RESUMEN
Keratins are one of the most abundant proteins in epithelial cells. They form a cytoskeletal filament network whose structural organization seriously conditions its function. Dynamic keratin particles and aggregates are often observed at the periphery of mutant keratinocytes related to the hereditary skin disorder epidermolysis bullosa simplex, which is due to mutations in keratins 5 and 14. To account for their emergence in mutant cells, we extended an existing mathematical model of keratin turnover in wild-type cells and developed a novel 2D phase-field model to predict the keratin distribution inside the cell. This model includes the turnover between soluble, particulate and filamentous keratin forms. We assumed that the mutation causes a slowdown in the assembly of an intermediate keratin phase into filaments, and demonstrated that this change is enough to account for the loss of keratin filaments in the cell's interior and the emergence of keratin particles at its periphery. The developed mathematical model is also particularly tailored to model the spatial distribution of keratins as the cell changes its shape.
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Expresión Génica , Queratinas/genética , Queratinas/metabolismo , Modelos Biológicos , Mutación , Algoritmos , Alelos , Sustitución de Aminoácidos , Línea Celular , Células Cultivadas , Epidermólisis Ampollosa Simple/genética , Epidermólisis Ampollosa Simple/metabolismo , Epidermólisis Ampollosa Simple/patología , Genes Reporteros , Humanos , Transporte de Proteínas , SolubilidadRESUMEN
Understanding the chemical and physical conditions for cell growth is important from biological and medical aspects. Many tissues and cell types (e.g., epithelial cells and neurons) naturally grow on surfaces that span in three-dimensions and offer structural or mechanical support. The scaffold surface has to promote adhesion and cell proliferation as well as support their weight and retain its structural integrity. Here, we present a flexible method that uses self-assembly of micrometer superparamagnetic particles to produce appropriate scaffold surfaces with controllable general appearance in three dimensions, such as oriented membranes, branched structure, or void network. As a proof of principle, the Chinese hamster ovary epithelial cell line was successfully grown for several days on inclined membranes. Robustness of the oriented membrane architecture was probed with optical tweezers. We measured the magnetic force holding one particle in a self-assembled upright hexagonal sheet and modeled it as a sum of pair interaction forces between spatially arrested static dipoles.
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Proliferación Celular , Coloides/química , Andamios del Tejido/química , Animales , Células CHO , Coloides/síntesis química , Cricetulus , Campos Magnéticos , Estructura MolecularRESUMEN
The formation of protrusions is necessary for numerous biological processes. It involves extension of the plasma membrane, and the force needed for this is provided by the actin cytoskeleton. Tether pulling with optical tweezers can mimic the formation of a protrusion, so we used this method to investigate the effects of modifying not only actin (with latrunculin A) but also microtubules (with nocodazole) and the plasma membrane itself (with methyl-ß-cyclodextrin) on the Chinese hamster ovary cell membrane. After these modifications, the membrane reservoir was supposed to redistribute. Caveolae constitute a small part of the reservoir, so the redistribution of caveolar proteins such as caveolin-1 and cavin-1 that represents caveolae per se was assessed. The main findings concerning protrusion force and membrane reservoir availability were as follows: (1) they correlated inversely, (2) their values underwent the greatest change after microtubule disruption, and (3) membrane composition had a major influence on the parameters studied. F-actin disruption and cholesterol depletion decreased, and microtubule disruption increased the amount of the caveolar proteins (caveolae). Caveolae presented just an example of the membrane reservoir, and from our findings, we suppose that the perturbations caused were too large to be related to caveolae redistribution alone. The integrity of the cytoskeleton and plasma membrane composition are important factors in the formation of protrusions and in determining the availability and distribution of the membrane reservoir.
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Caveolas/metabolismo , Membrana Celular/metabolismo , Colesterol/metabolismo , Citoesqueleto/metabolismo , Animales , Células CHO , Caveolina 1/metabolismo , Membrana Celular/química , Colesterol/química , Cricetulus , Citoesqueleto/química , HumanosRESUMEN
Controlled poration of lipid membranes is crucial for numerous biomimetic applications such as targeted drug delivery. Although several chemical and physical mechanisms have been proposed for the poration of synthetic membranes, achieving good temporal and spatial control remains a challenge. In this study, we introduce a novel method for membrane poration that utilizes the mechanical shockwave generated by the photo-acoustic effect, which occurs when an optically opaque microparticle is illuminated by a near-infrared laser of optical tweezers. We show that the shockwave effectively porates membranes of giant unilamellar vesicles in close proximity to the microparticle without damaging nearby cells, which is a desirable outcome for potential targeted drug delivery. The poration effect is nonspecific and operates on both liquid and gel phase membranes. Since the photo-acoustic effect can be triggered by standard optical tweezers, this method holds broad applicability in various experimental settings within the field of soft matter research.
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Introduction: Streptococcus agalactiae (Group B Streptococcus, GBS), a Gram-positive commensal in healthy adults, remains a major cause of neonatal infections, usually manifesting as sepsis, meningitis, or pneumonia. Intrapartum antibiotic prophylaxis has greatly reduced the incidence of early-onset disease. However, given the lack of effective measures to prevent the risk of late-onset disease and invasive infections in immunocompromised individuals, more studies investigating the GBS-associated pathogenesis and the interplay between bacteria and host immune system are needed. Methods: Here, we examined the impact of 12 previously genotyped GBS isolates belonging to different serotypes and sequence types on the immune response of THP-1 macrophages. Results: Flow cytometry analysis showed isolate-specific differences in phagocytic uptake, ranging from 10% for isolates of serotype Ib, which possess the virulence factor protein ß, to over 70% for isolates of serotype III. Different isolates also induced differential expression of co-stimulatory molecules and scavenger receptors with colonizing isolates inducing higher expression levels of CD80 and CD86 compared to invasive isolates. In addition, real-time measurements of metabolism revealed that macrophages enhanced both glycolysis and mitochondrial respiration after GBS infection, with isolates of serotype III being the most potent activators of glycolysis and glycolytic ATP production. Macrophages also showed differential resistance to GBS-mediated cell cytotoxicity as measured by LDH release and real-time microscopy. The differences were evident both between serotypes and between isolates obtained from different specimens (colonizing or invasive isolates) demonstrating the higher cytotoxicity of vaginal compared with blood isolates. Conclusions: Thus, the data suggest that GBS isolates differ in their potential to become invasive or remain colonizing. In addition, colonizing isolates appear to be more cytotoxic, whereas invasive isolates appear to exploit macrophages to their advantage, avoiding the immune recognition and antibiotics.
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To visualize fast-moving objects in microfluidic applications, the image acquisition time must be on the order of a microsecond or less. Commercial imaging systems capable of such short exposure times may be too expensive for many research laboratories. We have therefore developed a low-cost stroboscopic illumination for transmitted-light microscopy based on a high-power LED that can be coupled to a standard industrial camera and provides exposure times on the order of 500 ns. The system is designed to be easily mounted on a standard condenser of an inverted microscope. The illumination is controlled by a fast Arduino-compatible Teensy® 4.0 development board, and the illumination parameters can be set from a PC via a graphical user interface written in Python. The system has been successfully used for high-throughput cell phenotyping using deformability cytometry on a Nikon TE2000 microscope, as well as for droplet microfluidic on an old Olympus inverted microscope.
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Metastatic cancer cells can overcome detachment-induced cell death and can proliferate in anchorage-independent conditions. A recent study revealed that a co-treatment with two drugs that interfere with cell metabolism, metformin and 2-deoxy-D-glucose, promotes detachment of viable MDA-MB-231 breast cancer cells. In the present study, we analyzed if these detached viable MDA-MB-231 cells also exhibit other features related to cancer metastatic potential, i.e., if they are softer and more prone to adhere to epithelial cells. The cell mechanics of attached cells and floating cells were analyzed by optical tweezers and cell deformability cytometry, respectively. The adhesion was assessed on a confluent monolayer of HUVEC cells, with MDA-MB-231 cells either in static conditions or in a microfluidic flow. Additionally, to test if adhesion was affected by the state of the epithelial glycocalyx, HUVEC cells were treated with neuraminidase and tunicamycin. It was found that the treated MDA-MB-231 cells were more prone to adhere to HUVEC cells and that they were softer than the control, both in the floating state and after re-seeding to a substrate. The changes in the HUVEC glycocalyx, however, did not increase the adhesion potential of MDA-MB-231.
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We examined keratin aggregate formation and the possible mechanisms involved. With this aim, we observed the effect that different ratios between mutant and wild-type keratins expressed in cultured keratinocytes may have on aggregate formation in vitro, as well as how keratin aggregate formation affects the mechanical properties of cells at the cell cortex. To this end we prepared clones with expression rates as close as possible to 25%, 50% and 100% of the EGFP-K14 proteins (either WT or R125P and V270M mutants). Our results showed that only in the case of the 25% EGFP-K14 R125P mutant significant differences could be seen. Namely, we observed in this case the largest accumulation of keratin aggregates and a significant reduction in cell stiffness. To gain insight into the possible mechanisms behind this observation, we extended our previous mathematical model of keratin dynamics by implementing a more complex reaction network that considers the coexistence of wild-type and mutant keratins in the cell. The new model, consisting of a set of coupled, non-linear, ordinary differential equations, allowed us to draw conclusions regarding the relative amounts of intermediate filaments and aggregates in cells, and suggested that aggregate formation by asymmetric binding between wild-type and mutant keratins could explain the data obtained on cells grown in culture.
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Queratinocitos/metabolismo , Queratinas/química , Proteínas Mutantes/química , Agregado de Proteínas , Línea Celular , Simulación por Computador , Colorantes Fluorescentes/química , Proteínas Fluorescentes Verdes/metabolismo , Humanos , Queratinocitos/efectos de los fármacos , Modelos Biológicos , Inhibidores de Proteasoma/farmacología , Agregado de Proteínas/efectos de los fármacosRESUMEN
We have generated MLi004-A, a new induced pluripotent stem cell (iPSC) line derived from skin fibroblasts of a female patient with recessive dystrophic epidermolysis bullosa (RDEB). This iPSC line may be used as a model system for studies on skin integrity, the extracellular matrix and skin barrier function. The characterization of the MLi004-A cell line consisted of molecular karyotyping, next-generation sequencing of the COL7A1 alleles, pluripotency and differentiation potentials testing by immunofluorescence of associated markers in vitro. The MLi-004A line has been also tested for ability to differentiate into fibroblasts.
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Epidermólisis Ampollosa Distrófica , Células Madre Pluripotentes Inducidas , Colágeno Tipo VII/genética , Epidermólisis Ampollosa Distrófica/genética , Femenino , Fibroblastos , Humanos , Mutación , PielRESUMEN
The cell volume changes induced by hypotonic electrolyte and sucrose solutions were studied in Chinese-hamster-ovary epithelial cells. The effects in the solutions with osmolarities between 32 and 315 mosM/L and distilled water were analyzed using bright-field and fluorescence confocal microscopy. The changes of the cell volume, accompanied by the detachment of cells, the formation of blebs, and the occurrence of almost spherical vesicle-like cells ("cell-vesicles"), showed significant differences in the long-time responses of the cells in the electrolyte solutions compared with the sucrose-containing solutions. A theoretical model based on different permeabilities of ions and sucrose molecules and on the action of Na+/K+-ATPase pumps is applied. It is consistent with the observed temporal behavior of the cells' volume and the occurrence of tension-induced membrane ruptures and explains lower long-time responses of the cells in the sucrose solutions.
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Keratin intermediate filaments are the principal structural element of epithelial cells. Their importance in providing bulk cellular stiffness is well recognized, but their role in the mechanics of cell cortex is less understood. In this study, we therefore compared the cortical stiffness of three keratinocyte lines: primary wild type cells (NHEK2), immortalized wild type cells (NEB1) and immortalized mutant cells (KEB7). The cortical stiffness was measured by lateral indentation of cells with AOD-steered optical tweezers without employing any moving mechanical elements. The method was validated on fixed cells and Cytochalasin-D treated cells to ensure that the observed variations in stiffness within a single cell line were not a consequence of low measurement precision. The measurements of the cortical stiffness showed that primary wild type cells were significantly stiffer than immortalized wild type cells, which was also detected in previous studies of bulk elasticity. In addition, a small difference between the mutant and the wild type cells was detected, showing that mutation of keratin impacts also the cell cortex. Thus, our results indicate that the role of keratins in cortical stiffness is not negligible and call for further investigation of the mechanical interactions between keratins and elements of the cell cortex.
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Citoesqueleto de Actina/metabolismo , Filamentos Intermedios/metabolismo , Queratinocitos/metabolismo , Queratinas/metabolismo , Microtúbulos/metabolismo , Citoesqueleto de Actina/ultraestructura , Línea Celular , Citocalasina D/farmacología , Elasticidad/efectos de los fármacos , Expresión Génica , Dureza/efectos de los fármacos , Humanos , Filamentos Intermedios/ultraestructura , Queratinocitos/efectos de los fármacos , Queratinocitos/ultraestructura , Queratinas/genética , Microtúbulos/ultraestructura , Pinzas Ópticas , Especificidad de ÓrganosRESUMEN
The interest in studying the mechanical and adhesive properties of cells has increased in recent years. The cytoskeleton is known to play a key role in cell mechanics. However, the role of the microtubules in shaping cell mechanics is not yet well understood. We have employed Atomic Force Microscopy (AFM) together with confocal fluorescence microscopy to determine the role of microtubules in cytomechanics of Human Umbilical Vein Endothelial Cells (HUVECs). Additionally, the time variation of the adhesion between tip and cell surface was studied. The disruption of microtubules by exposing the cells to two colchicine concentrations was monitored as a function of time. Already, after 30 min of incubation the cells stiffened, their relaxation times increased (lower fluidity) and the adhesion between tip and cell decreased. This was accompanied by cytoskeletal rearrangements, a reduction in cell area and changes in cell shape. Over the whole experimental time, different behavior for the two used concentrations was found while for the control the values remained stable. This study underlines the role of microtubules in shaping endothelial cell mechanics.
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Adhesión Celular/fisiología , Colchicina/farmacología , Células Endoteliales de la Vena Umbilical Humana/fisiología , Mecanotransducción Celular/fisiología , Microtúbulos/metabolismo , Adhesión Celular/efectos de los fármacos , Técnicas de Cultivo de Célula , Línea Celular , Relación Dosis-Respuesta a Droga , Células Endoteliales de la Vena Umbilical Humana/efectos de los fármacos , Humanos , Microscopía Intravital , Microscopía de Fuerza Atómica , Microscopía Confocal , Microscopía FluorescenteRESUMEN
BACKGROUND: Cell based carriers are increasingly recognized as a good system for cargo delivery to cells. One of the reasons is their biocompatibility and low toxicity compared to artificial systems. Giant plasma membrane vesicles (GPMV) derive from the cell plasma membrane. Thus they offer the closest approximation to it, which makes them good candidates for potential drug delivery systems. To evaluate the applicability of GPMVs as carriers, we analyzed their basic biophysical properties to test their robustness in the face of changeable physiological conditions, as well as their ability to translocate across the membrane into cells. RESULTS: GPMVs formed from human umbilical vein endothelial cells (HUVEC) sustain a drastic osmotic challenge (50-500 mOsmoL/kg) unlike giant unilamelar vesicles (GUVs). In hyper-osmotic solutions the average volume decreases and membrane invaginations form, while in the hypo-osmolar buffer the volume of GPMVs increases and these changes were not reversible. The membranes of flaccid GPMVs started to wrinkle unevenly giving rise to buds after exposure to lipopolysaccharide (LPS). The shape changes in GUVs are reversible in contrast to GPMVs after LPS removal. GPMVs exposed to fluorescent LPS exhibited a signal that remained visible in some GPMVs even after LPS removal, which was never the case with GUVs. Calcein penetrated both into GUVs and GPMVs, however after the removal from the bulk solution some of the GPMVs still exhibited very bright signal, while in GUVs only a weak fluorescent signal was detected. We could also see that practically all GPMVs incorporated dextran initially, but after the dextran solution was changed with the initial non-fluorescent solution it remained only in 20% of them. The majority of HUVEC cells displayed a fluorescent signal after the incubation with GPMVs that contained fluorescently labeled dextran. CONCLUSION: Our findings indicate that GPMVs behave quite differently from artificially made giant phospholipid vesicles and the changes induced by the different treatments we subjected them to are not reversible. We also demonstrate that different substances can be both loaded into them and delivered into cells, so GPMVs may be of potential use as cargo/therapy delivery systems.
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A small library of 17 organoruthenium compounds with the general formula [RuII (fcl)(chel)(L)]n+ (in which fcl=face capping ligand, chel=chelating bidentate ligand, and L=monodentate ligand) were screened for inhibitory activity against cholinesterases and glutathione-S-transferases of human and animal origins. Compounds were selected to include different chelating ligands (i.e., N,N-, N,O-, O,O-, S,O-) and monodentate ligands that can modulate the aquation rate of the metal species. Compounds with a labile ruthenium chloride bond that provided rapid aquation were found to inhibit both sets of enzymes in reversible competitive modes and at pharmaceutically relevant concentrations. When applied at concentrations that completely abolish the activity of human acetylcholinesterase, the lead compound [(η6 -p-cymene)Ru(pyrithionato)Cl] (C1 a) showed no undesirable physiological responses on the neuromuscular system. Finally, C1 a was not cytotoxic against non-transformed cells at pharmaceutically relevant concentrations.
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Inhibidores de la Colinesterasa/farmacología , Complejos de Coordinación/farmacología , Glutatión Transferasa/antagonistas & inhibidores , Profármacos/farmacología , Rutenio/química , Acetilcolinesterasa/metabolismo , Animales , Butirilcolinesterasa/metabolismo , Línea Celular , Inhibidores de la Colinesterasa/síntesis química , Inhibidores de la Colinesterasa/química , Inhibidores de la Colinesterasa/toxicidad , Complejos de Coordinación/síntesis química , Complejos de Coordinación/química , Complejos de Coordinación/toxicidad , Diafragma/efectos de los fármacos , Electrophorus , Caballos , Humanos , Potenciales de la Membrana/efectos de los fármacos , Ratones , Contracción Muscular/efectos de los fármacos , Músculo Esquelético/efectos de los fármacos , Profármacos/síntesis química , Profármacos/química , Profármacos/toxicidad , Bibliotecas de Moléculas PequeñasRESUMEN
The responses of Chinese hamster ovary epithelial cells, caused by the pore-forming agent nystatin, were investigated using brightfield and fluorescence microscopy. Different phenomena, i.e., the detachment of cells, the formation of blebs, the occurrence of "cell-vesicles" and cell ruptures, were observed. These phenomena were compared to those discovered in giant lipid vesicles. A theoretical model, based on the osmotic effects that occur due to the size-discriminating nystatin transmembrane pores in lipid vesicles, was extended with a term that considers the conservation of the electric charge density in order to describe the cell's behavior. The increase of the cellular volume was predicted and correlated with the observed phenomena.