RESUMEN
Dissemination of food-borne L. monocytogenes in the host relies on internalin-mediated invasion, but the underlying invasion strategies remain elusive. Here we use live-cell microscopy to follow single cell interactions between individual human cells and L. monocytogenes and elucidate mechanisms associated with internalin B (InlB)-mediated invasion. We demonstrate that whilst a replicative invasion of nonphagocytic cells is a rare event even at high multiplicities of invasion, L. monocytogenes overcomes this by utilising a strategy relaying on PrfA-mediated ActA-based aggregation. We show that L. monocytogenes forms aggregates in extracellular host cell environment, which promote approximately 5-fold more host cell adhesions than the non-aggregating actA-ΔC mutant (which lacks the C-terminus coding region), with the adhering bacteria inducing 3-fold more intracellular invasions. Aggregation is associated with robust MET tyrosine kinase receptor clustering in the host cells, a hallmark of InlB-mediated invasion, something not observed with the actA-ΔC mutant. Finally, we show via RNA-seq analyses that aggregation involves a global adaptive response to host cell environment (including iron depletion), resulting in metabolic changes in L. monocytogenes and upregulation of the PrfA virulence regulon. Overall, our analyses provide new mechanistic insights into internalin-mediated host-pathogen interactions of L. monocytogenes.
Asunto(s)
Adhesión Bacteriana , Proteínas Bacterianas , Listeria monocytogenes , Listeria monocytogenes/genética , Listeria monocytogenes/patogenicidad , Listeria monocytogenes/fisiología , Listeria monocytogenes/metabolismo , Humanos , Proteínas Bacterianas/genética , Proteínas Bacterianas/metabolismo , Proteínas de la Membrana/metabolismo , Proteínas de la Membrana/genética , Interacciones Huésped-Patógeno , Listeriosis/microbiología , Factores de Terminación de Péptidos/metabolismo , Factores de Terminación de Péptidos/genética , Regulación Bacteriana de la Expresión Génica , Virulencia/genética , Factores de Virulencia/genética , Factores de Virulencia/metabolismoRESUMEN
Pathogens have developed intricate strategies to overcome the host's innate immune responses. In this paper we use live-cell microscopy with a single bacterium resolution to follow in real time interactions between the food-borne pathogen L. monocytogenes and host macrophages, a key event controlling the infection in vivo. We demonstrate that infection results in heterogeneous outcomes, with only a subset of bacteria able to establish a replicative invasion of macrophages. The fate of individual bacteria in the same host cell was independent from the host cell and non-cooperative, being independent from co-infecting bacteria. A higher multiplicity of infection resulted in a reduced probability of replication of the overall bacterial population. By use of internalisation assays and conditional probabilities to mathematically describe the two-stage invasion process, we demonstrate that the higher MOI compromises the ability of macrophages to phagocytose bacteria. We found that the rate of phagocytosis is mediated via the secreted Listeriolysin toxin (LLO), while the probability of replication of intracellular bacteria remained constant. Using strains expressing fluorescent reporters to follow transcription of either the LLO-encoding hly or actA genes, we show that replicative bacteria exhibited higher PrfA regulon expression in comparison to those bacteria that did not replicate, however elevated PrfA expression per se was not sufficient to increase the probability of replication. Overall, this demonstrates a new role for the population-level, but not single cell, PrfA-mediated activity to regulate outcomes of host pathogen interactions.
Asunto(s)
Listeria monocytogenes , Macrófagos , Fagocitosis , Microscopía , Inmunidad InnataRESUMEN
Cells respond to inflammatory stimuli such as cytokines by activation of the nuclear factor-κB (NF-κB) signalling pathway, resulting in oscillatory translocation of the transcription factor p65 between nucleus and cytoplasm in some cell types. We investigate the relationship between p65 and inhibitor-κB⺠(IκBα) protein levels and dynamic properties of the system, and how this interaction impacts on the expression of key inflammatory genes. Using bacterial artificial chromosomes, we developed new cell models of IκBâº-eGFP protein overexpression in a pseudo-native genomic context. We find that cells with high levels of the negative regulator IκBα remain responsive to inflammatory stimuli and maintain dynamics for both p65 and IκBα. In contrast, canonical target gene expression is dramatically reduced by overexpression of IκBα, but can be partially rescued by overexpression of p65. Treatment with leptomycin B to promote nuclear accumulation of IκB⺠also suppresses canonical target gene expression, suggesting a mechanism in which nuclear IκB⺠accumulation prevents productive p65 interaction with promoter binding sites. This causes reduced target promoter binding and gene transcription, which we validate by chromatin immunoprecipitation and in primary cells. Overall, we show how inflammatory gene transcription is modulated by the expression levels of both IκB⺠and p65. This results in an anti-inflammatory effect on transcription, demonstrating a broad mechanism to modulate the strength of inflammatory response.
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Immune cells fine tune their responses to infection and inflammatory cues. Here, using live-cell confocal microscopy and mathematical modelling, we investigate interferon-induced JAK-STAT signalling in innate immune macrophages. We demonstrate that transient exposure to IFN-γ stimulation induces a long-term desensitisation of STAT1 signalling and gene expression responses, revealing a dose- and time-dependent regulatory feedback that controls JAK-STAT responses upon re-exposure to stimulus. We show that IFN-α/ß1 elicit different level of desensitisation from IFN-γ, where cells refractory to IFN-α/ß1 are sensitive to IFN-γ, but not vice versa. We experimentally demonstrate that the underlying feedback mechanism involves regulation of STAT1 phosphorylation but is independent of new mRNA synthesis and cognate receptor expression. A new feedback model of the protein tyrosine phosphatase activity recapitulates experimental data and demonstrates JAK-STAT network's ability to decode relative changes of dose, timing, and type of temporal interferon stimulation. These findings reveal that STAT desensitisation renders cells with signalling memory of type I and II interferon stimulation, which in the future may improve administration of interferon therapy.
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Interferón-alfa , Proteínas Tirosina Quinasas , Antivirales , Retroalimentación , Interferón-alfa/metabolismo , Quinasas Janus/metabolismo , Proteínas Tirosina Fosfatasas/metabolismo , Proteínas Tirosina Quinasas/metabolismo , ARN Mensajero , Factores de Transcripción STAT/metabolismo , Transcripción GenéticaRESUMEN
Mitochondrial dysfunction is interconnected with cancer. Nevertheless, how defective mitochondria promote cancer is poorly understood. We find that mitochondrial dysfunction promotes DNA damage under conditions of increased apoptotic priming. Underlying this process, we reveal a key role for mitochondrial dynamics in the regulation of DNA damage and genome instability. The ability of mitochondrial dynamics to regulate oncogenic DNA damage centers upon the control of minority mitochondrial outer membrane permeabilization (MOMP), a process that enables non-lethal caspase activation leading to DNA damage. Mitochondrial fusion suppresses minority MOMP and its associated DNA damage by enabling homogeneous mitochondrial expression of anti-apoptotic BCL-2 proteins. Finally, we find that mitochondrial dysfunction inhibits pro-apoptotic BAX retrotranslocation, causing BAX mitochondrial localization and thereby promoting minority MOMP. Unexpectedly, these data reveal oncogenic effects of mitochondrial dysfunction that are mediated via mitochondrial dynamics and caspase-dependent DNA damage.
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Caspasas , Dinámicas Mitocondriales , Apoptosis/genética , Proteínas Reguladoras de la Apoptosis/genética , Caspasas/metabolismo , Daño del ADN , Inestabilidad Genómica , Humanos , Proteína X Asociada a bcl-2/metabolismoRESUMEN
Chronic inflammation underpins many human diseases. Morbidity and mortality associated with chronic inflammation are often mediated through metabolic dysfunction. Inflammatory and metabolic processes vary through circadian time, suggesting an important temporal crosstalk between these systems. Using an established mouse model of rheumatoid arthritis, we show that chronic inflammatory arthritis results in rhythmic joint inflammation and drives major changes in muscle and liver energy metabolism and rhythmic gene expression. Transcriptional and phosphoproteomic analyses revealed alterations in lipid metabolism and mitochondrial function associated with increased EGFR-JAK-STAT3 signaling. Metabolomic analyses confirmed rhythmic metabolic rewiring with impaired ß-oxidation and lipid handling and revealed a pronounced shunt toward sphingolipid and ceramide accumulation. The arthritis-related production of ceramides was most pronounced during the day, which is the time of peak inflammation and increased reliance on fatty acid oxidation. Thus, our data demonstrate that localized joint inflammation drives a time-of-daydependent build-up of bioactive lipid species driven by rhythmic inflammation and altered EGFR-STAT signaling.
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Artritis , Relojes Circadianos , Ritmo Circadiano/fisiología , Metabolismo Energético , Humanos , Inflamación/metabolismoRESUMEN
The mammalian circadian clock exerts control of daily gene expression through cycles of DNA binding. Here, we develop a quantitative model of how a finite pool of BMAL1 protein can regulate thousands of target sites over daily time scales. We used quantitative imaging to track dynamic changes in endogenous labelled proteins across peripheral tissues and the SCN. We determine the contribution of multiple rhythmic processes coordinating BMAL1 DNA binding, including cycling molecular abundance, binding affinities, and repression. We find nuclear BMAL1 concentration determines corresponding CLOCK through heterodimerisation and define a DNA residence time of this complex. Repression of CLOCK:BMAL1 is achieved through rhythmic changes to BMAL1:CRY1 association and high-affinity interactions between PER2:CRY1 which mediates CLOCK:BMAL1 displacement from DNA. Finally, stochastic modelling reveals a dual role for PER:CRY complexes in which increasing concentrations of PER2:CRY1 promotes removal of BMAL1:CLOCK from genes consequently enhancing ability to move to new target sites.
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Relojes Circadianos , Factores de Transcripción ARNTL/genética , Factores de Transcripción ARNTL/metabolismo , Animales , Proteínas CLOCK/genética , Proteínas CLOCK/metabolismo , Relojes Circadianos/genética , Ritmo Circadiano/genética , Mamíferos/metabolismoRESUMEN
Gene transcription occurs in short bursts interspersed with silent periods, and these kinetics can be altered by promoter structure. The effect of alternate promoter architecture on transcription bursting is not known. We studied the human prolactin (hPRL) gene that contains 2 promoters, a pituitary-specific promoter that requires the transcription factor Pit-1 and displays dramatic transcriptional bursting activity and an alternate upstream promoter that is active in nonpituitary tissues. We studied large hPRL genomic fragments with luciferase reporters, and used bacterial artificial chromosome recombineering to manipulate critical promoter regions. Stochastic switch mathematical modelling of single-cell time-lapse luminescence image data revealed that the Pit-1-dependent promoter showed longer, higher-amplitude transcriptional bursts. Knockdown studies confirmed that the presence of Pit-1 stabilized and prolonged periods of active transcription. Pit-1 therefore plays an active role in establishing the timing of transcription cycles, in addition to its cell-specific functions.
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Prolactina/genética , Regiones Promotoras Genéticas , Factor de Transcripción Pit-1/metabolismo , Transcripción Genética , Línea Celular , Regulación de la Expresión Génica , Humanos , Hipófisis/metabolismo , Prolactina/metabolismo , Factor de Transcripción Pit-1/genéticaRESUMEN
Pituitary cells have been reported to show spontaneous calcium oscillations and dynamic transcription cycles. To study both processes in the same living cell in real time, we used rat pituitary GH3 cells stably expressing human prolactin-luciferase or prolactin-EGFP reporter gene constructs loaded with a fluorescent calcium indicator and measured activity using single-cell time-lapse microscopy. We observed heterogeneity between clonal cells in the calcium activity and prolactin transcription in unstimulated conditions. There was a significant correlation between cells displaying spontaneous calcium spikes and cells showing spontaneous bursts in prolactin expression. Notably, cells showing no basal calcium activity showed low prolactin expression but elicited a significantly greater transcriptional response to BayK8644 compared to cells showing basal calcium activity. This suggested the presence of two subsets of cells within the population at any one time. Fluorescence-activated cell sorting was used to sort cells into two populations based on the expression level of prolactin-EGFP however, the bimodal pattern of expression was restored within 26 h. Chromatin immunoprecipitation showed that these sorted populations were distinct due to the extent of histone acetylation. We suggest that maintenance of a heterogeneous bimodal population is a fundamental characteristic of this cell type and that calcium activation and histone acetylation, at least in part, drive prolactin transcriptional competence.
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Calcio/metabolismo , Ensamble y Desensamble de Cromatina , Heterogeneidad Genética , Prolactina/genética , Transcripción Genética , Acetilación , Animales , Línea Celular , Histonas/metabolismo , Prolactina/metabolismo , Ratas , Análisis de la Célula IndividualRESUMEN
Evolutionarily conserved circadian clocks generate 24-hour rhythms in physiology and behaviour that adapt organisms to their daily and seasonal environments. In mammals, the suprachiasmatic nucleus (SCN) of the hypothalamus is the principal co-ordinator of the cell-autonomous clocks distributed across all major tissues. The importance of robust daily rhythms is highlighted by experimental and epidemiological associations between circadian disruption and human diseases. BMAL1 (a bHLH-PAS domain-containing transcription factor) is the master positive regulator within the transcriptional-translational feedback loops (TTFLs) that cell-autonomously define circadian time. It drives transcription of the negative regulators Period and Cryptochrome alongside numerous clock output genes, and thereby powers circadian time-keeping. Because deletion of Bmal1 alone is sufficient to eliminate circadian rhythms in cells and the whole animal it has been widely used as a model for molecular disruption of circadian rhythms, revealing essential, tissue-specific roles of BMAL1 in, for example, the brain, liver and the musculoskeletal system. Moreover, BMAL1 has clock-independent functions that influence ageing and protein translation. Despite the essential role of BMAL1 in circadian time-keeping, direct measures of its intra-cellular behaviour are still lacking. To fill this knowledge-gap, we used CRISPR Cas9 to generate a mouse expressing a knock-in fluorescent fusion of endogenous BMAL1 protein (Venus::BMAL1) for quantitative live imaging in physiological settings. The Bmal1Venus mouse model enabled us to visualise and quantify the daily behaviour of this core clock factor in central (SCN) and peripheral clocks, with single-cell resolution that revealed its circadian expression, anti-phasic to negative regulators, nuclear-cytoplasmic mobility and molecular abundance.
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Factores de Transcripción ARNTL/genética , Envejecimiento/genética , Ritmo Circadiano , Factores de Transcripción ARNTL/metabolismo , Envejecimiento/metabolismo , Animales , Proteínas Bacterianas/genética , Proteínas Bacterianas/metabolismo , Encéfalo/embriología , Células Cultivadas , Retroalimentación Fisiológica , Hígado/metabolismo , Proteínas Luminiscentes/genética , Proteínas Luminiscentes/metabolismo , Ratones , Microscopía Fluorescente/métodos , Músculo Esquelético/metabolismo , Biosíntesis de Proteínas , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Análisis de la Célula Individual/métodosRESUMEN
BACKGROUND: Ability to adapt to temperature changes trough the Heat Shock Response (HSR) pathways is one of the most fundamental and clinically relevant cellular response systems. Heat Shock (HS) affects the signalling and gene expression responses of the Nuclear Factor κB (NF-κB) transcription factor, a critical regulator of proliferation and inflammation, however, our quantitative understanding of how cells sense and adapt to temperature changes is limited. METHODS: We used live-cell time-lapse microscopy and mathematical modelling to understand the signalling of the NF-κB system in the human MCF7 breast adenocarcinoma cells in response to pro-inflammatory Interleukin 1ß (IL1ß) and Tumour Necrosis Factor α (TNFα) cytokines, following exposure to a 37-43 °C range of physiological and clinical temperatures. RESULTS: We show that exposure to 43 °C 1 h HS inhibits the immediate NF-κB signalling response to TNFα and IL1ß stimulation although uptake of cytokines is not impaired. Within 4 h after HS treatment IL1ß-induced NF-κB responses return to normal levels, but the recovery of the TNFα-induced responses is still affected. Using siRNA knock-down of Heat Shock Factor 1 (HSF1) we show that this stimulus-specificity is conferred via the Inhibitory κB kinase (IKK) signalosome where HSF1-dependent feedback regulates TNFα, but not IL1ß-mediated IKK recovery post HS. Furthermore, we demonstrate that through the temperature-dependent denaturation and recovery of IKK, TNFα and IL1ß-mediated signalling exhibit different temperature sensitivity and adaptation to repeated HS when exposed to a 37-43 °C temperature range. Specifically, IL1ß-mediated NF-κB responses are more robust to temperature changes in comparison to those induced by TNFα treatment. CONCLUSIONS: We demonstrate that the kinetics of the NF-κB system following temperature stress is cytokine specific and exhibit differential adaptation to temperature changes. We propose that this differential temperature sensitivity is mediated via the IKK signalosome, which acts as a bona fide temperature sensor trough the HSR cross-talk. This novel quantitative understanding of NF-κB and HSR interactions is fundamentally important for the potential optimization of therapeutic hyperthermia protocols. Video Abstract.
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Expresión Génica/efectos de los fármacos , Respuesta al Choque Térmico , Inflamación/metabolismo , Interleucina-1beta/farmacología , FN-kappa B/metabolismo , Factor de Necrosis Tumoral alfa/farmacología , Humanos , Células MCF-7RESUMEN
Glucocorticoids (GCs) act through the glucocorticoid receptor (GR, also known as NR3C1) to regulate immunity, energy metabolism and tissue repair. Upon ligand binding, activated GR mediates cellular effects by regulating gene expression, but some GR effects can occur rapidly without new transcription. Here, we show that GCs rapidly inhibit cell migration, in response to both GR agonist and antagonist ligand binding. The inhibitory effect on migration is prevented by GR knockdown with siRNA, confirming GR specificity, but not by actinomycin D treatment, suggesting a non-transcriptional mechanism. We identified a rapid onset increase in microtubule polymerisation following GC treatment, identifying cytoskeletal stabilisation as the likely mechanism of action. HDAC6 overexpression, but not knockdown of αTAT1, rescued the GC effect, implicating HDAC6 as the GR effector. Consistent with this hypothesis, ligand-dependent cytoplasmic interaction between GR and HDAC6 was demonstrated by quantitative imaging. Taken together, we propose that activated GR inhibits HDAC6 function, and thereby increases the stability of the microtubule network to reduce cell motility. We therefore report a novel, non-transcriptional mechanism whereby GCs impair cell motility through inhibition of HDAC6 and rapid reorganization of the cell architecture.This article has an associated First Person interview with the first author of the paper.
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Glucocorticoides , Receptores de Glucocorticoides , Movimiento Celular , Citosol , Expresión Génica , Glucocorticoides/farmacología , Histona Desacetilasa 6 , Receptores de Glucocorticoides/genéticaRESUMEN
The heterogeneous nature of inflammatory bowel disease (IBD) presents challenges, particularly when choosing therapy. Activation of the NF-κB transcription factor is a highly regulated, dynamic event in IBD pathogenesis. Using a lentivirus approach, NF-κB-regulated luciferase was expressed in patient macrophages, isolated from frozen peripheral blood mononuclear cell samples. Following activation, samples could be segregated into three clusters based on the NF-κB-regulated luciferase response. The ulcerative colitis (UC) samples appeared only in the hypo-responsive Cluster 1, and in Cluster 2. Conversely, Crohn's disease (CD) patients appeared in all Clusters with their percentage being higher in the hyper-responsive Cluster 3. A positive correlation was seen between NF-κB-induced luciferase activity and the concentrations of cytokines released into medium from stimulated macrophages, but not with serum or biopsy cytokine levels. Confocal imaging of lentivirally-expressed p65 activation revealed that a higher proportion of macrophages from CD patients responded to endotoxin lipid A compared to controls. In contrast, cells from UC patients exhibited a shorter duration of NF-κB p65 subunit nuclear localization compared to healthy controls, and CD donors. Analysis of macrophage cytokine responses and patient metadata revealed a strong correlation between CD patients who smoked and hyper-activation of p65. These in vitro dynamic assays of NF-κB activation in blood-derived macrophages have the potential to segregate IBD patients into groups with different phenotypes and may therefore help determine response to therapy.
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Núcleo Celular/inmunología , Colitis Ulcerosa/inmunología , Enfermedad de Crohn/inmunología , Macrófagos/inmunología , Transducción de Señal/inmunología , Factor de Transcripción ReIA/inmunología , Transporte Activo de Núcleo Celular/genética , Transporte Activo de Núcleo Celular/inmunología , Adulto , Animales , Núcleo Celular/genética , Colitis Ulcerosa/genética , Colitis Ulcerosa/patología , Enfermedad de Crohn/genética , Enfermedad de Crohn/patología , Femenino , Humanos , Macrófagos/patología , Masculino , Ratones , Ratones Noqueados , Persona de Mediana Edad , Transducción de Señal/genética , Factor de Transcripción ReIA/genéticaRESUMEN
Specific molecular interactions that underpin the switch between ER stress-triggered autophagy-mediated cellular repair and cellular death by apoptosis are not characterized. This study reports the unexpected interaction elicited by ER stress between the plasma membrane (PM)-localized apoptosis effector PERP and the ER Ca2+ pump SERCA2b. We show that the p53 effector PERP, which specifically induces apoptosis when expressed above a threshold level, has a heterogeneous distribution across the PM of un-stressed cells and is actively turned over by the lysosome. PERP is upregulated following sustained starvation-induced autophagy, which precedes the onset of apoptosis indicating that PERP protein levels are controlled by a lysosomal pathway that is sensitive to cellular physiological state. Furthermore, ER stress stabilizes PERP at the PM and induces its increasing co-localization with SERCA2b at ER-PM junctions. The findings highlight a novel crosstalk between pro-survival autophagy and pro-death apoptosis pathways and identify, for the first time, accumulation of an apoptosis effector to ER-PM junctions in response to ER stress.
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Prolactin is a major hormone product of the pituitary gland, the central endocrine regulator. Despite its physiological importance, the cell-level mechanisms of prolactin production are not well understood. Having significantly improved the resolution of real-time-single-cell-GFP-imaging, the authors recently revealed that prolactin gene transcription is highly dynamic and stochastic yet shows space-time coordination in an intact tissue slice. However, it still remains an open question as to what kind of cellular communication mediates the observed space-time organization. To determine the type of interaction between cells we developed a statistical model. The degree of similarity between two expression time series was studied in terms of two distance measures, Euclidean and geodesic, the latter being a network-theoretic distance defined to be the minimal number of edges between nodes, and this was used to discriminate between juxtacrine from paracrine signalling. The analysis presented here suggests that juxtacrine signalling dominates. To further determine whether the coupling is coordinating transcription or post-transcriptional activities we used stochastic switch modelling to infer the transcriptional profiles of cells and estimated their similarity measures to deduce that their spatial cellular coordination involves coupling of transcription via juxtacrine signalling. We developed a computational model that involves an inter-cell juxtacrine coupling, yielding simulation results that show space-time coordination in the transcription level that is in agreement with the above analysis. The developed model is expected to serve as the prototype for the further study of tissue-level organised gene expression for epigenetically regulated genes, such as prolactin.
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Comunicación Celular/genética , Modelos Biológicos , Comunicación Paracrina/genética , Animales , Comunicación Celular/fisiología , Biología Computacional , Regulación de la Expresión Génica/genética , Humanos , Masculino , Comunicación Paracrina/fisiología , Hipófisis/metabolismo , Prolactina/genética , Prolactina/metabolismo , Ratas , Ratas Transgénicas , Procesos EstocásticosRESUMEN
During embryogenesis cells make fate decisions within complex tissue environments. The levels and dynamics of transcription factor expression regulate these decisions. Here, we use single cell live imaging of an endogenous HES5 reporter and absolute protein quantification to gain a dynamic view of neurogenesis in the embryonic mammalian spinal cord. We report that dividing neural progenitors show both aperiodic and periodic HES5 protein fluctuations. Mathematical modelling suggests that in progenitor cells the HES5 oscillator operates close to its bifurcation boundary where stochastic conversions between dynamics are possible. HES5 expression becomes more frequently periodic as cells transition to differentiation which, coupled with an overall decline in HES5 expression, creates a transient period of oscillations with higher fold expression change. This increases the decoding capacity of HES5 oscillations and correlates with interneuron versus motor neuron cell fate. Thus, HES5 undergoes complex changes in gene expression dynamics as cells differentiate.
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Factores de Transcripción con Motivo Hélice-Asa-Hélice Básico/metabolismo , Células-Madre Neurales/metabolismo , Neurogénesis , Proteínas Represoras/metabolismo , Animales , Factores de Transcripción con Motivo Hélice-Asa-Hélice Básico/química , Factores de Transcripción con Motivo Hélice-Asa-Hélice Básico/genética , Femenino , Regulación del Desarrollo de la Expresión Génica , Masculino , Ratones/embriología , Ratones/metabolismo , Ratones Endogámicos ICR , Ratones Noqueados , Células-Madre Neurales/química , Células-Madre Neurales/citología , Proteínas Represoras/química , Proteínas Represoras/genética , Análisis de la Célula IndividualRESUMEN
Toll-like receptor (TLR) signaling regulates macrophage activation and effector cytokine propagation in the constrained environment of a tissue. In macrophage populations, TLR4 stimulates the dose-dependent transcription of nuclear factor κB (NF-κB) target genes. However, using single-RNA counting, we found that individual cells exhibited a wide range (three orders of magnitude) of expression of the gene encoding the proinflammatory cytokine tumor necrosis factor-α (TNF-α). The TLR4-induced TNFA transcriptional response correlated with the extent of NF-κB signaling in the cells and their size. We compared the rates of TNF-α production and uptake in macrophages and mouse embryonic fibroblasts and generated a mathematical model to explore the heterogeneity in the response of macrophages to TLR4 stimulation and the propagation of the TNF-α signal in the tissue. The model predicts that the local propagation of the TLR4-dependent TNF-α response and cellular NF-κB signaling are limited to small distances of a few cell diameters between neighboring tissue-resident macrophages. In our predictive model, TNF-α propagation was constrained by competitive uptake of TNF-α from the environment, rather than by heterogeneous production of the cytokine. We propose that the highly constrained architecture of tissues enables effective localized propagation of inflammatory cues while avoiding out-of-context responses at longer distances.
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Inflamación/inmunología , Activación de Macrófagos , Macrófagos/metabolismo , Transducción de Señal , Factor de Necrosis Tumoral alfa/metabolismo , Animales , Células Cultivadas , Células HEK293 , Humanos , Inflamación/metabolismo , Macrófagos/inmunología , Ratones , Ratones Transgénicos , FN-kappa B/metabolismo , Subunidad p50 de NF-kappa B/genética , Subunidad p50 de NF-kappa B/metabolismo , Células RAW 264.7 , Análisis de la Célula Individual , Receptor Toll-Like 4/genética , Receptor Toll-Like 4/metabolismo , Factor de Necrosis Tumoral alfa/inmunologíaRESUMEN
The interest in graphene and its translation into commercial products has been expanding at a high pace. Based on previously described pulmonary safety concerns for carbon nanomaterials, there is a great need to define parameters guiding interactions between graphene-based materials and the pulmonary system. The aim of the present study was to determine the importance of two critical parameters: lateral dimensions of the material and coating with proteins in relation to each other and their pulmonary impact. Endotoxin-free materials with distinct lateral dimensions, s-GO (50-200 nm) and l-GO (5-15 µm), were produced and thoroughly characterized. Exploiting intrinsic fluorescence of graphene oxide (GO) and using confocal live-cell imaging, the behavior of the cells in response to the material was visualized in real time. Although BEAS-2B cells internalized GO efficiently, l-GO was linked to higher plasma membrane interactions correlated with elevated reactive oxygen species (ROS) levels, pro-inflammatory response, and greater cytotoxicity, in agreement with the oxidative stress paradigm. For both GO types, the presence of serum alleviated lipid peroxidation of plasma membrane and decreased intracellular ROS levels. However, protein coating was not enough to entirely mitigate toxicity and inflammatory response induced by l-GO. In vitro results were validated in vivo, as l-GO was more prone to induce pulmonary granulomatous response in mice compared to s-GO. In conclusion, the lateral dimension of GO played a more important role than serum protein coating in determining biological responses to the material. It was also demonstrated that time-lapse imaging of live cells interacting with label-free GO sheets can be used as a tool to assess GO-induced cytotoxicity.
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Grafito/química , Animales , Células Cultivadas , Grafito/farmacología , Humanos , Ratones , Ratones Endogámicos C57BL , Estrés Oxidativo/efectos de los fármacos , Especies Reactivas de Oxígeno/análisis , Especies Reactivas de Oxígeno/metabolismoRESUMEN
Transcription in eukaryotic cells occurs in gene-specific bursts or pulses of activity. Recent studies identified a spectrum of transcriptionally active "on-states," interspersed with periods of inactivity, but these "off-states" and the process of transcriptional deactivation are poorly understood. To examine what occurs during deactivation, we investigate the dynamics of switching between variable rates. We measured live single-cell expression of luciferase reporters from human growth hormone or human prolactin promoters in a pituitary cell line. Subsequently, we applied a statistical variable-rate model of transcription, validated by single-molecule FISH, to estimate switching between transcriptional rates. Under the assumption that transcription can switch to any rate at any time, we found that transcriptional activation occurs predominantly as a single switch, whereas deactivation occurs with graded, stepwise decreases in transcription rate. Experimentally altering cAMP signalling with forskolin or chromatin remodelling with histone deacetylase inhibitor modifies the duration of defined transcriptional states. Our findings reveal transcriptional activation and deactivation as mechanistically independent, asymmetrical processes.
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Hormona de Crecimiento Humana/genética , Modelos Teóricos , Hipófisis/fisiología , Prolactina/genética , Transcripción Genética , Animales , Línea Celular , AMP Cíclico/metabolismo , Femenino , Genes Reporteros/genética , Histona Desacetilasas/metabolismo , Humanos , Luciferasas/genética , Regiones Promotoras Genéticas/genética , Ratas , Análisis de la Célula Individual , Activación TranscripcionalRESUMEN
The mammalian NUDT13 protein possesses a sequence motif characteristic of the NADH pyrophosphohydrolase subfamily of Nudix hydrolases. Due to the persistent insolubility of the recombinant product expressed in Escherichia coli, active mouse Nudt13 was expressed in insect cells from a baculovirus vector as a histidine-tagged recombinant protein. In vitro, it efficiently hydrolysed NADH to NMNH and AMP and NADPH to NMNH and 2',5'-ADP and had a marked preference for the reduced pyridine nucleotides. Much lower activity was obtained with other nucleotide substrates tested. K m and k cat values for NADH were 0.34 mM and 7 s-1 respectively. Expression of Nudt13 as an N-terminal fusion to green fluorescent protein revealed that it was targeted exclusively to mitochondria by the N-terminal targeting peptide, suggesting that Nudt13 may act to regulate the concentration of mitochondrial reduced pyridine nucleotide cofactors and the NAD(P)+/NAD(P)H ratio in this organelle and elsewhere. Future studies of the enzymology of pyridine nucleotide metabolism in relation to energy homeostasis, redox control, free radical production and cellular integrity should consider the possible regulatory role of Nudt13.