing biological tissue.


Assuntos
Bacillus subtilis/fisiologia , Biofilmes , Microscopia Intravital/métodos , Bacillus subtilis/ultraestrutura , Fenômenos Biomecânicos , Microscopia Eletrônica de Varredura/métodos , Microscopia de Fluorescência/métodos , Estresse Mecânico , Imagem com Lapso de Tempo/métodos
14.
Nat Commun ; 10(1): 2075, 2019 05 06.
Artigo em Inglês | MEDLINE | ID: mdl-31061418

RESUMO

Diverse processes-e.g. bioremediation, biofertilization, and microbial drug delivery-rely on bacterial migration in disordered, three-dimensional (3D) porous media. However, how pore-scale confinement alters bacterial motility is unknown due to the opacity of typical 3D media. As a result, models of migration are limited and often employ ad hoc assumptions. Here we reveal that the paradigm of run-and-tumble motility is dramatically altered in a porous medium. By directly visualizing individual Escherichia coli, we find that the cells are intermittently and transiently trapped as they navigate the pore space, exhibiting diffusive behavior at long time scales. The trapping durations and the lengths of "hops" between traps are broadly distributed, reminiscent of transport in diverse other disordered systems; nevertheless, we show that these quantities can together predict the long-time bacterial translational diffusivity. Our work thus provides a revised picture of bacterial motility in complex media and yields principles for predicting cellular migration.


Assuntos
Escherichia coli/fisiologia , Microscopia Intravital/métodos , Modelos Biológicos , Análise de Célula Única/métodos , Hidrogéis/química , Microscopia de Fluorescência/métodos , Movimento/fisiologia , Porosidade
15.
Nat Plants ; 5(5): 498-504, 2019 05.
Artigo em Inglês | MEDLINE | ID: mdl-31040442

RESUMO

Cotton (Gossypium hirsutum) fibres consist of single cells that grow in a highly polarized manner, assumed to be controlled by the cytoskeleton1-3. However, how the cytoskeletal organization and dynamics underpin fibre development remains unexplored. Moreover, it is unclear whether cotton fibres expand via tip growth or diffuse growth2-4. We generated stable transgenic cotton plants expressing fluorescent markers of the actin and microtubule cytoskeleton. Live-cell imaging revealed that elongating cotton fibres assemble a cortical filamentous actin network that extends along the cell axis to finally form actin strands with closed loops in the tapered fibre tip. Analyses of F-actin network properties indicate that cotton fibres have a unique actin organization that blends features of both diffuse and tip growth modes. Interestingly, typical actin organization and endosomal vesicle aggregation found in tip-growing cell apices were not observed in fibre tips. Instead, endomembrane compartments were evenly distributed along the elongating fibre cells and moved bi-directionally along the fibre shank to the fibre tip. Moreover, plus-end tracked microtubules transversely encircled elongating fibre shanks, reminiscent of diffusely growing cells. Collectively, our findings indicate that cotton fibres elongate via a unique tip-biased diffuse growth mode.


Assuntos
Fibra de Algodão , Citoesqueleto/ultraestrutura , Gossypium/ultraestrutura , Actinas/ultraestrutura , Proteínas de Fluorescência Verde , Imagem Tridimensional , Microscopia Intravital/métodos , Microtúbulos/ultraestrutura
16.
Nat Commun ; 10(1): 1877, 2019 04 23.
Artigo em Inglês | MEDLINE | ID: mdl-31015402

RESUMO

Many microorganisms have evolved chemotactic strategies to exploit the microscale heterogeneity that frequently characterizes microbial habitats. Chemotaxis has been primarily studied as an average characteristic of a population, with little regard for variability among individuals. Here, we adopt a classic tool from animal ecology - the T-maze - and implement it at the microscale by using microfluidics to expose bacteria to a sequence of decisions, each consisting of migration up or down a chemical gradient. Single-cell observations of clonal Escherichia coli in the maze, coupled with a mathematical model, reveal that strong heterogeneity in the chemotactic sensitivity coefficient exists even within clonal populations of bacteria. A comparison of different potential sources of heterogeneity reveals that heterogeneity in the T-maze originates primarily from the chemotactic sensitivity coefficient, arising from a distribution of pathway gains. This heterogeneity may have a functional role, for example in the context of migratory bet-hedging strategies.


Assuntos
Quimiotaxia/genética , Escherichia coli/fisiologia , Modelos Biológicos , Fenótipo , Dimetilpolisiloxanos/química , Microscopia Intravital/métodos , Técnicas Analíticas Microfluídicas/métodos , Microscopia de Contraste de Fase/métodos , Análise de Célula Única/métodos
17.
Methods Mol Biol ; 1968: 183-194, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-30929215

RESUMO

Two-photon intravital imaging (2P-IVM) of the murine trachea is a powerful technique for real-time imaging of immune cell recruitment and trafficking during airborne pathogen infections. Neutrophils are an important component of the innate immune response that are able to rapidly infiltrate the airway mucosa in response to Streptococcus pneumoniae infection. Here we describe a protocol to visualize in vivo neutrophil extravasation and cell dynamics in the tracheal tissue of a S. pneumoniae-infected mouse using 2P-IVM. To perform this protocol, we infected and imaged the trachea of a lysozyme M green fluorescent protein (LysM-GFP) mouse, in which neutrophils express GFP. Additionally, we used a custom-designed platform, which allowed the intubation and fixation of the trachea after surgical exposition, and we injected intravenously a fluorescently labeled dextran solution to visualize the blood vessels.


Assuntos
Microscopia Intravital/métodos , Leucócitos/metabolismo , Infecções Pneumocócicas/diagnóstico , Infecções Pneumocócicas/microbiologia , Traqueia/diagnóstico por imagem , Traqueia/metabolismo , Animais , Proteínas de Fluorescência Verde/metabolismo , Camundongos , Neutrófilos/metabolismo , Streptococcus pneumoniae/patogenicidade
18.
Chemosphere ; 227: 551-560, 2019 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-31004822

RESUMO

Precise in vivo toxicological assays to determine the cardiotoxicity of pharmaceuticals and their waste products are essential in order to evaluate their risks to humans and the environment following industrial release. In the present study, we aimed to develop the sensitive imaging-based cardiotoxicity assay and combined 3D light-sheet microscopy with a zebrafish model to identify hidden cardiovascular anomalies induced by valproic acid (VPA) exposure. The zebrafish model is advantageous for this assessment because its embryos remain transparent. The 3D spatial localization of fluorescence-labeled cardiac cells in and around the heart using light-sheet technology revealed dislocalization of the heart from the outflow tract in two-day-old zebrafish embryos treated with 50 µM and 100 µM VPA (P < 0.01) and those embryos exposed to 20 µM VPA presented hypoplastic distal ventricles (P < 0.01). These two observed phenotypes are second heart field-derived cardiac defects. Quantitative analysis of the light-sheet imaging demonstrated that folic acid (FA) supplementation significantly increased the numbers of endocardial and myocardial cells (P < 0.05) and the accretion of second heart field-derived cardiomyocytes to the arterial pole of the outflow tract. The heart rate increased in response to the cellular changes occurring in embryonic heart development (P < 0.05). The present study disclosed the cellular mechanism underlying the role of FA in spontaneous cellular changes in cardiogenesis and in VPA-associated cardiotoxicity. The 3D light-sheet assay may be the next-generation test to evaluate the risks of previously undetected pharmaceutical and environmental cardiotoxicities in both humans and animals.


Assuntos
Cardiotoxicidade/diagnóstico por imagem , Ácido Fólico/uso terapêutico , Cardiopatias Congênitas/induzido quimicamente , Ácido Valproico/toxicidade , Peixe-Zebra/embriologia , Animais , Bioensaio/métodos , Cardiotoxicidade/tratamento farmacológico , Cardiotoxicidade/etiologia , Diagnóstico por Imagem/métodos , Embrião não Mamífero/efeitos dos fármacos , Desenvolvimento Embrionário/efeitos dos fármacos , Microscopia Intravital/métodos , Miócitos Cardíacos/efeitos dos fármacos , Miócitos Cardíacos/patologia
19.
Nat Commun ; 10(1): 1799, 2019 04 17.
Artigo em Inglês | MEDLINE | ID: mdl-30996301

RESUMO

Chemoenzymatic modification of cell-surface glycan structures has emerged as a complementary approach to metabolic oligosaccharide engineering. Here, we identify Pasteurella multocida α2-3-sialyltransferase M144D mutant, Photobacterium damsela α2-6-sialyltransferase, and Helicobacter mustelae α1-2-fucosyltransferase, as efficient tools for live-cell glycan modification. Combining these enzymes with Helicobacter pylori α1-3-fucosyltransferase, we develop a host-cell-based assay to probe glycan-mediated influenza A virus (IAV) infection including wild-type and mutant strains of H1N1 and H3N2 subtypes. At high NeuAcα2-6-Gal levels, the IAV-induced host-cell death is positively correlated with haemagglutinin (HA) binding affinity to NeuAcα2-6-Gal. Remarkably, an increment of host-cell-surface sialyl Lewis X (sLeX) exacerbates the killing by several wild-type IAV strains and a previously engineered mutant HK68-MTA. Structural alignment of HAs from HK68 and HK68-MTA suggests formation of a putative hydrogen bond between Trp222 of HA-HK68-MTA and the C-4 hydroxyl group of the α1-3-linked fucose of sLeX, which may account for the enhanced host cell killing of that mutant.


Assuntos
Proteínas de Bactérias/metabolismo , Glicosiltransferases/metabolismo , Hemaglutininas/imunologia , Interações Hospedeiro-Patógeno/imunologia , Influenza Humana/imunologia , Oligossacarídeos/metabolismo , Animais , Proteínas de Bactérias/genética , Bioensaio/métodos , Células CHO , Cricetulus , Cães , Glicosiltransferases/genética , Voluntários Saudáveis , Helicobacter mustelae/genética , Helicobacter mustelae/metabolismo , Hemaglutininas/metabolismo , Humanos , Vírus da Influenza A Subtipo H1N1/genética , Vírus da Influenza A Subtipo H1N1/imunologia , Vírus da Influenza A Subtipo H3N2/genética , Vírus da Influenza A Subtipo H3N2/imunologia , Influenza Humana/virologia , Microscopia Intravital/métodos , Luciferases Bacterianas/genética , Luciferases Bacterianas/metabolismo , Pulmão/patologia , Células Madin Darby de Rim Canino , Engenharia Metabólica/métodos , Microscopia de Fluorescência/métodos , Imagem Molecular/métodos , Oligossacarídeos/imunologia , Pasteurella multocida/genética , Pasteurella multocida/metabolismo , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Coloração e Rotulagem/métodos
20.
Nat Commun ; 10(1): 1652, 2019 04 10.
Artigo em Inglês | MEDLINE | ID: mdl-30971691

RESUMO

Understanding the relationship between intracellular motion and macromolecular structure remains a challenge in biology. Macromolecular structures are assembled from numerous molecules, some of which cannot be labeled. Most techniques to study motion require potentially cytotoxic dyes or transfection, which can alter cellular behavior and are susceptible to photobleaching. Here we present a multimodal label-free imaging platform for measuring intracellular structure and macromolecular dynamics in living cells with a sensitivity to macromolecular structure as small as 20 nm and millisecond temporal resolution. We develop and validate a theory for temporal measurements of light interference. In vitro, we study how higher-order chromatin structure and dynamics change during cell differentiation and ultraviolet (UV) light irradiation. Finally, we discover cellular paroxysms, a near-instantaneous burst of macromolecular motion that occurs during UV induced cell death. With nanoscale sensitive, millisecond resolved capabilities, this platform could address critical questions about macromolecular behavior in live cells.


Assuntos
Apoptose/efeitos da radiação , Microscopia Intravital/métodos , Microscopia de Interferência/métodos , Imagem Multimodal/métodos , Raios Ultravioleta/efeitos adversos , Citoesqueleto de Actina/metabolismo , Diferenciação Celular , Cromatina/metabolismo , Células HeLa , Humanos , Microscopia Intravital/instrumentação , Células-Tronco Mesenquimais , Microscopia de Interferência/instrumentação , Imagem Multimodal/instrumentação , Nanosferas , Imagens de Fantasmas , Fosfatidilserinas/metabolismo , Fatores de Tempo
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