RESUMEN
Abnormal processing of stressed replication forks by nucleases can cause fork collapse, genomic instability, and cell death. Despite its importance, it is poorly understood how the cell properly controls nucleases to prevent detrimental fork processing. Here, we report a signaling pathway that controls the activity of exonuclease Exo1 to prevent aberrant fork resection during replication stress. Our results indicate that replication stress elevates intracellular Ca2+ concentration ([Ca2+]i), leading to activation of CaMKK2 and the downstream kinase 5' AMP-activated protein kinase (AMPK). Following activation, AMPK directly phosphorylates Exo1 at serine 746 to promote 14-3-3 binding and inhibit Exo1 recruitment to stressed replication forks, thereby avoiding unscheduled fork resection. Disruption of this signaling pathway results in excessive ssDNA, chromosomal instability, and hypersensitivity to replication stress inducers. These findings reveal a link between [Ca2+]i and the replication stress response as well as a function of the Ca2+-CaMKK2-AMPK signaling axis in safeguarding fork structure to maintain genome stability.
Asunto(s)
Proteínas Quinasas Activadas por AMP/genética , Quinasa de la Proteína Quinasa Dependiente de Calcio-Calmodulina/genética , Calcio/metabolismo , Enzimas Reparadoras del ADN/genética , Reparación del ADN , Replicación del ADN , Exodesoxirribonucleasas/genética , Proteínas 14-3-3/genética , Proteínas 14-3-3/metabolismo , Proteínas Quinasas Activadas por AMP/metabolismo , Animales , Señalización del Calcio/genética , Quinasa de la Proteína Quinasa Dependiente de Calcio-Calmodulina/metabolismo , Línea Celular Tumoral , Quinasa 1 Reguladora del Ciclo Celular (Checkpoint 1)/genética , Quinasa 1 Reguladora del Ciclo Celular (Checkpoint 1)/metabolismo , Cromatina/química , Cromatina/metabolismo , Daño del ADN , Enzimas Reparadoras del ADN/metabolismo , ADN de Cadena Simple/genética , ADN de Cadena Simple/metabolismo , Exodesoxirribonucleasas/metabolismo , Fibroblastos/citología , Fibroblastos/metabolismo , Células HEK293 , Células HeLa , Humanos , Isoenzimas/genética , Isoenzimas/metabolismo , Ratones , Osteoblastos/citología , Osteoblastos/metabolismo , Fosforilación , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismoRESUMEN
The process in which locally confined epithelial malignancies progressively evolve into invasive cancers is often promoted by unjamming, a phase transition from a solid-like to a liquid-like state, which occurs in various tissues. Whether this tissue-level mechanical transition impacts phenotypes during carcinoma progression remains unclear. Here we report that the large fluctuations in cell density that accompany unjamming result in repeated mechanical deformations of cells and nuclei. This triggers a cellular mechano-protective mechanism involving an increase in nuclear size and rigidity, heterochromatin redistribution and remodelling of the perinuclear actin architecture into actin rings. The chronic strains and stresses associated with unjamming together with the reduction of Lamin B1 levels eventually result in DNA damage and nuclear envelope ruptures, with the release of cytosolic DNA that activates a cGAS-STING (cyclic GMP-AMP synthase-signalling adaptor stimulator of interferon genes)-dependent cytosolic DNA response gene program. This mechanically driven transcriptional rewiring ultimately alters the cell state, with the emergence of malignant traits, including epithelial-to-mesenchymal plasticity phenotypes and chemoresistance in invasive breast carcinoma.
Asunto(s)
Actinas , Neoplasias , ADN , Nucleotidiltransferasas/genética , Nucleotidiltransferasas/metabolismo , Citosol/metabolismo , Transducción de SeñalRESUMEN
Wolfram Syndrome 1 (WFS1) protein is an endoplasmic reticulum (ER) factor whose deficiency results in juvenile-onset diabetes secondary to cellular dysfunction and apoptosis. The mechanisms guiding ß-cell outcomes secondary to WFS1 function, however, remain unclear. Here, we show that WFS1 preserves normal ß-cell physiology by promoting insulin biosynthesis and negatively regulating ER stress. Depletion of Wfs1 in vivo and in vitro causes functional defects in glucose-stimulated insulin secretion and insulin content, triggering Chop-mediated apoptotic pathways. Genetic proof of concept studies coupled with RNA-seq reveal that increasing WFS1 confers a functional and a survival advantage to ß-cells under ER stress by increasing insulin gene expression and downregulating the Chop-Trib3 axis, thereby activating Akt pathways. Remarkably, WFS1 and INS levels are reduced in type-2 diabetic (T2DM) islets, suggesting that WFS1 may contribute to T2DM ß-cell pathology. Taken together, this work reveals essential pathways regulated by WFS1 to control ß-cell survival and function primarily through preservation of ER homeostasis.
Asunto(s)
Células Secretoras de Insulina , Proteínas de la Membrana , Animales , Glucemia/análisis , Glucemia/metabolismo , Línea Celular , Células Cultivadas , Estrés del Retículo Endoplásmico/fisiología , Humanos , Insulina/análisis , Insulina/metabolismo , Células Secretoras de Insulina/citología , Células Secretoras de Insulina/metabolismo , Proteínas de la Membrana/genética , Proteínas de la Membrana/metabolismo , Proteínas de la Membrana/fisiología , Ratones Noqueados , Transducción de Señal/fisiología , Síndrome de WolframRESUMEN
The advent of fluorescent proteins (FPs) for genetic labeling of molecules and cells has revolutionized fluorescence microscopy. Genetic manipulations have created a vast array of bright and stable FPs spanning blue to red spectral regions. Common to autofluorescent FPs is their tight ß-barrel structure, which provides the rigidity and chemical environment needed for effectual fluorescence. Despite the common structure, each FP has unique properties. Thus, there is no single 'best' FP for every circumstance, and each FP has advantages and disadvantages. To guide decisions about which FP is right for a given application, we have quantitatively characterized the brightness, photostability, pH stability and monomeric properties of more than 40 FPs to enable straightforward and direct comparison between them. We focus on popular and/or top-performing FPs in each spectral region.
Asunto(s)
Proteínas Luminiscentes/análisis , Microscopía Fluorescente/métodos , Proteínas Recombinantes de Fusión/análisis , Espectrometría de Fluorescencia/métodos , Fluorescencia , Células HeLa , HumanosRESUMEN
The islet of Langerhans plays a key role in glucose homeostasis through regulated secretion of the hormones insulin and glucagon. Islet research has focused on the insulin-secreting ß-cells, even though aberrant glucagon secretion from α-cells also contributes to the aetiology of diabetes. Despite its importance, the mechanisms controlling glucagon secretion remain controversial. Proper α-cell function requires the islet milieu, where ß- and δ-cells drive and constrain α-cell dynamics. The response of glucagon to glucose is similar between isolated islets and that measured in vivo, so it appears that the glucose dependence requires only islet-intrinsic factors and not input from blood flow or the nervous system. Elevated intracellular free Ca2+ is needed for α-cell exocytosis, but interpreting Ca2+ data is tricky since it is heterogeneous among α-cells at all physiological glucose levels. Total Ca2+ activity in α-cells increases slightly with glucose, so Ca2+ may serve a permissive, rather than regulatory, role in glucagon secretion. On the other hand, cAMP is a more promising candidate for controlling glucagon secretion and is itself driven by paracrine signalling from ß- and δ-cells. Another pathway, juxtacrine signalling through the α-cell EphA receptors, stimulated by ß-cell ephrin ligands, leads to a tonic inhibition of glucagon secretion. We discuss potential combinations of Ca2+ , cAMP, paracrine and juxtacrine factors in the regulation of glucagon secretion, focusing on recent data in the literature that might unify the field towards a quantitative understanding of α-cell function.
Asunto(s)
Calcio/metabolismo , Células Secretoras de Glucagón/metabolismo , Glucagón/metabolismo , Glucemia/metabolismo , Glucemia/fisiología , Comunicación Celular/fisiología , AMP Cíclico/fisiología , Glucagón/antagonistas & inhibidores , Humanos , Transducción de Señal/fisiologíaRESUMEN
The observation of ionic signaling dynamics in intact pancreatic islets has contributed greatly to our understanding of both α- and ß-cell function. Insulin secretion from ß-cells depends on the firing of action potentials and consequent rises of intracellular calcium activity ([Ca(2+)]i). Zinc (Zn(2+)) is cosecreted with insulin, and has been postulated to play a role in cell-to-cell cross talk within an islet, in particular inhibiting glucagon secretion from α-cells. Thus, measuring [Ca(2+)]i and Zn(2+) dynamics from both α- and ß-cells will elucidate mechanisms underlying islet hormone secretion. [Ca(2+)]i and intracellular Zn(2+) can be measured using fluorescent biosensors, but the most efficient sensors have overlapping spectra that complicate their discrimination. Hyperspectral imaging can be used to distinguish signals from multiple fluorophores, but available hyperspectral implementations are either too slow to measure the dynamics of ionic signals or not suitable for thick samples. We have developed a five-dimensional (x,y,z,t,λ) imaging system that leverages a snapshot hyperspectral imaging method, image mapping spectrometry, and light-sheet microscopy. This system provides subsecond temporal resolution from deep within multicellular structures. Using a single excitation wavelength (488 nm) we acquired images from triply labeled samples with two biosensors and a genetically expressing fluorescent protein (spectrally overlapping with one of the biosensors) with high temporal resolution. Measurements of [Ca(2+)]i and Zn(2+) within both α- and ß-cells as a function of glucose concentration show heterogeneous uptake of Zn(2+) into α-cells that correlates to the known heterogeneities in [Ca(2+)]i. These differences in intracellular Zn(2+) among α-cells may contribute to the inhibition in glucagon secretion observed at elevated glucose levels.
Asunto(s)
Islotes Pancreáticos/citología , Imagen Molecular , Transducción de Señal , Animales , Calcio/metabolismo , Supervivencia Celular , Espacio Intracelular/metabolismo , Islotes Pancreáticos/metabolismo , Ratones , Zinc/metabolismoRESUMEN
We demonstrate three-dimensional (3D) super-resolution live-cell imaging through thick specimens (50-150 µm), by coupling far-field individual molecule localization with selective plane illumination microscopy (SPIM). The improved signal-to-noise ratio of selective plane illumination allows nanometric localization of single molecules in thick scattering specimens without activating or exciting molecules outside the focal plane. We report 3D super-resolution imaging of cellular spheroids.
Asunto(s)
Imagenología Tridimensional/métodos , Microscopía Fluorescente/métodos , Esferoides Celulares/citología , Línea Celular , Supervivencia Celular , Humanos , Nanocápsulas , Fantasmas de ImagenRESUMEN
The cell cortex is a dynamic assembly formed by the plasma membrane and underlying cytoskeleton. As the main determinant of cell shape, the cortex ensures its integrity during passive and active deformations by adapting cytoskeleton topologies through yet poorly understood mechanisms. The spectrin meshwork ensures such adaptation in erythrocytes and neurons by adopting different organizations. Erythrocytes rely on triangular-like lattices of spectrin tetramers, whereas in neurons they are organized in parallel, periodic arrays. Since spectrin is ubiquitously expressed, we exploited Expansion Microscopy to discover that, in fibroblasts, distinct meshwork densities co-exist. Through biophysical measurements and computational modeling, we show that the non-polarized spectrin meshwork, with the intervention of actomyosin, can dynamically transition into polarized clusters fenced by actin stress fibers that resemble periodic arrays as found in neurons. Clusters experience lower mechanical stress and turnover, despite displaying an extension close to the tetramer contour length. Our study sheds light on the adaptive properties of spectrin, which participates in the protection of the cell cortex by varying its densities in response to key mechanical features.
Asunto(s)
Espectrina , Espectrina/metabolismo , Animales , Fibroblastos/metabolismo , Actomiosina/metabolismo , Ratones , Citoesqueleto/metabolismo , Estrés Mecánico , Membrana Celular/metabolismo , Forma de la Célula , Actinas/metabolismo , Fibras de Estrés/metabolismo , HumanosRESUMEN
Micronuclei (MN) can form through many mechanisms, including the breakage of aberrant cytokinetic chromatin bridges. The frequent observation of MN in tumors suggests that they might not merely be passive elements but could instead play active roles in tumor progression. Here, we propose a mechanism through which the presence of micronuclei could induce specific phenotypic and functional changes in cells and increase the invasive potential of cancer cells. Through the integration of diverse in vitro imaging and molecular techniques supported by clinical samples from patients with prostate cancer (PCa) defined as high-risk by the D'Amico classification, we demonstrate that the resolution of chromosome bridges can result in the accumulation of Emerin and the formation of Emerin-rich MN. These structures are negative for Lamin A/C and positive for the Lamin-B receptor and Sec61ß. MN can act as a protein sinks and result in the pauperization of Emerin from the nuclear envelope. The Emerin mislocalization phenotype is associated with a molecular signature that is correlated with a poor prognosis in PCa patients and is enriched in metastatic samples. Emerin mislocalization corresponds with increases in the migratory and invasive potential of tumor cells, especially in a collagen-rich microenvironment. Our study demonstrates that the mislocalization of Emerin to MN results in increased cell invasiveness, thereby worsening patient prognosis.
RESUMEN
In this work we report the advantages provided by two photon excitation (2PE) implemented in a selective plane illumination microscopy (SPIM) when imaging thick scattering samples. In particular, a detailed analysis of the effects induced on the real light sheet excitation intensity distribution is performed. The comparison between single-photon and two-photon excitation profiles shows the reduction of the scattering effects and sample-induced aberrations provided by 2PE-SPIM. Furthermore, uniformity of the excitation distribution and the consequent improved image contrast is shown when imaging scattering phantom samples in depth by 2PE-SPIM. These results show the advantages of 2PE-SPIM and suggest how this combination can further enhance the SPIM performance. Phantom samples have been designed with optical properties compatible with biological applications of interest.
Asunto(s)
Microscopía de Fluorescencia por Excitación Multifotónica/métodos , Dispersión de Radiación , Células Epiteliales/citología , Humanos , Fantasmas de ImagenRESUMEN
Nuclear factors rapidly scan the genome for their targets, but the role of nuclear organization in such search is uncharted. Here we analyzed how multiple factors explore chromatin, combining live-cell single-molecule tracking with multifocal structured illumination of DNA density. We find that factors displaying higher bound fractions sample DNA-dense regions more exhaustively. Focusing on the tumor-suppressor p53, we demonstrate that it searches for targets by alternating between rapid diffusion in the interchromatin compartment and compact sampling of chromatin dense regions. Efficient targeting requires balanced interactions with chromatin: fusing p53 with an exogenous intrinsically disordered region potentiates p53-mediated target gene activation at low concentrations, but leads to condensates at higher levels, derailing its search and downregulating transcription. Our findings highlight the role of disordered regions on factors search and showcase a powerful method to generate traffic maps of the eukaryotic nucleus to dissect how its organization guides nuclear factors action.
Asunto(s)
Cromatina , Proteína p53 Supresora de Tumor , Proteína p53 Supresora de Tumor/genética , Proteína p53 Supresora de Tumor/metabolismo , Cromatina/genética , Cromatina/metabolismo , ADN/metabolismo , Cromosomas/metabolismo , Activación Transcripcional , Núcleo Celular/genética , Núcleo Celular/metabolismoRESUMEN
Alternative lengthening of telomeres (ALT) is a telomere maintenance mechanism activated in ~10-15% of cancers, characterized by telomeric damage. Telomeric damage-induced long non-coding RNAs (dilncRNAs) are transcribed at dysfunctional telomeres and contribute to telomeric DNA damage response (DDR) activation and repair. Here we observed that telomeric dilncRNAs are preferentially elevated in ALT cells. Inhibition of C-rich (teloC) dilncRNAs with antisense oligonucleotides leads to DNA replication stress responses, increased genomic instability, and apoptosis induction selectively in ALT cells. Cell death is dependent on DNA replication and is increased by DNA replication stress. Mechanistically, teloC dilncRNA inhibition reduces RAD51 and 53BP1 recruitment to telomeres, boosts the engagement of BIR machinery, and increases C-circles and telomeric sister chromatid exchanges, without increasing telomeric non-S phase synthesis. These results indicate that teloC dilncRNA is necessary for a coordinated recruitment of DDR factors to ALT telomeres and it is essential for ALT cancer cells survival.
Asunto(s)
Telomerasa , Homeostasis del Telómero , Homeostasis del Telómero/genética , Replicación del ADN , ARN , Supervivencia Celular/genética , Telómero/genética , Telómero/metabolismo , Telomerasa/genética , Telomerasa/metabolismoRESUMEN
Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is the RNA virus responsible for the coronavirus disease 2019 (COVID-19) pandemic. Although SARS-CoV-2 was reported to alter several cellular pathways, its impact on DNA integrity and the mechanisms involved remain unknown. Here we show that SARS-CoV-2 causes DNA damage and elicits an altered DNA damage response. Mechanistically, SARS-CoV-2 proteins ORF6 and NSP13 cause degradation of the DNA damage response kinase CHK1 through proteasome and autophagy, respectively. CHK1 loss leads to deoxynucleoside triphosphate (dNTP) shortage, causing impaired S-phase progression, DNA damage, pro-inflammatory pathways activation and cellular senescence. Supplementation of deoxynucleosides reduces that. Furthermore, SARS-CoV-2 N-protein impairs 53BP1 focal recruitment by interfering with damage-induced long non-coding RNAs, thus reducing DNA repair. Key observations are recapitulated in SARS-CoV-2-infected mice and patients with COVID-19. We propose that SARS-CoV-2, by boosting ribonucleoside triphosphate levels to promote its replication at the expense of dNTPs and by hijacking damage-induced long non-coding RNAs' biology, threatens genome integrity and causes altered DNA damage response activation, induction of inflammation and cellular senescence.
Asunto(s)
COVID-19 , Animales , Ratones , SARS-CoV-2 , Senescencia Celular , Daño del ADNRESUMEN
Translation initiation is a major rate-limiting step for protein synthesis. However, recent studies strongly suggest that the efficiency of protein synthesis is additionally regulated by multiple factors that impact the elongation phase. To assess the influence of early elongation on protein synthesis, we employed a library of more than 250,000 reporters combined with in vitro and in vivo protein expression assays. Here we report that the identity of the amino acids encoded by codons 3 to 5 impact protein yield. This effect is independent of tRNA abundance, translation initiation efficiency, or overall mRNA structure. Single-molecule measurements of translation kinetics revealed pausing of the ribosome and aborted protein synthesis on codons 4 and 5 of distinct amino acid and nucleotide compositions. Finally, introduction of preferred sequence motifs only at specific codon positions improves protein synthesis efficiency for recombinant proteins. Collectively, our data underscore the critical role of early elongation events in translational control of gene expression.
Asunto(s)
Codón/genética , Extensión de la Cadena Peptídica de Translación/genética , Ribosomas/metabolismo , Aminoácidos/genética , Subunidades alfa de la Proteína de Unión al GTP Gi-Go/genética , Subunidades alfa de la Proteína de Unión al GTP Gi-Go/metabolismo , Biblioteca de Genes , Genes Reporteros/genética , Proteínas Luminiscentes/genética , Proteínas Luminiscentes/metabolismo , Nucleótidos/metabolismo , Iniciación de la Cadena Peptídica Traduccional , Proteínas RGS/genética , Proteínas RGS/metabolismo , ARN de Transferencia/metabolismo , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Imagen Individual de MoléculaRESUMEN
The intracellular life of insulin secretory granules (ISGs) from biogenesis to secretion depends on their structural (e.g. size) and dynamic (e.g. diffusivity, mode of motion) properties. Thus, it would be useful to have rapid and robust measurements of such parameters in living ß-cells. To provide such measurements, we have developed a fast spatiotemporal fluctuation spectroscopy. We calculate an imaging-derived Mean Squared Displacement (iMSD), which simultaneously provides the size, average diffusivity, and anomalous coefficient of ISGs, without the need to extract individual trajectories. Clustering of structural and dynamic quantities in a multidimensional parametric space defines the ISGs' properties for different conditions. First, we create a reference using INS-1E cells expressing proinsulin fused to a fluorescent protein (FP) under basal culture conditions and validate our analysis by testing well-established stimuli, such as glucose intake, cytoskeleton disruption, or cholesterol overload. After, we investigate the effect of FP-tagged ISG protein markers on the structural and dynamic properties of the granule. While iMSD analysis produces similar results for most of the lumenal markers, the transmembrane marker phogrin-FP shows a clearly altered result. Phogrin overexpression induces a substantial granule enlargement and higher mobility, together with a partial de-polymerization of the actin cytoskeleton, and reduced cell responsiveness to glucose stimulation. Our data suggest a more careful interpretation of many previous ISG-based reports in living ß-cells. The presented data pave the way to high-throughput cell-based screening of ISG structure and dynamics under various physiological and pathological conditions.
Asunto(s)
Glucosa/farmacología , Proteínas Fluorescentes Verdes/metabolismo , Secreción de Insulina/fisiología , Células Secretoras de Insulina/fisiología , Proteínas Tirosina Fosfatasas Clase 8 Similares a Receptores/metabolismo , Vesículas Secretoras/fisiología , Animales , Secreción de Insulina/efectos de los fármacos , Células Secretoras de Insulina/citología , Células Secretoras de Insulina/efectos de los fármacos , Ratas , Proteínas Tirosina Fosfatasas Clase 8 Similares a Receptores/genética , Vesículas Secretoras/efectos de los fármacos , Edulcorantes/farmacologíaRESUMEN
Pro-inflammatory cytokines are important mediators of islet inflammation, leading to beta cell death in type 1 diabetes. Although alterations in both endoplasmic reticulum (ER) and cytosolic free calcium levels are known to play a role in cytokine-mediated beta cell death, there are currently no treatments targeting cellular calcium homeostasis to combat type 1 diabetes. Here we show that modulation of cellular calcium homeostasis can mitigate cytokine- and ER stress-mediated beta cell death. The calcium modulating compounds, dantrolene and sitagliptin, both prevent cytokine and ER stress-induced activation of the pro-apoptotic calcium-dependent enzyme, calpain, and partly suppress beta cell death in INS1E cells and human primary islets. These agents are also able to restore cytokine-mediated suppression of functional ER calcium release. In addition, sitagliptin preserves function of the ER calcium pump, sarco-endoplasmic reticulum Ca2+-ATPase (SERCA), and decreases levels of the pro-apoptotic protein thioredoxin-interacting protein (TXNIP). Supporting the role of TXNIP in cytokine-mediated cell death, knock down of TXNIP in INS1-E cells prevents cytokine-mediated beta cell death. Our findings demonstrate that modulation of dynamic cellular calcium homeostasis and TXNIP suppression present viable pharmacologic targets to prevent cytokine-mediated beta cell loss in diabetes.
Asunto(s)
Calcio/metabolismo , Muerte Celular , Citocinas/farmacología , Inflamación/prevención & control , Células Secretoras de Insulina/efectos de los fármacos , Animales , Células Cultivadas , Retículo Endoplásmico/efectos de los fármacos , Retículo Endoplásmico/metabolismo , Retículo Endoplásmico/patología , Estrés del Retículo Endoplásmico/efectos de los fármacos , Homeostasis , Inflamación/metabolismo , Inflamación/patología , Células Secretoras de Insulina/metabolismo , Células Secretoras de Insulina/patología , Ratas , ATPasas Transportadoras de Calcio del Retículo Sarcoplásmico/metabolismoRESUMEN
In the last decade light sheet fluorescence microscopy techniques, such as selective plane illumination microscopy (SPIM), has become a well established method for developmental biology. However, conventional SPIM architectures hardly permit imaging of certain tissues since the common sample mounting procedure, based on gel embedding, could interfere with the sample morphology. In this work we propose an inverted selective plane microscopy system (iSPIM), based on non-linear excitation, suitable for 3D tissue imaging. First, the iSPIM architecture provides flexibility on the sample mounting, getting rid of the gel-based mounting typical of conventional SPIM, permitting 3D imaging of hippocampal slices from mouse brain. Moreover, all the advantages brought by two photon excitation (2PE) in terms of reduction of scattering effects and contrast improvement are exploited, demonstrating an improved image quality and contrast compared to single photon excitation. The system proposed represents an optimal platform for tissue imaging and it smooths the way to the applicability of light sheet microscopy to a wider range of samples including those that have to be mounted on non-transparent surfaces.
RESUMEN
Small nucleolar RNAs (snoRNAs) are non-coding RNAs that form ribonucleoproteins to guide covalent modifications of ribosomal and small nuclear RNAs in the nucleus. Recent studies have also uncovered additional non-canonical roles for snoRNAs. However, the physiological contributions of these small RNAs are largely unknown. Here, we selectively deleted four snoRNAs encoded within the introns of the ribosomal protein L13a (Rpl13a) locus in a mouse model. Loss of Rpl13a snoRNAs altered mitochondrial metabolism and lowered reactive oxygen species tone, leading to increased glucose-stimulated insulin secretion from pancreatic islets and enhanced systemic glucose tolerance. Islets from mice lacking Rpl13a snoRNAs demonstrated blunted oxidative stress responses. Furthermore, these mice were protected against diabetogenic stimuli that cause oxidative stress damage to islets. Our study illuminates a previously unrecognized role for snoRNAs in metabolic regulation.
Asunto(s)
Diabetes Mellitus Experimental/metabolismo , Glucosa/metabolismo , Intrones , Islotes Pancreáticos/metabolismo , Mitocondrias/metabolismo , Estrés Oxidativo , ARN Nuclear Pequeño/metabolismo , Proteínas Ribosómicas , Animales , Diabetes Mellitus Experimental/genética , Diabetes Mellitus Experimental/patología , Glucosa/genética , Islotes Pancreáticos/patología , Ratones , Ratones Noqueados , Mitocondrias/genética , Mitocondrias/patología , ARN Nuclear Pequeño/genética , Especies Reactivas de Oxígeno/metabolismoRESUMEN
Light-sheet microscopy is a useful tool for performing biological investigations of thick samples and it has recently been demonstrated that it can also act as a suitable architecture for super-resolution imaging of thick biological samples by means of individual molecule localization. However, imaging in depth is still limited since it suffers from a reduction in image quality caused by scattering effects. This paper sets out to investigate the advantages of non-linear photoactivation implemented in a selective plane illumination configuration when imaging scattering samples. In particular, two-photon excitation is proven to improve imaging capabilities in terms of imaging depth and is expected to reduce light-sample interactions and sample photo-damage. Here, two-photon photoactivation is coupled to individual molecule localization methods based on light-sheet illumination (IML-SPIM), allowing super-resolution imaging of nuclear pH2AX in NB4 cells.