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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.

Mesenchymal stem cells (MSCs) are broadly applied in regenerative therapy to replace cells that are lost or impaired during disease. The low survival rate of MSCs after transplantation is one of the major limitations heavily influencing the success of the therapy. Unfavorable microenvironments with inflammation and oxidative stress in the damaged regions contribute to MSCs loss. Most of the strategies developed to overcome this obstacle are aimed to prevent stress-induced apoptosis, with little attention paid to senescence-another common stress reaction of MSCs. Here, we proposed the strategy to prevent oxidative stress-induced senescence of human endometrial stem cells (hMESCs) based on deferoxamine (DFO) application. DFO prevented DNA damage and stress-induced senescence of hMESCs, as evidenced by reduced levels of reactive oxygen species, lipofuscin, cyclin D1, decreased SA-β-Gal activity, and improved mitochondrial function. Additionally, DFO caused accumulation of HIF-1α, which may contribute to the survival of H 2 O 2 -treated cells. Importantly, cells that escaped senescence due to DFO preconditioning preserved all the properties of the initial hMESCs. Therefore, once protecting cells from oxidative damage, DFO did not alter further hMESCs functioning. The data obtained may become the important prerequisite for development of a new strategy in regenerative therapy based on MSCs preconditioning using DFO.

Accumulating evidence suggests that the senescence-messaging secretome (SMS) factors released by senescent cells play a key role in cellular senescence and physiological aging. Phenomenon of the senescence induction in human endometrium-derived mesenchymal stem cells (MESCs) in response to SMS factors has not yet been described. In present study, we examine a hypothesis whether the conditioned medium from senescent cells (CM-old) may promote premature senescence of young MESCs. In this case, we assume that SMS factors, containing in CM-old are capable to trigger senescence mechanism in a paracrine manner. A long-term cultivation MESCs in the presence of CM-old caused deceleration of cell proliferation along with emerging senescence phenotype, including increase in both the cell size and SA-β-Gal activity. The phosphorylation of p53 and MAPKAPK-2, a direct target of p38MAPK, as well as the expression of p21Cip1 and p16Ink4a were increased in CM-old treated cells with senescence developing whereas the Rb phosphorylation was diminished. The senescence progression was accompanied by both enhanced ROS generation and persistent activation of DNA damage response, comprising protein kinase ATM, histone H2A.X, and adapter protein 53BP1. Thus, we suggest that a senescence inducing signal is transmitted through p16/MAPKAPK-2/Rb and DDR-mediated p53/p21/Rb signaling pathways. This study is the first to demonstrate that the SMS factors secreted in conditioned medium of senescent MESCs trigger a paracrine mechanism of premature senescence in young cells., (Copyright © 2018 Elsevier Inc. All rights reserved.)

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., (Copyright © 2016 Elsevier Inc. All rights reserved.)

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.

According to current model, stimulation of EGF-receptor endocytosis results in recruitment onto early endosomes cytosolic tether protein EEA1 necessary for their further fusions. However, EEA1-positive vesicles are found in the cells not treated with growth factor, that were incubated in serum-free conditions. It is known also that prolonged serum deprivation induces autophagosomes formation, the process possibly involving endocytic compartments. To check whether EEA1-positive vesicles seen in serum-deprived HeLa cells are autophagosomes, we here evaluated colocalization of EEA1 and autophagosome marker LC3 and studied dynamics of the EEA1- and LC3-vesicles’ number and size during 12—36 h cell cultivation in serum-free medium. It was found that the number of autophagosomes per cell is significantly less than the number of EEA1-vesicles. We show that serum starvation results in increase of only mean autophagosomes’ size, while the number and size of EEA1-vesicles did not changed. Colocalization of EEA1 and LC3 in serum-free cells was very low during first 12—18 h of starvation and increased insignificantly only by 36 h. Biosynthetic pathway inhibition by Golgi apparatus disruption by brefeldin A, decreased the number and increased the size of EEA1-vesicles. LC3-vesicles also demonstrated an increase of mean size and growth of colocalization with EEA1. Thus, we conclude that the majority of EEA1-vesicles in serum-starved cells are not autophagosomes. More pronounced effect of brefeldin A indicates that blockade of biosynthetic pathway is more strong stress factor comparing to serum deprivation in HeLa cells. This also suggests that this pathway is involved in EEA1-vesicles biogenesis.

Confocal immunofluorescent analysis of interphase HeLa cells has demonstrated that involved in regulation of homotypic fusions early endosomal autoantigene EEA1 is associated with vesicles represented by two populations differing in apparent size, localization and the level of bound EEA1. Before analysis the cells have been preincubated in serum-deprived medium for 12 h to minimize ligand-dependent endocytosis of serum growth factors. The first subpopulation is mainly represented by large vesicles strongly decorated with EEA1. These vesicles are localized presumably in juxtanuclear region. Microtubule depolimerization experiments have shown that this localization is maintained by tubulin cytoskeleton. The second subpopulation consists of numerous small vesicles slightly stained by EEA1 antibody and localized more peripherally. Double indirect immunofluorescent ananlysis of fixed cell images has revealed that juxtanuclear vesicles enriched in EEA1 are fully colocalized with key protein of early endosomes small GTPase Rab5, whereas about 50% of slightly decorated peripheral vesicles are Rab5-negative. It is found that the number of Rab5-positive vesicles per cell is higher than that of EEA1-positive vesicles. Thus, in serum-deprivated HeLa cells with low endocytic activity two subpopulations of EEA1-vesicles are revealed: the first one carries the both EEA1 at high level and Rab5 (EEA1+++/Rab5+), and the second subpopulation oconsists of weakly decorated EEA1-vesicles, that can be both Rab5-positive and -negative (EEA1+/Rab5- and EEA1+/Rab5+). Besides, there are vesicles carrying Rab5 only (EEA1-/Rab5+). The data obtained favor different functional role of all these subpopulations, which are associated with proteins widely considered as equivalent markers of early endosomes.

Disturbances at the childhood age increase risk of the appearance of cardiovascular diseases decades later. The nature of this interconnection called ontogenetic programming is not completely understood. Valuable sources of knowledge about mechanisms of ontogenetic programming are data of interspecies study of biology of the body life cycles and of heart physiological capabilities. Taken into account the interspecies differences, these data allow finding the correct direction of experimental investigations. Results of studies of almost 100 homoiothermal species have shown the slow growth and a high loading on the heart at postnatal development to decrease its aerobic capability in adults. Basing on these data, we suggested that the neonatal gastroenteritis causing tachyarrhythmia, malabsorption, and the growth deceleration might lead to pathological changes in the heart. Our task was to evaluate the effect of cryptosporidial gastroenteritis of different degrees of severity on heart of the neonatal rats. By using methods of Real-Time PCR, immunocytochemistry, image analysis, and study of interatrial septum, we have established that a gradual increase of intensity of infestation with Cryptosporidium parvum oocytes causes sharp changes corresponding to the "all or nothing" response. At a weak infestation the interatrial septum was close (like in control), while significant changes in expression of isoforms of heavy chains of alpha- and beta-myosin were absent. At the intermediate and severe infestation, in the interatrial septum the foramen ovale was visualized and there were observed cardiac atrophy and a strong shift of ration of expression of myosin heavy chains toward the low-velocity beta chain. Thus, by disturbing the frequency-strength ratios and causing outflow of resources from the formed heart, the neonatal gastroenteritis produces pathological changes of the organ molecular and anatomical structures. Our results can be interest to evolutionary biologists and physicians, as they show importance of knowledge of evolutionary-comparative investigations for the search for novel risk factors of heart diseases and demonstrate interconnection between gastroenteritis, pathology of interatrial septum, and a change of composition of the main contractile proteins in cardiomyocytes.

Endocytosis of signaling receptors EGF receptor in particular, starting at the plasma membrane and finishing in perinuclear lysosomes implies endosomes' multiple interactions with homotypic endosomes and vesicles of other origin (lysosomes, trans-Golgi network), which results in endosomal size changes. In addition, the characteristics of endocytic pathway is endosomes' translocation from cell periphery to juxtanuclear region. Thus, endocytosis as a highly dynamic process develops in time and space. Obviously one of the most productive approach is light immunofluorescent microscopy that allows of estimating endocytes dynamics at the level of one or several cells. Different impacts influencing endocytic regulator components are inevitably reflected on dynamics of endosome size and (or) its translocation. This provide possibility to uncover the both crucial and secondary components of regulatory machinery. However, visual estimation of such effects is often too subjective and does not allow getting statistically reliable data. Comparison of different experiments even in the case of the same series is also impeded. In this work we use such parameters as apparent vesicle size (diameter, area or volume) and vesicle number per cell to provide quantitative estimation of fusion efficacy, as well as the coefficient reflecting vesicles' clasterization in perinuclear region as a measure of their translocation along microtubules toward nucleus (D(clust)) and present these parameters application using EGF receptor endocytosis as an example.

Infectious gastroenteritis is one of the common causes of tachyarrythmia, malabsorbtion and growth retardation in children. Our recent studies have indicated that neonatal.cryptosporidial gastroenteritis is associated with long-term cardiomyocyte abnormalities. The aim of the present study was to find out how neonatal cryptosporidiosis of various severities affects cardiac anatomy and cardiomyocyte polyploidization, remodeling and HIF-1α expression. Using real-time PCR, cytometry, immunohistochemistry, image analysis and interatrial septum visual examination, we revealed that gradual increase in cryptosporidial invasion was associated with threshold changes. At weak parasitic infection, interatrial septum was entire and there was no statistically significant change in cardiomyocytes. At moderate and severe infection, all changes in cardiac anatomy and cardiomyocytes were statistically significant and demonstrated approximately similar degree. Compared to control, heart were atrophied and elongated, interatrial septum contained a small window (patentforamrn ovale), and cardiomyocytes lost protein, became elongated, thin and accumulated additional genomes. Also we found HIF-1α mRNA hyperexpression. Notable, the threshold response to gradual stimulus is an important criterion of development programming since such a response is commonly a consequence of abnormal anatomic structure formation and cell differentiation failure. Our results can be interesting for physicians because they indicate that even moderate cryptosporidiosis can be dangerous for neonatal heart and can trigger neonatal programming of cardiovascular pathology. Also, our results for the first time demonstrate the association between gastroenteritis, patent foramen ovale and cardiomyocyte malfunction.

Cardiovascular diseases are the most common case of human death in developed countries. Thus, the discovering of their new risk factors is of primary importance. Based on epidemiology studies, vertebrate life-history traits comparison and cross-species cardiomyocyte transcriptome analysis, we suggest that one of these factors could be infectious gastroenteritis. This disease outflows recourses from cardio-vascular system and triggers pathological stimuli, like tachyarrhythmia, inflammation, malapsorption and energy depletion thereby disturbing cardiomyocyte metabolism and function. To test this hypothesis, we challenged gastroenteritis in neonatal rats with widespread human parasite Cryptosporidium parvum (Apicomplexa, Sporozoa). The results obtained by the methods of immunocytochemistry, quantitative morphometry and real-time PCR, indicate that moderate cryptosporidiosis lasting four days induces dramatic shift in myosin isoform expression ration toward isoform beta (with low ATPase activity) both at mRNA (by 1.7-4.5 folds) and protein (by 2.5-6 folds) levels. Antithetical manner of this shift and coherence between changes in mRNA and protein suggest that cryptosporidiosis affects all main steps of a complex myosin heavy chain regulatory network. Since the overexpression of myosin heavy chain beta (showing several times lower ATPase activity than myosin heavy chain alfa) is a generally accepted marker of human cardiac failure, we can consider cryptosporidial gastroenteritis as a new risk factor of cardiac contractile ability impairment. Our data can be interesting for clinicians.

The aim of the study was to generate dopaminergic (DA) neurons from human embryonic stem cells (ESC) in vitro. It was shown that human ESCs are able to differentiated into DA neurons without co-culture with stromal cells. Terminal differentiation into DA neurons was reached by successive application of noggin and bFGF growth factors on collagen and matrigel substrates during 3-4 weeks. Differentiation efficiency was evaluated by the number of colonies with cells expressing tyrosine hydroxylase (TH), a DA neuron marker, and by the number of TH-positive cells in cell suspension using flow cytometry. No cells with pluripotent markers were detected in DA-differentiated cultures. It makes possible to propose that the protocol of human ESC differentiation might be applied to generate DA neurons for their transplantation into the animals modeling neurodegenerative (Parkinson) disease without the risk of tumor growth.

Acetylation of microtubules (MT) during endocytosis of epidermal growth factor receptor, c-ErbB1, was studied by confocal immunofluorescence microscopy. It was found that stimulation of endocytosis of c-ErbB1 complexed with the epidermal growth factor (EGF), resulted in continuous raising of MT acetylation that reached its maximum at 60-90 min and then went down to the control level. Simultaneously, the receptor-containing endosomes grew in size and were redistributed into juxtranuclear region. Enlarged endosomes formed dense clusters around MTOC in 60-90 min. Another native c-ErbB1 ligand, transforming growth factor-alpha (TGF-alpha) and unlike EGF causing the receptor recycling, also initiated a wave of MT acetylation, but the effect was expressed more poorly. In this case, endosomes did not grow in size and did not form dense clusters near the MTOC. Cell treatment with deacetylase inhibitor trichostatin A (TSA) caused acetylation of the whole cellular MT population. Under these conditions, translocations of EGF-c-ErbB1-containing endosomes had the same pattern as in the cells untreated with the inhibitor, but the size of endosomes didn't increase during their redistribution into juxtranuclear region. Acetylation was especially pronounced in strongly bent MT regions positioned proximally to MTOC and forming there a dense meshwork whereas peripheral MT plus-ends were basically straight and not modified. We assume that MT acetylation is not so much crucial for preferential interaction with dynein or kinesin and, accordingly, for organization of endosome translocations in a certain direction. It is rather the result of stabilization of some MT pool which supports homotypic fusion of endosomes at early stage of their maturation.

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.

The idea of microtubules (MTs) as of passive railway tracks, along which transport vesicles travel by use of motor proteins, is widely accepted. In the present work the organization of MT system during EGF-receptor endocytosis was investigated by indirect double immunofluorescence in HeLa and A431 cell lines. Stimulation of cells with EGF resulted in formation of EGF receptor-containing peripheral vesicular endosomes. During time course of endocytosis the endosomes tended to concentrate in juxtranuclear region close to MTOC. This translocation was dependent on MTs since nocodazole treatment resulted in endosomes' scattering throughout the cytoplasm. Parallel staining of the cells with tubulin antibody has revealed significant remodeling of MTs organization during endocytosis. At early stages MTs demonstrated slight retraction at the cell periphery and the increasing intensity of tubulin fluorescence in the juxtranuclear region. Later on, long individual MTs disappeared and peripheral cytoplasm show diffuse staining in combination with a meshwork of short MT fragments. This stage correlated with EGFR localization in juxtranuclear endosomes. Disappearance of EGFR-positive staining due to its lysosomal degradation occurred in parallel to reestablishment of radial MT system. Possible functional significance of described alterations in organization of tubulin cytoskeleton is discussed.

The previous data (Zheleznova et al., 2001) did not enable the authors to conclude which particular wortmannin sensitive PI-3-kinase--p85/p110 (I class PI-3-K) or hVPS34 (III class PI-3-K)--may be involved in the regulation of EGF-receptor endocytosis. In the present work, we have shown that upon stimulation of EGF-receptor endocytosis additional structures stained with antibody against p85 appear in A431 cells, but the p85-positive compartment never co-localized with EGF-receptor-containing compartments either in control or in wortmannin-treated cells. At the same time, wortmannin treatment prevented association of hVPS34 with endosomal membranes. We have also found that early endosomal markers--Rab5 and EEA1 (membrane association of the latter depends on Rab5 and hVPS34)--co-localized with EGF-receptor in the juxtranuclear region during late stages of endocytosis, both in control and upon wortmannin treatment. These observations favor our suggestions that the transition of EGF-receptors from early to late endosomes may occur directly in this juxtranuclear region and be tightly associated with the formation of so called multivesicular bodies (MVB), which are late endosomes per se. We suggest that wortmannin may have no effect on early EEA1-dependent stage of the receptor endocytosis but blocks a transition of EGF-receptor complexes into the late endosomes by inhibiting activity of hVPS34 and removing it from membranes. The hVPS34 product PI-3-K, according to the known data, is involved in the formation of internal vesicles of MVB. Accumulation of EGF-receptors in these vesicles is believed to be necessary for the receptor degradation.

Hypertonic sucrose and amiloride or derivatives of the latter are commonly used to selectively inhibit clathrin-dependent (usually considered as a synonim of receptor-mediated endocytosis) or clathrin-independent endocytosis. Though these approaches are widely used in experimental practice, their limitations have not been studied in detail. In the present work, an attempt was made to evaluate possible side effects and selectivity of these agents towards the type of endocytosis. It was found that the incubation of A431 cells in 0.4 M sucrose resulted in a decrease in both intracellular accumulation and surface binding of the RME marker 125I-EGF. However, while the binding drops by about 3 times, the internalization of EGF in low concentrations was inhibited by more than 30 times, and that of high EGF concentrations by 5-10 times. It may evidence that at high EGF concentrations about 10-20% of ligand-receptor complexes can enter the cells through clathrin-independent pathway. Nevertheless, these results cannot be interpreted so unambigously, because we found that at the incubation longer than 30 min a significant portion of cells became dead or damaged, yielding about 50% of the whole population. By immunofluorescent assay, 5-(N,N-hexametylen)-amiloride commonly used to inhibit fluid phase endocytosis was demonstrated to reduce the staining of fluorescein-containing pinocytic vesicles, but it did not inhibit totally the entering of this marker. Under simultaneous stimulation of fluorescein and EGF endocytosis in the presence of the amiloride derivative, such a residual fluorescence shifted with time to the juxtranuclear region, which is characteristic of the late steps of RME. We suppose that a significant portion of extracellular fluid phase can be included in clathrin-dependent vesicles, whose formation is not disturbed by amiloride. It means that conclusions about the degree of pinocytosis inhibition are to be corrected, taking into account the constitutive clathrin-dependent endocytosis.