The Dual Luminescence Lifetime pH/Oxygen Sensor: Evaluation of Applicability for Intravital Analysis of 2D- and 3D-Cultivated Human Endometrial Mesenchymal Stromal Cells.

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.

EGF, TGF-α and Amphiregulin Differently Regulate Endometrium-Derived Mesenchymal Stromal/Stem Cells.

The prototypical receptor tyrosine kinase epidermal growth factor receptor (EGFR) is regulated by a set of its ligands, which determines the specificity of signaling and intracellular fate of the receptor. The EGFR signaling system is well characterized in immortalized cell lines such as HeLa derived from tumor tissues, but much less is known about EGFR function in untransformed multipotent stromal/stem cells (MSCs). We compared the effect of epidermal growth factor (EGF), transforming growth factor-α (TGF-α) and amphiregulin (AREG) on physiological responses in endometrial MSCs (enMSC) and HeLa cells. In addition, using Western blotting and confocal microscopy, we studied the internalization and degradation of EGFR stimulated by the three ligands in these cell lines. We demonstrated that unlike HeLa, EGF and TGF-α, but not AREG, stimulated enMSC proliferation and prevented decidual differentiation in an EGFR-dependent manner. In HeLa cells, EGF targeted EGFR for degradation, while TGF-α stimulated its recycling. Surprisingly, in enMSC, both ligands caused EGFR degradation. In both cell lines, AREG-EGFR internalization was not registered. In HeLa cells, EGFR was degraded within 2 h, restoring its level in 24 h, while in enMSC, degradation took more than 4-8 h, and the low EGFR level persisted for several days. This indicates that EGFR homeostasis in MSCs may differ significantly from that in immortalized cell lines.

Microenvironmental Impact on InP/ZnS-Based Quantum Dots in In Vitro Models and in Living Cells: Spectrally- and Time-Resolved Luminescence Analysis.

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.

Time- and Spectrally-Resolved Photoluminescence Study of Alloyed CdxZn1-xSeyS1-y/ZnS Quantum Dots and Their Nanocomposites with SPIONs in Living Cells.

Magnetic-luminescent composites based on semiconductor quantum dots (QDs) and superparamagnetic iron oxide nanoparticles (SPIONs) can serve as a platform combining visualization and therapy. Here, we report the construction of QD-SPION nanocomposites based on synthesized SPIONs and alloyed QDs (CdxZn1-xSeyS1-y)/ZnS solubilized with L-cysteine molecules. The study of the spectral-luminescence characteristics, the kinetics of luminescence decay show the composite's stability in a solution. After incubation with HeLa cells, QDs, SPIONs, and their composites form clusters on the cell surface and associate with endosomes inside the cells. Component-wise analysis of the photoluminescence decay of cell-associated QDs/SPIONs provides information about their localization and aggregate status.

Machine Learning Assisted Classification of Cell Lines and Cell States on Quantitative Phase Images.

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.

Biocompatible Ir(III) Complexes as Oxygen Sensors for Phosphorescence Lifetime Imaging.

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.

Positioning of endoplasmic reticulum exit sites around the Golgi depends on BicaudalD2 and Rab6 activity.

The endoplasmic reticulum (ER) is involved in biogenesis, modification and transport of secreted and membrane proteins. The ER membranes are spread throughout the cell cytoplasm as well as the export domains known as ER exit sites (ERES). A subpopulation of ERES is centrally localized proximal to the Golgi apparatus. The significance of this subpopulation on ER-to-Golgi transport remains unclear. Transport carriers (TCs) form at the ERES via a COPII-dependent mechanism and move to Golgi on microtubule (MT) tracks. It was shown previously that ERES are distributed along MTs and undergo chaotic short-range movements and sporadic rapid long-range movements. The long-range movements of ERES are impaired by either depolymerization of MTs or inhibition of dynein, suggesting that ERES central concentration is mediated by dynein activity. We demonstrate that the processive movements of ERES are frequently coupled with the TC departure. Using the Sar1a[H79G]-induced ERES clustering at the perinuclear region, we identified BicaudalD2 (BicD2) and Rab6 as components of the dynein adaptor complex which drives perinuclear ERES concentration at the cell center. BicD2 partially colocalized with ERES and with TC. Peri-Golgi ERES localization was significantly affected by inhibition of BicD2 function with its N-terminal fragment or inhibition of Rab6 function with its dominant-negative mutant. Golgi accumulation of secretory protein was delayed by inhibition of Rab6 and BicD2. Thus, we conclude that a BicD2/Rab6 dynein adaptor is required for maintenance of Golgi-associated ERES. We propose that Golgi-associated ERES may enhance the efficiency of the ER-to-Golgi transport.

Functional cycle of EEA1-positive early endosome: Direct evidence for pre-existing compartment of degradative pathway.

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.

EGF receptor lysosomal degradation is delayed in the cells stimulated with EGF-Quantum dot bioconjugate but earlier key events of endocytic degradative pathway are similar to that of native EGF.

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.

Quantitative analysis of the heterogeneous population of endocytic vesicles.

The quantitative characterization of endocytic vesicles in images acquired with microscope is critically important for deciphering of endocytosis mechanisms. Image segmentation is the most important step of quantitative image analysis. In spite of availability of many segmentation methods, the accurate segmentation is challenging when the images are heterogeneous with respect to object shapes and signal intensities what is typical for images of endocytic vesicles. We present a Morphological reconstruction and Contrast mapping segmentation method (MrComas) for the segmentation of the endocytic vesicle population that copes with the heterogeneity in their shape and intensity. The method uses morphological opening and closing by reconstruction in the vicinity of local minima and maxima respectively thus creating the strong contrast between their basins of attraction. As a consequence, the intensity is flattened within the objects and their edges are enhanced. The method accurately recovered quantitative characteristics of synthetic images that preserve characteristic features of the endocytic vesicle population. In benchmarks and quantitative comparisons with two other popular segmentation methods, namely manual thresholding and Squash plugin, MrComas shows the best segmentation results on real biological images of EGFR (Epidermal Growth Factor Receptor) endocytosis. As a proof of feasibility, the method was applied to quantify the dynamical behavior of Early Endosomal Autoantigen 1 (EEA1)-positive endosome subpopulations during EGF-stimulated endocytosis.

Mobility of tethering factor EEA1 on endosomes is decreased upon stimulation of EGF receptor endocytosis in HeLa cells.

Tethering factor EEA1, mediating homotypic fusion of early endosomes, was shown to be localized in membrane-bound state both in serum-deprived and stimulated for EGF receptor endocytosis cells. However, it is not known whether dynamics behavior of EEA1 is affected by EGF stimulation. We investigated EEA1 cytosol-to-membrane exchange rate in interphase HeLa cells by FRAP analysis. The data obtained fitted two-states binding model, with the bulk of membrane-associated EEA1 protein represented by the mobile fraction both in serum-starved and EGF-stimulated cells. Fast recovery state had similar half-times in the two cases: about 1.6 s and 2.8 s, respectively. However, the recovery half-time of slowly cycled EEA1 fraction significantly increased in EGF-stimulated comparing to serum-starved cells (from 21 to 99 s). We suppose that the retardation of EEA1 fluorescence recovery upon EGF-stimulation may be due to the increase of activated Rab5 on endosomal membranes, the growth of the number of tethering events between EEA1-positive vesicles and their clustering.

Quantum dots implementation as a label for analysis of early stages of EGF receptor endocytosis: a comparative study on cultured cells.

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.

Termination of tyrphostin AG1478 application results in different recovery of EGF receptor tyrosine residues 1045 and 1173 phosphorylation in A431 cells.

Tyrphostin AG1478 is known as a specific and reversible inhibitor of TK (tyrosine kinase) activity of the EGFR [EGF (epidermal growth factor) receptor]. It is attractive as an anticancer agent for cancers with elevated EGFR TK levels. However, post-application effects of AG1478 are not well studied. We have analysed EGFR phosphorylation after termination of AG1478 application using human epidermoid carcinoma A431 cells. It was found that AG1478 inhibitory action is fast, but not fully reversible: removal of tyrphostin resulted in incomplete restoration of the overall EGFR phosphorylation. Analysing the state of two individual autophosphorylation sites of internalized EGFR, Tyr1045 and Tyr1173, we demonstrated that phosphorylation of Tyr1173 involved in stimulation of the MAPK (mitogen-activated protein kinase) cascade was restored much more efficiently than that in position 1045, which binds the ubiquitin ligase c-Cbl and is necessary for targeting the receptor for lysosomal degradation. c-Cbl association with EGFR abolished by AG1478 was not reestablished after tyrphostin cessation. As a consequence, ubiquitination-dependent EGFR delivery to lysosomes was blocked, while phosphorylation of ERK1/2 (extracellular-signal-regulated kinase 1/2) was even increased. Thus, after termination of AG1478, the intracellular level of the inhibitor can be reached at which mitogenic signalling will be restored, whereas the EGFR negative regulation due to lysosomal degradation will not.

Epidermal growth factor (EGF) receptor endocytosis is accompanied by reorganization of microtubule system in HeLa cells.

Translocation of endosomes along microtubules (MTs) from the cell periphery toward the juxtranuclear region proximal to MTOC is well established. During this translocation the radial MT system is believed to retain its organization. Here we demonstrate that epidermal growth factor receptor (EGFR) endocytosis in HeLa cells is accompanied by dramatic remodeling of the MT system. Synchronized endocytosis was stimulated by warming the cells after EGF prebinding to EGFR on ice. Soon after that MTs were fully reestablished and EGFR was found in EE aligned along peripheral MTs. By the beginning of EE-to-LE sorting, the number of long MTs decreased and MTs appeared like an entangled meshwork of disorientated fragments and were partially depolymerized. Simultaneously, tubulin staining increased in juxtranuclear region, and at the time of LE-Lys interaction, enlarged EGFR-containing endosomes were localized there. Radial MTs were re-established when EGF-EGFR degradation started in lysosomes. In EGF absence, no alterations occurred upon MTs re-establishment. We conclude that MT remodeling is endocytosis-dependent.

Two different stages of epidermal growth factor (EGF) receptor endocytosis are sensitive to free ubiquitin depletion produced by proteasome inhibitor MG132.

Numerous studies implicate proteasomes in the regulation of EGF receptor (EGFR) endocytosis on the basis of the ability of inhibitors to decrease EGFR degradation, but the exact mechanisms remain obscure. We demonstrated that EGFR itself is not a direct target for proteasome, since it is delivered to lysosomes intact. Evidence is presented that the inhibitory effect of MG132 on EGF degradation is due mostly to free ubiquitin depletion resultant from the suppression of proteasomal functioning by MG132. By subcellular fractionation, we show two MG132-sensitive steps in the EGFR degradation pathway: sorting from early (EE) to late (LE) endosomes, and late stage of LE maturation. MG132 treatment resulted in stabilization of EGFR tyrosine phosphorylation and its association with c-Cbl. Nevertheless, ubiquitination of EGFR at late stages of endocytosis was significantly lower than that in control cells. Highly ubiquitinated forms of EGFR demonstrated more sensitivity to MG132 treatment.