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Cell death is a phenomenon, frequently perceived as an absolute event for cell, tissue and the organ. However, the rising popularity and complexity of such 3D multicellular 'tissue building blocks' as heterocellular spheroids, organoids, and 'assembloids' prompts to revise the definition and quantification of cell viability and death. It raises several questions on the overall viability of all the cells within 3D volume and on choosing the appropriate, continuous, and non-destructive viability assay enabling for a single-cell analysis. In this review, we look at cell viability and cell death modalities with attention to the intrinsic features of such 3D models as spheroids, organoids, and bioprints. Furthermore, we look at emerging and promising methodologies, which can help define and understand the balance between cell viability and death in dynamic and complex 3D environments. We conclude that the recent innovations in biofabrication, biosensor probe development, and fluorescence microscopy can help answer these questions.
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Organoides , Esferoides Celulares , Sobrevivência Celular , Morte CelularRESUMO
Monitoring of cell metabolism represents an important application area for fluorescence lifetime imaging microscopy (FLIM). In particular, assessment of mitochondrial membrane potential (MMP) in complex three-dimensional multicellular in vitro, ex vivo, and in vivo models would enable improved segmentation and functional discrimination of cell types, directly report on the mitochondrial function and complement the quenched-phosphorescence detection of cellular O2 and two-photon excited FLIM of endogenous NAD(P)H. Here, we report the green and orange-emitting fluorescent dyes SYTO and tetramethylrhodamine methyl ester (TMRM) as potential FLIM probes for MMP. In addition to nuclear, SYTO 16 and 24 dyes also display mitochondrial accumulation. FLIM with the culture of human colon cancer HCT116 cells allowed observation of the heterogeneity of mitochondrial polarization during the cell cycle progression. The dyes also demonstrated good performance with 3D cultures of Lgr5-GFP mouse intestinal organoids, providing efficient and quick cell staining and compatibility with two-photon excitation. Multiplexed imaging of Lgr5-GFP, proliferating cells (Hoechst 33342-aided FLIM), and TMRM-FLIM allowed us to identify the population of metabolically active cells in stem cell niche. TMRM-FLIM enabled to visualize the differences in membrane potential between Lgr5-positive and other proliferating and differentiated cell types. Altogether, SYTO 24 and TMRM dyes represent promising markers for advanced FLIM-based studies of cell bioenergetics with complex 3D and in vivo models. © 2019 International Society for Advancement of Cytometry.
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Corantes Fluorescentes , Organoides , Animais , Corantes Fluorescentes/metabolismo , Humanos , Potencial da Membrana Mitocondrial , Camundongos , Microscopia de Fluorescência , Nicho de Células-TroncoRESUMO
The imaging of real-time fluxes of K+ ions in live cell with high dynamic range (5-150 mM) is of paramount importance for neuroscience and physiology of the gastrointestinal tract, kidney and other tissues. In particular, the research on high-performance deep-red fluorescent nanoparticle-based biosensors is highly anticipated. We found that BODIPY-based FI3 K+-sensitive fluoroionophore encapsulated in cationic polymer RL100 nanoparticles displays unusually strong efficiency in staining of broad spectrum of cell models, such as primary neurons and intestinal organoids. Using comparison of brightness, photostability and fluorescence lifetime imaging microscopy (FLIM) we confirmed that FI3 nanoparticles display distinctively superior intracellular staining compared to the free dye. We evaluated FI3 nanoparticles in real-time live cell imaging and found that it is highly useful for monitoring intra- and extracellular K+ dynamics in cultured neurons. Proof-of-concept in vivo brain imaging confirmed applicability of the biosensor for visualization of epileptic seizures. Collectively, this data makes fluoroionophore FI3 a versatile cross-platform fluorescent biosensor, broadly compatible with diverse experimental models and that crown ether-based polymer nanoparticles can provide a new venue for design of efficient fluorescent probes.
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Colonic inflammation is associated with decreased tissue oxygenation, significantly affecting gut homeostasis. However, the crosstalk between O2 consumption and supply in the inflamed tissue are not fully understood. Using a murine model of colitis, we analysed O2 in freshly prepared samples of healthy and inflamed colon tissue. We developed protocols for efficient ex vivo staining of mouse distal colon mucosa with a cell-penetrating O2 sensitive probe Pt-Glc and high-resolution imaging of O2 concentration in live tissue by confocal phosphorescence lifetime-imaging microscopy (PLIM). Microscopy analysis revealed that Pt-Glc stained mostly the top 50-60 µm layer of the mucosa, with high phosphorescence intensity in epithelial cells. Measured O2 values in normal mouse tissue ranged between 5 and 35 µM (4-28 Torr), tending to decrease in the deeper tissue areas. Four-day treatment with dextran sulphate sodium (DSS) triggered colon inflammation, as evidenced by an increase in local IL6 and mKC mRNA levels, but did not affect the gross architecture of colonic epithelium. We further observed an increase in oxygenation, partial activation of hypoxia inducible factor (HIF) 1 signalling, and negative trends in pyruvate dehydrogenase activity and O2 consumption rate in the colitis mucosa, suggesting a decrease in mitochondrial respiration, which is known to be regulated via HIF-1 signalling and pyruvate oxidation rate. These results along with efficient staining with Pt-Glc of rat and human colonic mucosa reveal high potential of PLIM platform as a powerful tool for the high-resolution analysis of the intestinal tissue oxygenation in patients with inflammatory bowel disease and other pathologies, affecting tissue respiration.
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Colite/patologia , Colo/patologia , Mucosa Intestinal/patologia , Oxigênio/análise , Animais , Células CACO-2 , Colite/imunologia , Colo/imunologia , Humanos , Mucosa Intestinal/imunologia , Medições Luminescentes , Masculino , Camundongos Endogâmicos C57BL , Microscopia Confocal , Imagem Óptica , Oxigênio/imunologia , Ratos Sprague-Dawley , Coloração e RotulagemRESUMO
Dynamics of oxygenation of tissue and stem cell niches are important for understanding physiological function of the intestine in normal and diseased states. Only a few techniques allow live visualization of tissue hypoxia at cellular level and in three dimensions. We describe an optimized protocol, which uses cell-penetrating O2-sensitive probe, Pt-Glc and phosphorescence lifetime imaging microscopy (PLIM), to analyze O2 distribution in mouse intestinal organoids. Unlike the other indirect and end-point hypoxia stains, or point measurements with microelectrodes, this method provides high-resolution real-time visualization of O2 in organoids. Multiplexing with conventional fluorescent live cell imaging probes such as the Hoechst 33342-based FLIM assay of cell proliferation, and immunofluorescence staining of endogenous proteins, allows analysis of key physiologic parameters under O2 control in organoids. The protocol is useful for gastroenterology and physiology of intestinal tissue, hypoxia research, regenerative medicine, studying host-microbiota interactions and bioenergetics.
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Células Epiteliais/ultraestrutura , Imunofluorescência/métodos , Hipóxia/metabolismo , Imagem Óptica/métodos , Organoides/ultraestrutura , Oxigênio/análise , Animais , Ciclo Celular/genética , Proliferação de Células , Respiração Celular , Células Epiteliais/metabolismo , Imunofluorescência/instrumentação , Mucosa Intestinal/metabolismo , Intestinos/citologia , Substâncias Luminescentes/química , Camundongos , Imagem Óptica/instrumentação , Organoides/metabolismo , Oxigênio/metabolismo , Platina/química , Porfirinas/química , Coloração e Rotulagem/métodosRESUMO
Abnormal accumulation of oncometabolite fumarate and succinate is associated with inhibition of mitochondrial function and carcinogenesis. By competing with α-ketoglutarate, oncometabolites also activate hypoxia inducible factors (HIFs), which makes oncometabolite mimetics broadly utilised in hypoxia research. We found that dimethyloxalylglycine (DMOG), a synthetic analogue of α-ketoglutarate, commonly used to induce HIF signalling, inhibits O2 consumption in cancer cell lines HCT116 and PC12, well before activation of HIF pathways. A rapid suppression of cellular respiration was accompanied by a decrease in histone H4 lysine 16 acetylation and not abolished by double knockdown of HIF-1α and HIF-2α. In agreement with this, production of NADH and state 3 respiration in isolated mitochondria were down-regulated by the de-esterified DMOG derivative, N-oxalylglycine. Exploring the roles of DMOG as a putative inhibitor of glutamine/α-ketoglutarate metabolic axis, we found that the observed suppression of OxPhos and compensatory activation of glycolytic ATP flux make cancer cells vulnerable to combined treatment with DMOG and inhibitors of glycolysis. On the other hand, DMOG treatment impairs deep cell deoxygenation in 3D tissue culture models, demonstrating a potential to relieve functional stress imposed by deep hypoxia on tumour, ischemic or inflamed tissues. Indeed, using a murine model of colitis, we found that O2 availability in the inflamed colon tissue rapidly increased after application of DMOG, which could contribute to the known therapeutic effect of this compound. Overall, our results provide new insights into the relationship between mitochondrial function, O2 availability, metabolic reprogramming and associated diseases.
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MSL1 protein regulates global histone H4 acetylation at residue K16 in stem and cancer cells, through interaction with KAT8. The functional significance of mammalian MSL1 isoforms, involved in various protein interactions, is poorly understood. We report the identification of a novel nuclear localization signal (NLS), common to all MSL1 isoforms, in addition to previously known bipartite NLS, located in domain PEHE. Isoforms having both NLS localize to sub-nuclear foci where they can target co-chaperone protein TTC4. However, all MSL1 isoforms also have ability to affect H4K16 acetylation. Thus, presence of two NLS in MSL1 protein can mediate activity of KAT8 in vivo.
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Núcleo Celular/metabolismo , Histona Acetiltransferases/metabolismo , Sinais de Localização Nuclear/genética , Proteínas Supressoras de Tumor/metabolismo , Acetilação , Animais , Células HCT116 , Células HEK293 , Células HeLa , Histona Acetiltransferases/genética , Histonas/metabolismo , Humanos , Camundongos , Células NIH 3T3 , Isoformas de Proteínas/química , Isoformas de Proteínas/metabolismo , Proteínas Supressoras de Tumor/genéticaRESUMO
Lysyl oxidase (LOX) is an amine oxidase involved in protein cross-linking of the extracellular matrix. Less well characterized is the role that LOX plays among nuclear proteins, and molecular mechanisms of its transport to the nucleus are currently unknown. Here, we have employed yeast two-hybrid library screening and found that the LOX catalytic domain interacts with the transcription repressor p66ß. This interaction has been confirmed in vitro and has been found to be accomplished through the CR2-containing domain of p66ß. Moreover, co-expression of p66ß and LOX in living tumor cells leads to the nuclear accumulation of LOX. Thus, p66ß might be important for the regulation of LOX in the nucleus.
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Núcleo Celular/metabolismo , Proteína-Lisina 6-Oxidase/metabolismo , Proteínas Repressoras/metabolismo , Animais , Domínio Catalítico , Linhagem Celular , Humanos , Modelos Biológicos , Ligação Proteica , Mapeamento de Interação de Proteínas , Transporte Proteico , Proteína-Lisina 6-Oxidase/química , Técnicas do Sistema de Duplo-HíbridoRESUMO
Assessment of hypoxia, nutrients, metabolite gradients, and other hallmarks of the tumor microenvironment within 3D multicellular spheroid and organoid models represents a challenging analytical task. Here, we report red/near-infrared (NIR) emitting cell staining with O2-sensitive nanoparticles, which enable measurements of spheroid oxygenation on a conventional fluorescence microscope. Nanosensor probes, termed "MMIR" (multimodal infrared), incorporate an NIR O2-sensitive metalloporphyrin (PtTPTBPF) and deep red aza-BODIPY reference dyes within a biocompatible polymer shell, allowing for oxygen gradient quantification via fluorescence ratio and phosphorescence lifetime readouts. We optimized staining techniques and evaluated the nanosensor probe characteristics and cytotoxicity. Subsequently, we applied nanosensors to the live spheroid models based on HCT116, DPSCs, and SKOV3 cells, at rest, and treated with drugs affecting cell respiration. We found that the growth medium viscosity, spheroid size, and formation method influenced spheroid oxygenation. Some spheroids produced from HCT116 and dental pulp stem cells exhibited "inverted" oxygenation gradients, with higher core oxygen levels than the periphery. This contrasted with the frequently encountered "normal" gradient of hypoxia toward the core caused by diffusion. Further microscopy analysis of spheroids with an "inverted" gradient demonstrated metabolic stratification of cells within spheroids: thus, autofluorescence FLIM of NAD(P)H indicated the formation of a glycolytic core and localization of OxPhos-active cells at the periphery. Collectively, we demonstrate a strong potential of NIR-emitting ratiometric nanosensors for advanced microscopy studies targeting live and quantitative real-time monitoring of cell metabolism and hypoxia in complex 3D tissue models.
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Nanopartículas , Oxigênio , Esferoides Celulares , Humanos , Esferoides Celulares/metabolismo , Esferoides Celulares/efeitos dos fármacos , Oxigênio/metabolismo , Oxigênio/química , Nanopartículas/química , Microscopia de Fluorescência , Raios Infravermelhos , Metaloporfirinas/química , Metaloporfirinas/farmacologiaRESUMO
Live cell microscopies of in vitro, ex vivo, and in vivo experimental intestinal models enable visualizing cell proliferation, differentiation, and functional cellular status in response to intrinsic and extrinsic (e.g., in the presence of microbiota) factors. While the use of transgenic animal models expressing biosensor fluorescent proteins can be laborious and not compatible with clinical samples and patient-derived organoids, the use of fluorescent dye tracers is an attractive alternative. In this protocol, we describe how the differentiation-dependent intestinal cell membrane composition can be labeled using fluorescent cholera toxin subunit B (CTX) derivatives. By using the culture of mouse adult stem cell-derived small intestinal organoids, we show that CTX can bind specific plasma membrane domains in differentiation-dependent manner. Green (Alexa Fluor 488) and red (Alexa Fluor 555) fluorescent CTX derivatives also display additional contrast in a fluorescence lifetime domain, when probed by the fluorescence lifetime imaging microscopy (FLIM), and can be used together with other fluorescent dyes and cell tracers. Importantly, CTX staining remains confined to specific regions in the organoids after fixation, which enables using it in both live cell and fixed tissue immunofluorescence microscopies.
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Toxina da Cólera , Organoides , Animais , Camundongos , Microscopia de Fluorescência , Diferenciação Celular , Corantes FluorescentesRESUMO
Multicellular spheroids are important tools for studying tissue and cancer physiology in 3D and are frequently used in tissue engineering as tissue assembling units for biofabrication. While the main power of the spheroid model is in mimicking physical-chemical gradients at the tissue microscale, the real physiological environment (including dynamics of metabolic activity, oxygenation, cell death, and proliferation) inside the spheroids is generally ignored. At the same time, the effects of the growth medium composition and the formation method on the resulting spheroid phenotype are well documented. Thus, characterization and standardization of spheroid phenotype are required to ensure the reproducibility and transparency of the research results. The analysis of average spheroid oxygenation and the value of O2 gradients in three dimensions (3D) can be a simple and universal way for spheroid phenotype characterization, pointing at their metabolic activity, overall viability, and potential to recapitulate in vivo tissue microenvironment. The visualization of 3D oxygenation can be easily combined with multiparametric analysis of additional physiological parameters (such as cell death, proliferation, and cell composition) and applied for continuous oxygenation monitoring and/or end-point measurements. The loading of the O2 probe is performed during the stage of spheroid formation and is compatible with various protocols of spheroid generation. The protocol includes a high-throughput method of spheroid generation with introduced red and near-infrared emitting ratiometric fluorescent O2 nanosensors and the description of multi-parameter assessment of spheroid oxygenation and cell death before and after bioprinting. The experimental examples show comparative O2 gradients analysis in homo- and hetero-cellular spheroids as well as spheroid-based bioprinted constructs. The protocol is compatible with a conventional fluorescence microscope having multiple fluorescence filters and a light-emitting diode as a light source.
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Bioimpressão , Esferoides Celulares , Microscopia , Oxigênio/metabolismo , Reprodutibilidade dos Testes , Esferoides Celulares/metabolismoRESUMO
Lysyl oxidase (LOX) is implicated in several extracellular matrix related disorders, including fibrosis and cancer. Methods of inhibition of LOX in vivo include antibodies, copper sequestration and toxic small molecules such as ß-aminopropionitrile. Here, we propose a novel approach to modulation of LOX activity based on the kinetic isotope effect (KIE). We show that 6,6-d(2)-lysine is oxidised by LOX at substantially lower rate, with apparent deuterium effect on V(max)/K(m) as high as 4.35 ± 0.22. Lys is an essential nutrient, so dietary ingestion of D(2)Lys and its incorporation via normal Lys turnover suggests new approaches to mitigating LOX-associated pathologies.
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Lisina/química , Proteína-Lisina 6-Oxidase/antagonistas & inibidores , Animais , Deutério/química , Marcação por Isótopo , Cinética , Camundongos , Oxirredução , Proteína-Lisina 6-Oxidase/metabolismo , Proteínas Recombinantes/antagonistas & inibidores , Proteínas Recombinantes/metabolismo , Ovinos , Especificidade por SubstratoRESUMO
Stem cells and the niche in which they reside feature a complex microenvironment with tightly regulated homeostasis, cell-cell interactions and dynamic regulation of metabolism. A significant number of organoid models has been described over the last decade, yet few methodologies can enable single cell level resolution analysis of the stem cell niche metabolic demands, in real-time and without perturbing integrity. Here, we studied the redox metabolism of Lgr5-GFP intestinal organoids by two emerging microscopy approaches based on luminescence lifetime measurement - fluorescence-based FLIM for NAD(P)H, and phosphorescence-based PLIM for real-time oxygenation. We found that exposure of stem (Lgr5-GFP) and differentiated (no GFP) cells to high and low glucose concentrations resulted in measurable shifts in oxygenation and redox status. NAD(P)H-FLIM and O2-PLIM both indicated that at high 'basal' glucose conditions, Lgr5-GFP cells had lower activity of oxidative phosphorylation when compared with cells lacking Lgr5. However, when exposed to low (0.5 mM) glucose, stem cells utilized oxidative metabolism more dynamically than non-stem cells. The high heterogeneity of complex 3D architecture and energy production pathways of Lgr5-GFP organoids were also confirmed by the extracellular flux (XF) analysis. Our data reveals that combined analysis of NAD(P)H-FLIM and organoid oxygenation by PLIM represents promising approach for studying stem cell niche metabolism in a live readout.
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Proteínas de Fluorescência Verde/metabolismo , Mucosa Intestinal/citologia , Organoides/citologia , Receptores Acoplados a Proteínas G/genética , Animais , Comunicação Celular , Glucose/farmacologia , Proteínas de Fluorescência Verde/genética , Mucosa Intestinal/efeitos dos fármacos , Mucosa Intestinal/metabolismo , Análise do Fluxo Metabólico , Camundongos , Microscopia de Fluorescência , Técnicas de Cultura de Órgãos , Organoides/efeitos dos fármacos , Organoides/metabolismo , Fosforilação Oxidativa , Receptores Acoplados a Proteínas G/metabolismo , Proteínas Recombinantes/metabolismo , Nicho de Células-Tronco/efeitos dos fármacos , Células-Tronco/citologia , Células-Tronco/efeitos dos fármacos , Células-Tronco/metabolismoRESUMO
Fluorescence lifetime imaging microscopy (FLIM), enabling live quantitative multiparametric analyses, is an emerging bioimaging approach in tissue engineering and regenerative medicine. When combined with stem cell-derived intestinal organoid models, FLIM allows for tracing stem cells and monitoring of their proliferation, metabolic fluxes, and oxygenation. It is compatible with the use of live Matrigel-grown intestinal organoids produced from primary adult stem cells, crypts, and transgenic Lgr5-GFP mice. In this chapter we summarize available experimental protocols, imaging platforms (one- and two-photon excited FLIM, phosphorescence lifetime imaging microscopy (PLIM)) and provide the anticipated data for FLIM imaging of the live intestinal organoids, focusing on labeling of cell proliferation, its colocalization with the stem cell niche, measured local oxygenation, autofluorescence, and some other parameters. The protocol is illustrated with examples of multiparameter imaging, employing spectral and "time domain"-based separation of dyes, probes, and assays.
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Microscopia de Fluorescência/métodos , Imagem Óptica/métodos , Animais , Proliferação de Células/fisiologia , Camundongos , Organoides/citologia , Software , Nicho de Células-Tronco/fisiologia , Engenharia TecidualRESUMO
The importance of extracellular gradients of biomolecules is increasingly appreciated in the processes of tissue development and regeneration, in health and disease. In particular, the dynamics of extracellular calcium concentration is rarely studied. Here, we present a low affinity Ca2+ biosensor based on Twitch-2B fluorescent protein fused with the cellulose- and collagen-binding peptides. These recombinant chimeric proteins can bind cellulose and collagen scaffolds and enable scaffold-based biosensing of Ca2+ in the proximity of cells in live 3D tissue models. We found that the Twitch-2B mutant is compatible with intensity-based ratiometric and fluorescence lifetime imaging microscopy (FLIM) measurement formats, under one- and two-photon excitation modes. Furthermore, the donor fluorescence lifetime of the biosensor displays response to [Ca2+] over a range of â¼2-2.5 ns, making it attractive for multiplexed FLIM assays. To evaluate the performance of this biosensor in physiological measurements, we applied it to the live Lgr5-GFP mouse intestinal organoid culture and measured its responses to the changes in extracellular Ca2+ upon chelation with EGTA. When combined with spectrally resolved FLIM of lipid droplets using Nile red dye, we observed changes in cytoplasmic and basal membrane-associated lipid droplet composition in response to the extracellular Ca2+ depletion, suggesting that the intestinal epithelium can respond to and compensate such treatment. Altogether, our results demonstrate Twitch-2B as a prospective Ca2+ sensor for multiplexed FLIM analysis in a complex 3D tissue environment.
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TTC4 (tetratricopeptide repeat domain protein 4) is a putative tumor suppressor involved in the transformation of melanocytes. At present, the relationships between TTC4 and DNA replication proteins are largely unknown, as are the tissue distribution and subcellular localization of TTC4. Using reverse transcription with the polymerase chain reaction, we have observed that the murine TTC4 gene is ubiquitously expressed. Analysis of the TTC4 subcellular localization has shown that, upon overexpression, TTC4 localizes to the cytoplasm. Interestingly, co-expression with a known protein interaction partner, hampin/MSL1, results in the nuclear translocation of the TTC4 protein. The subcellular localization of endogenous TTC4 depends, however, on the cell cycle: it is mostly nuclear in the G1 and S phases and is evenly distributed between the nucleus and cytoplasm in G2. The nuclear transport of TTC4 is apparently a complex process dependent on interactions with other proteins during the progression of the cell cycle. Thus, the dynamic character of the nuclear accumulation of TTC4 might be a potential link with regard to its function in tumor suppression.
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Ciclo Celular , Núcleo Celular/metabolismo , Proteínas Supressoras de Tumor/metabolismo , Transporte Ativo do Núcleo Celular , Animais , Linhagem Celular , Fase G1 , Humanos , Carioferinas/metabolismo , Camundongos , Proteínas Nucleares/metabolismo , Especificidade de Órgãos , Ratos , Receptores Citoplasmáticos e Nucleares/metabolismo , Proteínas Recombinantes de Fusão/metabolismo , Fase S , Frações Subcelulares/metabolismo , Proteína Exportina 1RESUMO
BACKGROUND: Lysyl oxidases (LOX) have been extensively studied in mammals, whereas properties and functions of recently found homologues in prokaryotic genomes remain enigmatic. METHODS: LOX open reading frame was cloned from Haloterrigena turkmenica in an E. coli expression vector. Recombinant Haloterrigena turkmenica lysyl oxidase (HTU-LOX) proteins were purified using metal affinity chromatography under denaturing conditions followed by refolding. Amine oxidase activity has been measured fluorometrically as hydrogen peroxide release coupled with the oxidation of 10-acetyl-3,7-dihydroxyphenoxazine in the presence of horseradish peroxidase. Rabbit polyclonal antibodies were obtained and used in western blotting. RESULTS: Cultured H. turkmenica has no detectable amine oxidase activity. HTU-LOX may be expressed in E. coli with a high protein yield. The full-length protein gives no catalytic activity. For this reason, we hypothesized that the hydrophobic N-terminal region may interfere with proper folding and its removal may be beneficial. Indeed, truncated His-tagged HTU-LOX lacking the N-terminal hydrophobic signal peptide purified under denaturing conditions can be successfully refolded into an active enzyme, and a larger N-terminal truncation further increases the amine oxidase activity. Refolding is optimal in the presence of Cu2+ at pH 6.2 and is not sensitive to salt. HTU-LOX is sensitive to LOX inhibitor 3-aminopropionitrile. HTU-LOX deaminates usual substrates of mammalian LOX such as lysine-containing polypeptides and polymers. The major difference between HTU-LOX and mammalian LOX is a relaxed substrate specificity of the former. HTU-LOX readily oxidizes various primary amines including such compounds as taurine and glycine, benzylamine being a poor substrate. Of note, HTU-LOX is also active towards several aminoglycoside antibiotics and polymyxin. Western blotting indicates that epitopes for the anti-HTU-LOX polyclonal antibodies coincide with a high molecular weight protein in H. turkmenica cells. CONCLUSION: H. turkmenica contains a lysyl oxidase gene that was heterologously expressed yielding an active recombinant enzyme with important biochemical features conserved between all known LOXes, for example, the sensitivity to 3-aminopropionitrile. However, the native function in the host appears to be cryptic. SIGNIFICANCE: This is the first report on some properties of a lysyl oxidase from Archaea and an interesting example of evolution of enzymatic properties after hypothetical horizontal transfers between distant taxa.
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Quantitative measurement of pH and metabolite gradients by microscopy is one of the challenges in the production of scaffold-grown organoids and multicellular aggregates. Herein, we used the cellulose-binding domain (CBD) of the Cellulomonas fimi CenA protein for designing biosensor scaffolds that allow measurement of pH and Ca2+ gradients by fluorescence intensity and lifetime imaging (FLIM) detection modes. By fusing CBD with pH-sensitive enhanced cyan fluorescent protein (CBD-ECFP), we achieved efficient labeling of cellulose-based scaffolds based on nanofibrillar, bacterial cellulose, and decellularized plant materials. CBD-ECFP bound to the cellulose matrices demonstrated pH sensitivity comparable to untagged ECFP (1.9-2.3â¯ns for pH 6-8), thus making it compatible with FLIM-based analysis of extracellular pH. By using 3D culture of human colon cancer cells (HCT116) and adult stem cell-derived mouse intestinal organoids, we evaluated the utility of the produced biosensor scaffold. CBD-ECFP was sensitive to increases in extracellular acidification: the results showed a decline in 0.2-0.4â¯pH units in response to membrane depolarization by the protonophore FCCP. With the intestinal organoid model, we demonstrated multiparametric imaging by combining extracellular acidification (FLIM) with phosphorescent probe-based monitoring of cell oxygenation. The described labeling strategy allows for the design of extracellular pH-sensitive scaffolds for multiparametric FLIM assays and their use in engineered live cancer and stem cell-derived tissues. Collectively, this research can help in achieving the controlled biofabrication of 3D tissue models with known metabolic characteristics. STATEMENT OF SIGNIFICANCE: We designed biosensors consisting of a cellulose-binding domain (CBD) and pH- and Ca2+-sensitive fluorescent proteins. CBD-tagged biosensors efficiently label various types of cellulose matrices including nanofibrillar cellulose and decellularized plant materials. Hybrid biosensing cellulose scaffolds designed in this study were successfully tested by multiparameter FLIM microscopy in 3D cultures of cancer cells and mouse intestinal organoids.
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Celulose/química , Imagem Óptica/métodos , Engenharia Tecidual/métodos , Alicerces Teciduais/química , Ácidos/química , Animais , Materiais Biocompatíveis/farmacologia , Técnicas Biossensoriais , Células HCT116 , Humanos , Concentração de Íons de Hidrogênio , Intestinos/fisiologia , Camundongos Endogâmicos C57BL , Organoides/metabolismo , Coloração e RotulagemRESUMO
Intestinal organoids are widely applied in stem cell research, regenerative medicine, toxicology, pharmacology, and host-microbe interactions research. The variability of oxidative metabolism for stem and differentiated cell types constituting organoid is known to be important but so far it has not been studied in details. Here, we report the use of live cell microscopy of oxygen via the phosphorescence lifetime imaging microscopy (PLIM) method to address the oxygenation and variability of aerobic metabolism of individual organoids in the culture. Using the cell-penetrating phosphorescent O2-sensitive probe, we found inhomogeneous O2 distribution in live organoids, with areas of relatively high oxygenation (up to 73 µM in organoid compared to an average 40 µM O2) and trans-epithelial O2 microgradients (up to 0.6-0.83 µM/µm). We also demonstrated that intestinal organoid culture consists of units with different respiration activity and oxygenation (from 27 to 92 µM, equal to 2.8-9.7% O2), depending on age of the culture and drug treatment. Collectively, our results indicate that ignoring the metabolic heterogeneity of organoid culture can be critical for proper data interpretation. The live cell imaging PLIM method demonstrates a clear advantage of using individual organoids as separate experimental units rather than 'bulk' organoid cultures.
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Diferenciação Celular/fisiologia , Organoides/metabolismo , Animais , Técnicas de Cultura de Células/métodos , Mucosa Intestinal/metabolismo , Camundongos , Oxigênio/metabolismoRESUMO
It is well known that sensitivity of quenched-phosphorescence O2 sensors can be tuned by changing the nature of indicator dye and host polymer acting as encapsulation and quenching mediums. Here, we describe a new type of sensor materials based on nanostructured hard elastic polymeric substrates. With the example of hard elastic polypropylene films impregnated with Pt-benzoporphyrin dye, we show that such substrates enable simple one-step fabrication of O2 sensors by standard and scalable polymer processing technologies. In addition, the resulting sensor materials show prominent response to tensile drawing via changes in phosphorescence intensity and lifetime and O2 quenching constant, Kq. The mechanosensitive response shows reversibility and hysteresis, which are related to macroscopic changes in the nanoporous structure of the polymer. Such multifunctional materials can find use as mechanically tunable O2 sensors, as well as strain/deformation sensors operating in a phosphorescence-lifetime-based detection mode.