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Synthesizing biomimetic prototissues with predictable physical properties is a promising tool for the study of cellular tissues, as they would enable to test systematically the role of individual physical mechanisms on complex biological processes. The aim of this study is to design a biomimetic cohesive tissue with tunable mechanical properties by the controlled assembly of giant unillamelar vesicles (GUV). GUV-GUV specific adhesion is mediated by the inclusion of the streptavidin-biotin pair, or DNA complementary strands. Using a simple assembly protocol, we are capable of synthesizing vesicle prototissues of spheroidal or sheet-like morphologies, with predictable cell-cell adhesion strengths, typical sizes, and degree of compaction.
Assuntos
Lipossomas Unilamelares , Adesão CelularRESUMO
The airway epithelium represents a fragile environmental interface potentially disturbed by cigarette smoke (CS), the major risk factor for developing chronic obstructive pulmonary disease (COPD). CS leads to bronchial epithelial damage on ciliated, goblet, and club cells, which could involve calcium (Ca2+) signaling. Ca2+ is a key messenger involved in virtually all fundamental physiological functions, including mucus and cytokine secretion, cilia beating, and epithelial repair. In this study, we analyzed Ca2+ signaling in air-liquid interface-reconstituted bronchial epithelium from control subjects and smokers (with and without COPD). We further aimed to determine how smoking impaired Ca2+ signaling. First, we showed that the endoplasmic reticulum (ER) depletion of Ca2+ stores was decreased in patients with COPD and that the Ca2+ influx was decreased in epithelial cells from smokers (regardless of COPD status). In addition, acute CS exposure led to a decrease in ER Ca2+ release, significant in smoker subjects, and to a decrease in Ca2+ influx only in control subjects. Furthermore, the differential expression of 55 genes involved in Ca2+ signaling highlighted that only ORAI3 expression was significantly altered in smokers (regardless of COPD status). Finally, we incubated epithelial cells with an ORAI antagonist (GSK-7975A). GSK-7975A altered Ca2+ influx and ciliary beating, but not mucus and cytokine secretion or epithelial repair, in control subjects. Our data suggest that Ca2+ signaling is impaired in smoker epithelia (regardless of COPD status) and involves ORAI3. Moreover, ORAI3 is additionally involved in ciliary beating.
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Brônquios/citologia , Canais de Cálcio/fisiologia , Cálcio/metabolismo , Doença Pulmonar Obstrutiva Crônica/metabolismo , Mucosa Respiratória/metabolismo , Fumar/metabolismo , Adulto , Idoso , Benzamidas/farmacologia , Brônquios/metabolismo , Canais de Cálcio/biossíntese , Canais de Cálcio/genética , Sinalização do Cálcio , Células Cultivadas , Cílios/efeitos dos fármacos , Cílios/fisiologia , Citocinas/metabolismo , Retículo Endoplasmático/metabolismo , Células Epiteliais/efeitos dos fármacos , Células Epiteliais/metabolismo , Feminino , Regulação da Expressão Gênica , Humanos , Interleucina-8/biossíntese , Masculino , Pessoa de Meia-Idade , Mucina-5AC/biossíntese , Muco/metabolismo , Pirazóis/farmacologia , Mucosa Respiratória/patologia , Transdução de Sinais/fisiologia , Fumaça , FumantesRESUMO
Vegetable oil based hybrid films were developed thanks to a novel solvent- and heating-free method at the air-water interface using silylated castor oil cross-linked via a sol-gel reaction. To understand the mechanism of the hybrid film formation, the reaction kinetics was studied in detail by using complementary techniques: rheology, thermogravimetric analysis, and infrared spectroscopy. The mechanical properties of the final films were investigated using nano-indentation, whereas their structure was studied using a combination of wide-angle X-ray scattering, electron diffraction, and atomic force microscopy. We found that solid and transparent films form in 24 hours and, by changing the silica precursor to castor oil ratio, their mechanical properties are tunable in the MPa-range by about a factor of twenty. In addition to that, a possible optimization of the cross-linking reaction with different catalysts was explored, and finally cytotoxicity tests were performed on fibroblasts proving the absence of film toxicity. The results of this work pave the way to a straightforward synthesis of castor-oil films with tunable mechanical properties: hybrid films cross-linked at the air-water interface combine an easy and cheap spreading protocol with the features of their thermal history optimized for possible future micro/nano drug loading, thus representing excellent candidates for the replacement of non-environmentally friendly petroleum-based materials.
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Several techniques to assemble artificial lipid bilayers involve the zipping of monolayers. Their efficiency is determined by the renewal of the saturated monolayers to be zipped and this proceeds by adsorption of lipids dispersed in oil as aggregates. The size of these lipids aggregates is a key parameter to ensure both the stability of the suspension and a fast release of lipids at the interface. We propose a new method inspired from the solvent-shifting nucleation process allowing to control and tune the lipid aggregates size and that improves the production of artificial membranes. It is simpler and faster than current methods starting from a dry lipid film, which are highly sensitive to environmental conditions. This method opens the route to bilayer production processes with new potentialities in membrane composition.
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Bicamadas Lipídicas/síntese química , Adsorção , Bicamadas Lipídicas/química , Membranas Artificiais , Óleos de Silicone/químicaRESUMO
Sickle cell anemia is a blood disorder, known to affect the microcirculation and is characterized by painful vaso-occlusive crises in deep tissues. During the last three decades, many scenarios based on the enhanced adhesive properties of the membrane of sickle red blood cells have been proposed, all related to a final decrease in vessels lumen by cells accumulation on the vascular walls. Up to now, none of these scenarios considered the possible role played by the geometry of the flow on deposition. The question of the exact locations of occlusive events at the microcirculatory scale remains open. Here, using microfluidic devices where both geometry and oxygen levels can be controlled, we show that the flow of a suspension of sickle red blood cells around an acute corner of a triangular pillar or of a bifurcation, leads to the enhanced deposition and aggregation of cells. Thanks to our devices, we follow the growth of these aggregates in time and show that their length does not depend on oxygenation levels; instead, we find that their morphology changes dramatically to filamentous structures when using autologous plasma as a suspending fluid. We finally discuss the possible role played by such aggregates in vaso-occlusive events.
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Anemia Falciforme/sangue , Eritrócitos/patologia , Técnicas Analíticas Microfluídicas , Anemia Falciforme/patologia , Plaquetas/patologia , Agregação Eritrocítica , Hemoglobinas/metabolismo , Hemólise , Humanos , Leucócitos/patologia , Oxigênio/metabolismoRESUMO
When a straight rod is bent and suddenly released on one end, a burst of dispersive flexural waves propagates down the material as predicted by linear beam theories. However, we show that for ribbons with a longitudinal natural radius of curvature a0, geometrical constraints lead to strain localization which controls the dynamics. This localized region of deformation selects a specific curling deformation front which travels down the ribbon when initially flattened and released. Performing experiments on different ribbons, in air and in water, we show that initially, on length scales on the order of a0, the curling front moves as a power law of time with an exponent ranging from 0.5 to 2 for increasing values of the ribbons' width. At longer time scales, the material wraps itself at a constant speed Vr into a roll of radius R ≠ a0. The relationship between Vr and R is calculated by a balance between kinetic, elastic and gravitational energy and both internal and external powers dissipated. When gravity and drag are negligible, we observe that a0/R reaches a limiting value of 0.48 that we predict by solving the Elastica on the curled ribbon considering the centrifugal forces due to rotation. The solution we propose represents a solitary traveling curvature wave which is reminiscent to propagating instabilities in mechanics.
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Large groups of active cilia collectively beat in a fluid medium as metachronal waves, essential for some microorganisms motility and for flow generation in mucociliary clearance. Several models can predict the emergence of metachronal waves, but what controls the properties of metachronal waves is still unclear. Here, we numerically investigate the respective impacts of active beating and viscous dissipation on the properties of metachronal waves in a collection of oscillators, using a simple model for cilia in the presence of noise on regular lattices in one and two dimensions. We characterize the wave using spatial correlation and the frequency of collective beating. Our results clearly show that the viscosity of the fluid medium does not affect the wavelength; the activity of the cilia does. These numerical results are supported by a dimensional analysis, which shows that the result of wavelength invariance is robust against the model taken for sustained beating and the structure of hydrodynamic coupling. Interestingly, the enhancement of cilia activity increases the wavelength and decreases the beating frequency, keeping the wave velocity almost unchanged. These results might have significance in understanding paramecium locomotion and mucociliary clearance diseases.
Assuntos
Cílios , Hidrodinâmica , Modelos Biológicos , Cílios/fisiologia , ViscosidadeRESUMO
The culminating step of the intraerythrocytic development of Plasmodium falciparum, the causative agent of malaria, is the spectacular release of multiple invasive merozoites on rupture of the infected erythrocyte membrane. This work reports for the first time that the whole process, taking place in time scales as short as 400 milliseconds, is the result of an elastic instability of the infected erythrocyte membrane. Using high-speed differential interference contrast (DIC) video microscopy and epifluorescence, we demonstrate that the release occurs in 3 main steps after osmotic swelling of the infected erythrocyte: a pore opens in ~ 100 milliseconds, ejecting 1-2 merozoites, an outward curling of the erythrocyte membrane is then observed, ending with a fast eversion of the infected erythrocyte membrane, pushing the parasites forward. It is noteworthy that this last step shows slight differences when infected erythrocytes are adhering. We rationalize our observations by considering that during the parasite development, the infected erythrocyte membrane acquires a spontaneous curvature and we present a subsequent model describing the dynamics of the curling rim. Our results show that sequential erythrocyte membrane curling and eversion is necessary for the parasite efficient angular dispersion and might be biologically essential for fast and numerous invasions of new erythrocytes.
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Eritrócitos/parasitologia , Malária Falciparum/parasitologia , Merozoítos/crescimento & desenvolvimento , Plasmodium falciparum/crescimento & desenvolvimento , Animais , Forma Celular/fisiologia , Elasticidade/fisiologia , Membrana Eritrocítica/parasitologia , Eritrócitos/citologia , Humanos , Técnicas In Vitro , Merozoítos/metabolismo , Pressão Osmótica/fisiologia , Elastase Pancreática/metabolismo , Plasmodium falciparum/metabolismoRESUMO
Precisely how malaria parasites exit from infected red blood cells to further spread the disease remains poorly understood. It has been shown recently, however, that these parasites exploit the elasticity of the cell membrane to enable their egress. Based on this work, showing that parasites modify the membrane's spontaneous curvature, initiating pore opening and outward membrane curling, we develop a model of the dynamics of the red blood cell membrane leading to complete parasite egress. As a result of the three-dimensional, axisymmetric nature of the problem, we find that the membrane dynamics involve two modes of elastic-energy release: 1), at short times after pore opening, the free edge of the membrane curls into a toroidal rim attached to a membrane cap of roughly fixed radius; and 2), at longer times, the rim radius is fixed, and lipids in the cap flow into the rim. We compare our model with the experimental data of Abkarian and co-workers and obtain an estimate of the induced spontaneous curvature and the membrane viscosity, which control the timescale of parasite release. Finally, eversion of the membrane cap, which liberates the remaining parasites, is driven by the spontaneous curvature and is found to be associated with a breaking of the axisymmetry of the membrane.
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Membrana Eritrocítica/metabolismo , Membrana Eritrocítica/parasitologia , Malária/parasitologia , Modelos Biológicos , Plasmodium falciparum/crescimento & desenvolvimento , Fenômenos Biomecânicos , Merozoítos/crescimento & desenvolvimento , Merozoítos/fisiologia , Plasmodium falciparum/fisiologiaRESUMO
A new methodology for an antibacterial assay based on capillary electrophoresis with multiple UV detection points has been proposed. The possible antibacterial activity of cationic molecules on bacteria (Gram-positive and Gram-negative) is studied by detecting the bacteria before, during, and after their meeting with the cationic antibacterial compound. For that, a UV area imaging detector having two loops and three detection windows was used with a 95 cm ×100 µm i.d. capillary. In the antibacterial assay, the bacteria (negatively charged) and the cationic molecules were injected separately from each end of the capillary. The bacteria were mobilized by anionic ITP mode while cationic molecules migrate in the opposite direction under conditions close to CZE. The cationic molecules were injected into the capillary as a broad band (injected volume about 16% of the volume of the capillary) to prevent dilution of the sample during the electrophoretic process. Bacteriolytic activity, as well as strong interactions between the small antibacterial molecules and the bacteria, can be investigated within a few minutes. The assay was used to study the antibacterial activity of dendrigraft poly-L-lysines on Micrococcus luteus and Erwinia carotovora. Because dendrigraft poly-L-lysines are nonimmunogenic and have low toxicity, this new class of dendritic biomacromolecules is very promising for antibacterial applications.
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Antibacterianos/farmacologia , Eletroforese Capilar/métodos , Espectrofotometria Ultravioleta/métodos , Antibacterianos/química , Benzoatos/química , Benzilaminas/química , Micrococcus luteus/química , Micrococcus luteus/efeitos dos fármacos , Pectobacterium carotovorum/química , Pectobacterium carotovorum/efeitos dos fármacos , Polilisina/química , Polilisina/farmacologiaRESUMO
Mucus is a viscoelastic aqueous fluid that participates in the protective barrier of many mammals' epithelia. In the airways, together with cilia beating, mucus rheological properties are crucial for lung mucociliary function, and, when impaired, potentially participate in the onset and progression of chronic obstructive pulmonary disease (COPD). Samples of human mucus collected in vivo are inherently contaminated and are thus poorly characterized. Human bronchial epithelium (HBE) cultures, differentiated from primary cells at an air-liquid interface, are highly reliable models to assess non-contaminated mucus. In this paper, the viscoelastic properties of HBE mucus derived from healthy subjects, patients with COPD and from smokers are measured. Hallmarks of shear-thinning and elasticity are obtained at the macroscale, whereas at the microscale mucus appears as a heterogeneous medium showing an almost Newtonian behaviour in some extended regions and an elastic behaviour close to boundaries. In addition, we developed an original method to probe mucus adhesion at the microscopic scale using optical tweezers. The measured adhesion forces and the comparison with mucus-simulants rheology as well as mucus imaging collectively support a structure composed of a network of elastic adhesive filaments with a large mesh size, embedded in a very soft gel.
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Background: Chronic Obstructive Pulmonary Disease (COPD), a major cause of mortality and disability, is a complex disease with heterogeneous and ill-understood biological mechanisms. Human induced pluripotent stem cells (hiPSCs) are a promising tool to model human disease, including the impact of genetic susceptibility. Methods: We developed a simple and reliable method for reprogramming peripheral blood mononuclear cells into hiPSCs and to differentiate them into air−liquid interface bronchial epithelium within 45 days. Importantly, this method does not involve any cell sorting step. We reprogrammed blood cells from one healthy control and three patients with very severe COPD. Results: The mean cell purity at the definitive endoderm and ventral anterior foregut endoderm (vAFE) stages was >80%, assessed by quantifying C-X-C Motif Chemokine Receptor 4/SRY-Box Transcription Factor 17 (CXCR4/SOX17) and NK2 Homeobox 1 (NKX2.1) expression, respectively. vAFE cells from all four hiPSC lines differentiated into bronchial epithelium in air−liquid interface conditions, with large zones covered by beating ciliated, basal, goblets, club cells and neuroendocrine cells, as found in vivo. The hiPSC-derived airway epithelium (iALI) from patients with very severe COPD and from the healthy control were undistinguishable. Conclusions: iALI bronchial epithelium is ready for better understanding lung disease pathogenesis and accelerating drug discovery.
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Células-Tronco Pluripotentes Induzidas , Doença Pulmonar Obstrutiva Crônica , Epitélio/metabolismo , Humanos , Leucócitos Mononucleares/patologia , Doença Pulmonar Obstrutiva Crônica/metabolismo , Mucosa Respiratória/patologiaRESUMO
Cilia are elastic hairlike protuberances of the cell membrane found in various unicellular organisms and in several tissues of most living organisms. In some tissues such as the airway tissues of the lung, the coordinated beating of cilia induces a fluid flow of crucial importance as it allows the continuous cleaning of our bronchia, known as mucociliary clearance. While most of the models addressing the question of collective dynamics and metachronal wave consider homogeneous carpets of cilia, experimental observations rather show that cilia clusters are heterogeneously distributed over the tissue surface. The purpose of this paper is to investigate the role of spatial heterogeneity on the coherent beating of cilia using a very simple one-dimensional model for cilia known as the rower model. We systematically study systems consisting of a few rowers to hundreds of rowers and we investigate the conditions for the emergence of collective beating. When considering a small number of rowers, a phase drift occurs, hence, a bifurcation in beating frequency is observed as the distance between rower clusters is changed. In the case of many rowers, a distribution of frequencies is observed. We found in particular the pattern of the patchy structure that shows the best robustness in collective beating behavior, as the density of cilia is varied over a wide range.
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Cílios/fisiologia , Modelos Biológicos , Animais , Brônquios/citologia , Brônquios/fisiologia , Movimento Celular , Elasticidade , Humanos , Hidrodinâmica , Depuração Mucociliar/fisiologia , PeriodicidadeRESUMO
We show the existence of a flow at the surface of strongly adhering giant lipid vesicles submitted to an external shear flow. The surface flow is divided into two symmetric quadrants and presents two stagnation points (SP) on each side of the vesicle meridian plane. The position of these stagnation points depends strongly on the adhesion strength, characterized by the ratio of the contact zone diameter to the vesicle diameter. Contrary to the case of non-adhesive vesicles, streamlines do not lie in the shear plane. By avoiding the motionless contact zone, streamlines result in three-dimensional paths, strongly asymmetric away from the SP. Additional shearing dissipation may occur on the membrane surface as we observed that the mean rotational velocity of the membrane increases towards the vesicle SP, and is mainly determined by the adhesion induced vesicle shape.
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We describe a model of cytoskeletal mechanics based on the force-induced conformational change of protein cross-links in a stressed polymer network. Slow deformation of simulated networks containing cross-links that undergo repeated, serial domain unfolding leads to an unusual state-with many cross-links accumulating near the critical force for further unfolding. This state is robust to thermalization and does not occur in similar protein unbinding based simulations. Moreover, we note that the unusual configuration of near-critical protein cross-links in the fragile state provides a physical mechanism for the chemical transduction of cell-level mechanical strain and extra-cellular matrix stiffness.
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Proteínas do Citoesqueleto/química , Proteínas do Citoesqueleto/fisiologia , Citoesqueleto/química , Citoesqueleto/fisiologia , Mecanotransdução Celular/fisiologia , Modelos Biológicos , Simulação por Computador , Elasticidade , Modelos Químicos , Modelos Moleculares , Movimento (Física) , Dobramento de Proteína , Estresse MecânicoRESUMO
Morphological transformations of living cells, such as shape adaptation to external stimuli, blebbing, invagination, or tethering, result from an intricate interplay between the plasma membrane and its underlying cytoskeleton, where molecular motors generate forces. Cellular complexity defies a clear identification of the competing processes that lead to such a rich phenomenology. In a synthetic biology approach, designing a cell-like model assembled from a minimal set of purified building blocks would allow the control of all relevant parameters. We reconstruct actomyosin vesicles in which the coupling of the cytoskeleton to the membrane, the topology of the cytoskeletal network, and the contractile activity can all be precisely controlled and tuned. We demonstrate that tension generation of an encapsulated active actomyosin network suffices for global shape transformation of cell-sized lipid vesicles, which are reminiscent of morphological adaptations in living cells. The observed polymorphism of our cell-like model, such as blebbing, tether extrusion, or faceted shapes, can be qualitatively explained by the protein concentration dependencies and a force balance, taking into account the membrane tension, the density of anchoring points between the membrane and the actin network, and the forces exerted by molecular motors in the actin network. The identification of the physical mechanisms for shape transformations of active cytoskeletal vesicles sets a conceptual and quantitative benchmark for the further exploration of the adaptation mechanisms of cells.
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Citoesqueleto de Actina/química , Actinas/química , Citoesqueleto/química , Proteínas Motores Moleculares/química , Actinas/metabolismo , Actomiosina/química , Biomimética , Membrana Celular/química , Microtúbulos/química , Contração Muscular/fisiologiaRESUMO
We use the random phase approximation to compute the structure factor S(q) of a solution of chains interacting through a soft and short range repulsive potential V. Above a threshold polymer concentration, whose magnitude is essentially controlled by the range of the potential, S(q) exhibits a peak whose position depends on the concentration. We take advantage of the close analogy between polymers and wormlike micelles and apply our model, using a Gaussian function for V, to quantitatively analyze experimental small angle neutron scattering profiles of solutions of hairy wormlike micelles. These samples, which consist in surfactant self-assembled flexible cylinders decorated by amphiphilic copolymer, provide indeed an appropriate experimental model system to study the structure of sterically interacting polymer solutions.
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The results of mechanical measurements on single cultured epithelial cells using both magnetic twisting cytometry (MTC) and laser tracking microrheology (LTM) are described. Our unique approach uses laser deflection for high-performance tracking of cell-adhered magnetic beads either in response to an oscillatory magnetic torque (MTC) or due to random Brownian or ATP-dependent forces (LTM). This approach is well suited for accurately determining the rheology of single cells, the study of temporal and cell-to-cell variations in the MTC signal amplitude, and assessing the statistical character of the tracers' random motion in detail. The temporal variation of the MTC rocking amplitude is surprisingly large and manifests as a frequency-independent multiplicative factor having a 1/f spectrum in living cells, which disappears upon ATP depletion. In the epithelial cells we study, random bead position fluctuations are Gaussian to the limits of detection both in the Brownian and ATP-dependent cases, unlike earlier studies on other cell types.
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Biofísica/métodos , Biologia Celular , Reologia/métodos , Trifosfato de Adenosina/química , Trifosfato de Adenosina/metabolismo , Animais , Adesão Celular , Linhagem Celular Tumoral , Chlorocebus aethiops , Células Epiteliais/metabolismo , Lasers , Magnetismo , Camundongos , Células NIH 3T3 , Distribuição Normal , Oscilometria , Fagocitose , Transdução de Sinais , Fatores de TempoRESUMO
Although actin and myosin are important contributors to cell-force generation, shape change, and motility, their contributions to cell stiffness and frequency-dependent rheology have not been conclusively determined. We apply several pharmacological interventions to cultured epithelial cells to elucidate the roles of actin and myosin in the mechanical response of cells and intracellular fluctuations. A suite of different methods is used to separately examine the mechanics of the deep cell interior and cortex, in response to depletion of intracellular ATP, depolymerization of F-actin, and inhibition of myosin II. Comparison of these results shows that F-actin plays a significant role in the mechanics of the cortical region of epithelial cells, but its disruption has no discernable effect on the rheology of the deeper interior. Moreover, we find that myosins do not contribute significantly to the rheology or ATP-dependent, non-Brownian motion in the cell interior. Finally, we investigate the broad distribution of apparent stiffness values reported by some microrheology methods, which are not observed with two-point microrheology. Based on our findings and a simple model, we conclude that heterogeneity of the tracer-cytoskeleton contacts, rather than the network itself, can explain the broad distribution of apparent stiffnesses.