RESUMEN
The oxygenation of cells and tissues and acidification of the cellular endolysosomal system are among the major factors that ensure normal functioning of an organism and are violated in various pathologies. Recording of these parameters and their changes under various conditions is an important task for both basic research and clinical applications. In the present work, we utilized internalizable dual pH/O2 lifetime sensor (Ir-HSA-FITC) based on the covalent conjugation of human serum albumin (HSA) with fluorescein isothiocyanate (FITC) as pH sensor and an orthometalated iridium complex as O2 sensor. The probe was tested for simultaneous detection of acidification level and oxygen concentration in endolysosomes of endometrial mesenchymal stem/stromal cells (enMSCs) cultivated as 2D monolayers and 3D spheroids. Using a combined FLIM/PLIM approach, we found that due to high autofluorescence of enMSCs FITC lifetime signal in control cells was insufficient to estimate pH changes. However, using flow cytometry and confocal microscopy, we managed to detect the FITC signal response to inhibition of endolysosomal acidification by Bafilomycin A1. The iridium chromophore phosphorescence was detected reliably by all methods used. It was demonstrated that the sensor, accumulated in endolysosomes for 24 h, disappeared from proliferating 2D enMSCs by 72 h, but can still be recorded in non-proliferating spheroids. PLIM showed high sensitivity and responsiveness of iridium chromophore phosphorescence to experimental hypoxia both in 2D and 3D cultures. In spheroids, the phosphorescence signal was detected at a depth of up to 60 µm using PLIM and showed a gradient in the intracellular O2 level towards their center.
Asunto(s)
Luminiscencia , Células Madre Mesenquimatosas , Humanos , Iridio/química , Fluoresceína-5-Isotiocianato , Oxígeno , Concentración de Iones de HidrógenoRESUMEN
Quantum dots (QDs) have attracted great attention as tools for theranostics that combine the possibility of simultaneous biological target visualization and medicine delivery. Here, we address whether core/shell InP/ZnS QDs (InP-QDs) may be an alternative to toxic Cd-based QDs. We analyze InP-QD photophysical characteristics in cell culture medium, salt solutions, and directly in the cells. It was demonstrated that InP-QDs were internalized into endolysosomes in HeLa and A549 cells with dynamics similar to Cd-based QDs of the same design, but the two cell lines accumulated them with different efficiencies. InP-QDs were reliably detected in the endosomes despite their low quantum yields. Cell culture medium efficiently decreased the InP-QD photoluminescence lifetime by 50%, acidic pH (4.0) had a moderate effect (20-25% reduction), and quenching by salt solutions typical of intra-endosomal medium composition resulted in a decrease of about 10-15%. The single-vesicle fluorescence-lifetime imaging microscopy analysis of QDs inside and outside the cells shows that the scatter between endosomes in the same cell can be significant, which indicates the complex impact of the abovementioned factors on the state of InP-QDs. The PI test and MTT test demonstrate that InP-QDs are toxic for both cell lines at concentrations higher than 20 nM. Possible reasons for InP-QD toxicity are discussed.
Asunto(s)
Puntos Cuánticos , Puntos Cuánticos/química , Luminiscencia , Cadmio , Sulfuros/farmacología , Sulfuros/químicaRESUMEN
In this report, we present implementation and validation of machine-learning classifiers for distinguishing between cell types (HeLa, A549, 3T3 cell lines) and states (live, necrosis, apoptosis) based on the analysis of optical parameters derived from cell phase images. Validation of the developed classifier shows the accuracy for distinguishing between the three cell types of about 93% and between different cell states of the same cell line of about 89%. In the field test of the developed algorithm, we demonstrate successful evaluation of the temporal dynamics of relative amounts of live, apoptotic and necrotic cells after photodynamic treatment at different doses.
Asunto(s)
Línea Celular Tumoral/clasificación , Células HeLa/metabolismo , Aprendizaje Automático/normas , Microscopía de Contraste de Fase/métodos , HumanosRESUMEN
Synthesis of biocompatible near infrared phosphorescent complexes and their application in bioimaging as triplet oxygen sensors in live systems are still challenging areas of organometallic chemistry. We have designed and synthetized four novel iridium [Ir(N^C)2(N^N)]+ complexes (N^C-benzothienyl-phenanthridine based cyclometalated ligand; N^N-pyridin-phenanthroimidazol diimine chelate), decorated with oligo(ethylene glycol) groups to impart these emitters' solubility in aqueous media, biocompatibility, and to shield them from interaction with bio-environment. These substances were fully characterized using NMR spectroscopy and ESI mass-spectrometry. The complexes exhibited excitation close to the biological "window of transparency", NIR emission at 730 nm, and quantum yields up to 12% in water. The compounds with higher degree of the chromophore shielding possess low toxicity, bleaching stability, absence of sensitivity to variations of pH, serum, and complex concentrations. The properties of these probes as oxygen sensors for biological systems have been studied by using phosphorescence lifetime imaging experiments in different cell cultures. The results showed essential lifetime response onto variations in oxygen concentration (2.0-2.3 µs under normoxia and 2.8-3.0 µs under hypoxia conditions) in complete agreement with the calibration curves obtained "in cuvette". The data obtained indicate that these emitters can be used as semi-quantitative oxygen sensors in biological systems.
Asunto(s)
Materiales Biocompatibles/química , Iridio/química , Luminiscencia , Oxígeno/análisis , Animales , Células CHO , Cricetulus , Células HeLa , Humanos , Conformación Molecular , Espectroscopía de Protones por Resonancia Magnética , Fracciones Subcelulares/metabolismoRESUMEN
Early endosomes, regarded as the main sorting station on endocytic pathway, are characterized by high frequency of homotypic fusions mediated by tethering protein EEA1. Despite intensive investigations, biogenesis of endosomes, boundaries between early and late endosomes, and process of cargo transition though them remain obscure. Here, using EGF/EGFR endocytosis as a model and confocal microscopy of fixed and live cells, we provide evidence favoring EEA1-vesicles being pre-existed vesicular compartment, that maintains its resident proteins' level and is sensitive to biosynthetic, but not endocytic pathway disturbance. EEA1-vesicles directly fuse with incoming EGF/EGFR-vesicles into hybrid endosomes with separated EEA1- and EGFR-domains, thus providing a platform for rapid achievement of an excess of surface-derived membrane that is used to form intraluminal vesicles (ILVs). Thus, multivesicular structures colocalized with EEA1 are still early endosomes. "EEA1-cycle" ends by exclusion of EGFR-containing domains with ILVs inside that turns into MVE and restoration of initial EEA1-vesicles population.
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Endosomas/metabolismo , Proteínas de Transporte Vesicular/metabolismo , Vías Biosintéticas , Medio de Cultivo Libre de Suero , Vesículas Citoplasmáticas/metabolismo , Endocitosis , Receptores ErbB/metabolismo , Proteínas Fluorescentes Verdes/metabolismo , Células HeLa , Humanos , Microscopía Confocal , Modelos Biológicos , Transporte de Proteínas , Proteínas Recombinantes de Fusión/metabolismoRESUMEN
Quantum dots (QDs) complexed to ligands recognizing surface receptors undergoing internalization are an attractive tool for live cell imaging of ligand-receptor complexes behavior and for specific tracking of the cells of interest. However, conjugation of quasi-multivalent large QD-particle to monovalent small growth factors like EGF that bound their tyrosine-kinase receptors may affect key endocytic events tightly bound to signaling. Here, by means of confocal microscopy we have addressed the key endocytic events of lysosomal degradative pathway stimulated by native EGF or EGF-QD bioconjugate. We have demonstrated that the decrease in endosome number, increase in mean endosome integrated density and the pattern of EEA1 co-localization with EGF-EGFR complexes at early stages of endocytosis were similar for the both native and QD-conjugated ligands. In both cases enlarged hollow endosomes appeared after wortmannin treatment. This indicates that early endosomal fusions and their maturation proceed similar for both ligands. EGF-QD and native EGF similarly accumulated in juxtanuclear region, and live cell imaging of endosome motion revealed the behavior described elsewhere for microtubule-facilitated motility. Finally, EGF-QD and the receptor were found in lysosomes. However, degradation of receptor part of QD-EGF-EGFR-complex was delayed compared to native EGF, but not inhibited, while QDs fluorescence was detected in lysosomes even after 24 hours. Importantly, in HeLa and A549 cells the both ligands behaved similarly. We conclude that during endocytosis EGF-QD behaves as a neutral marker for degradative pathway up to lysosomal stage and can also be used as a long-term cell marker.
Asunto(s)
Factor de Crecimiento Epidérmico/farmacología , Receptores ErbB/metabolismo , Lisosomas/metabolismo , Transducción de Señal/efectos de los fármacos , Línea Celular Tumoral , Endocitosis/efectos de los fármacos , Endosomas/metabolismo , Células HeLa , Humanos , Imagen Molecular , Unión Proteica , Transporte de Proteínas , Proteolisis , Puntos CuánticosRESUMEN
EGF complexed to fluorescent photostable quantum dots by biotin-streptavidin system (bEGF-savQD) is attractive for both the basic research and therapeutic application such as targeted drug delivery in EGF-receptor (EGFR) expressing cancers. However, compared to native EGF, the large size of QD and its quasi-multivalency can have unpredictable effects on EGFR endocytosis changing the internalization portal and/or endosomal processing tightly bound to EGF signaling. We have found that bEGF-savQDs enter HeLa cells via the temperature-dependent clathrin-mediated EGF-receptor-specific pathway characteristic for native EGF. We also found that EGF-to-QD concentration ratios used for the complex preparation and the level of EGF receptor expression affect the number and integral densities of the formed endosomes. So, at EGF-to-QD ratio from 4:1 to 12:1 (at nanomolar bEGF concentrations) on average 100 bright endosomes per HeLa cell were formed 15 min after the complex addition, while 1:1 ratio resulted in formation of very few dim endosomes. However, in A431 cells overexpressing EGFR 1:1 ratio was effective. Using dynamin inhibition and Na-acidic washout we showed that bEGF-savQDs bind surface receptors and enter clathrin-coated pits slower than the same ligands without QD. Yet, the bEGF-savQD demonstrated similar to native EGF and bEGF-savCy3 co-localization dynamics with tethering protein EEA1 and HRS, the key component of sorting ESCRT0 complex. In conclusion, our comparative study reveals that in respect to entrapment into coated pits, endosomal recruitment, endosome fusions, and the initial steps of endosomal maturation, bEGF-savQD behaves like native EGF and QD implementation does not affect these important events.