RESUMEN
The understanding of endothelium-extracellular matrix interactions during the initiation of new blood vessels is of great medical importance; however, the mechanobiological principles governing endothelial protrusive behaviours in 3D microtopographies remain imperfectly understood. In blood capillaries submitted to angiogenic factors (such as vascular endothelial growth factor, VEGF), endothelial cells can transiently transdifferentiate in filopodia-rich cells, named tip cells, from which angiogenesis processes are locally initiated. This protrusive state based on filopodia dynamics contrasts with the lamellipodia-based endothelial cell migration on 2D substrates. Using two-photon polymerization, we generated 3D microstructures triggering endothelial phenotypes evocative of tip cell behaviour. Hexagonal lattices on pillars ("open"), but not "closed" hexagonal lattices, induced engagement from the endothelial monolayer with the generation of numerous filopodia. The development of image analysis tools for filopodia tracking allowed to probe the influence of the microtopography (pore size, regular vs. elongated structures, role of the pillars) on orientations, engagement and filopodia dynamics, and to identify MLCK (myosin light-chain kinase) as a key player for filopodia-based protrusive mode. Importantly, these events occurred independently of VEGF treatment, suggesting that the observed phenotype was induced through microtopography. These microstructures are proposed as a model research tool for understanding endothelial cell behaviour in 3D fibrillary networks.
Asunto(s)
Células Endoteliales/citología , Quinasa de Cadena Ligera de Miosina/metabolismo , Seudópodos/metabolismo , Células Endoteliales/metabolismo , Células Endoteliales de la Vena Umbilical Humana , Humanos , Mecanotransducción Celular , Neovascularización Fisiológica , Andamios del TejidoRESUMEN
Epithelial-mesenchymal transition (EMT) is important for the initial steps of metastasis. Although it is well accepted that the nucleoside diphosphate kinase NME1 is a metastasis suppressor, its effect on EMT remains poorly documented, as does that of its closely related isoform, NME2. Here, by using gene silencing, inactivation and overexpression strategies in a variety of cellular models of cancer, we show that NME1 is a powerful inhibitor of EMT. Genetic manipulation of NME2, by contrast, had no effect on the EMT phenotype of cancer cells, indicating a specific function of NME1 in EMT regulation. Loss of NME1 in epithelial cancer cells resulted in a hybrid phenotype intermediate between epithelial and mesenchymal cells, which is known to be associated with cells with a highly metastatic character. Conversely, overexpression of NME1 in mesenchymal cancer cells resulted in a more epithelial phenotype. We found that NME1 expression was negatively associated with EMT markers in many human cancers and was reduced in human breast tumor cell lines with the aggressive 'triple-negative' phenotype when compared to human breast tumor cell lines positive for estrogen receptor. We show that NME1, but not NME2, is an inhibitor of essential concerted intracellular signaling pathways involved in inducing EMT, including the AKT and MAPK (ERK, p38, and JNK) pathways. Additionally, NME1 depletion considerably altered the distribution of E-cadherin, a gatekeeper of the epithelial phenotype, shifting it from the plasma membrane to the cytosol and resulting in less E-cadherin on the cell surface than in control cells. Functional aggregation and dispersion assays demonstrated that inactivation of NME1 decreases E-cadherin-mediated cell-cell adhesion. We conclude that NME1, but not NME2, acts specifically to inhibit EMT and prevent the earliest stages of metastasis.
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Transición Epitelial-Mesenquimal , Nucleósido Difosfato Quinasas NM23/metabolismo , Animales , Cadherinas/metabolismo , Adhesión Celular , Línea Celular Tumoral , Femenino , Edición Génica , Humanos , Sistema de Señalización de MAP Quinasas , Ratones Desnudos , Neoplasias de la Mama Triple Negativas/metabolismoRESUMEN
Polycystins 1 and 2, which are mutated in Autosomal Polycystic Kidney Disease, are involved in mechanotransduction through various mechanisms. In renal cells, polycystins not only have an important mechanotransductive role in primary cilia but are also present in intercellular contacts but their role there remains unclear. Here, we address the hypothesis that polycystins are involved in mechanotransduction via intercellular junctions, which would be expected to have consequences on tissue organization. We focused on the role of polycystin 2, which could be involved in mechanical organization at junctions either by its channel activity or by the direct recruitment of cytoskeleton components such as the F-actin cross-linker α-actinin. After mechanical stimulation of intercellular junctions in MDCK renal epithelial cells, α-actinin is rapidly recruited but this is inhibited upon overexpression of PC2 or the D509V mutant that lacks channel activity, and is also decreased upon PC2 silencing. This suggests that a precise dosage of PC2 is necessary for an adequate mechanosensitive α-actinin recruitment at junctions. At the multicellular level, a change in PC2 expression was associated with changes in velocity in confluent epithelia and during wound healing together with a loss of orientation. This study suggests that the mechanosensitive regulation of cytoskeleton by polycystins in intercellular contacts may be important in the context of ADPKD.
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Actinina/metabolismo , Uniones Intercelulares/metabolismo , Mecanotransducción Celular , Canales Catiónicos TRPP/metabolismo , Animales , Calcio/metabolismo , Movimiento Celular , Perros , Humanos , Células de Riñón Canino Madin Darby , Estrés MecánicoRESUMEN
Kidney tubular cells are submitted to two distinct mechanical forces generated by the urine flow: shear stress and hydrostatic pressure. In addition, the mechanical properties of the surrounding extracellular matrix modulate tubule deformation under constraints. These mechanical factors likely play a role in the pathophysiology of kidney diseases as exemplified by autosomal dominant polycystic kidney disease, in which pressure, flow and matrix stiffness have been proposed to modulate the cystic dilation of tubules with PKD1 mutations. The lack of in vitro systems recapitulating the mechanical environment of kidney tubules impedes our ability to dissect the role of these mechanical factors. Here we describe a perfused kidney-on-chip with tunable extracellular matrix mechanical properties and hydrodynamic constraints, that allows a decoupling of shear stress and flow. We used this system to dissect how these mechanical cues affect Pkd1 -/- tubule dilation. Our results show two distinct mechanisms leading to tubular dilation. For PCT cells (proximal tubule), overproliferation mechanically leads to tubular dilation, regardless of the mechanical context. For mIMCD-3 cells (collecting duct), tube dilation is associated with a squamous cell morphology but not with overproliferation and is highly sensitive to extracellular matrix properties and hydrodynamic constraints. Surprisingly, flow alone suppressed Pkd1 -/- mIMCD-3 tubule dilation observed in static conditions, while the addition of luminal pressure restored it. Our in vitro model emulating nephron geometrical and mechanical organization sheds light on the roles of mechanical constraints in ADPKD and demonstrates the importance of controlling intraluminal pressure in kidney tubule models.
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The organ-on-chip model offers versatility and modularity of in vitro models while approaching the biological fidelity of in vivo models. We propose a method to build a perfusable kidney-on-chip aiming at reproducing key features of the densely packed segments of nephrons in vitro; such as their geometry, their extracellular matrix, and their mechanical properties. The core of the chip is made of parallel tubular channels molded into collagen I that are as small as 80 µm in diameter and as close as 100 µm apart. These channels can further be coated with basement membrane components and seeded by perfusion of a suspension of cells originating from a given segment of the nephron. We optimized the design of our microfluidic device to achieve high reproducibility regarding the seeding density of the channels and high fluidic control of the channels. This chip was designed as a versatile tool to study nephropathies in general, contributing to building ever better in vitro models. It could be particularly interesting for pathologies such as polycystic kidney diseases where mechanotransduction of the cells and their interaction with adjacent extracellular matrix and nephrons may play a key role.
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Enfermedades Renales , Mecanotransducción Celular , Humanos , Reproducibilidad de los Resultados , Riñón , Nefronas , Dispositivos Laboratorio en un ChipRESUMEN
E-cadherin plays a key role at adherens junctions between epithelial cells, but the mechanisms controlling its assembly, maintenance, and dissociation from junctions remain poorly understood. In particular, it is not known to what extent the number of E-cadherins engaged at junctions is regulated by endocytosis, or by dissociation of adhesive bonds and redistribution within the membrane from a pool of diffusive cadherins. To determine whether cadherin levels at mature junctions are regulated by endocytosis or dissociation and membrane diffusion, the dynamics of E-cadherin were quantitatively analyzed by a new approach combining 2-photon fluorescence recovery after photobleaching (FRAP) and fast 3D wide-field fluorescence microscopy. Image analysis of fluorescence recovery indicates that most E-cadherin did not diffuse in the membrane along mature junctions, but followed a first order turn-over process that was rate-limited by endocytosis. In confluent cultures of MCF7 or MDCK cells, stably expressed EGFP-E-cadherin was rapidly recycled with spatially uniform kinetics (50 s in MCF7 and 4 min in MDCK). In addition, when endocytosis was pharmacologically blocked by dynasore or MiTMAB, no fluorescence recovery was observed, suggesting that no endocytosis-independent membrane redistribution was occurring. Our data show that membrane redistribution of E-cadherin molecules engaged in mature junctions requires endocytosis and subsequent exocytosis, and lead to the notion that E-cadherins engaged at junctions do not directly revert to free membrane diffusion. Our results point to the possibility that a direct mechanical coupling between endocytosis efficiency and cadherin-mediated forces at junctions could help to regulate intercellular adhesion and locally stabilize epithelia.
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Uniones Adherentes/metabolismo , Cadherinas/metabolismo , Endocitosis , Animales , Cadherinas/genética , Línea Celular , Perros , Recuperación de Fluorescencia tras Fotoblanqueo , Humanos , Factores de TiempoRESUMEN
Dynamics is the essence of life, from the most macroscopic scale of evolution theories or ecology, down to the submicroscopic scale of the molecular mechanisms underlying for instance the activity of enzymes on their chemical target or their mechanical work in our muscles. Thanks to new methods, most phenomena in cell biology must now be reported not only in terms of their biochemistry, but also with a description of their geometric and temporal characteristics using quantitative imaging. This is leading to a dramatic accumulation of data, with little understanding of its deep physiological significance. Based on recent results, we raise here a few questions regarding the possible role of time as a key determinant of the metabolic energy budget, that impacts living cells from the organisation of macromolecular assemblies up to the morphogenesis and stabilisation of tissues.
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Fenómenos Fisiológicos Celulares , Tiempo , Animales , Ciclo Celular , Forma de la Célula , Células/metabolismo , Células/ultraestructura , Citoplasma/metabolismo , Citoplasma/ultraestructura , Citoesqueleto/ultraestructura , Metabolismo Energético , Recuperación de Fluorescencia tras Fotoblanqueo , Colorantes Fluorescentes/análisis , Humanos , Sustancias Macromoleculares , Microscopía Electrónica , Microscopía Fluorescente , Modelos Biológicos , Morfogénesis , Proteínas/análisisRESUMEN
Skin pigmentation is dependent on cellular processes including melanosome biogenesis, transport, maturation and transfer to keratinocytes. However, how the cells finely control these processes in space and time to ensure proper pigmentation remains unclear. Here, we show that a component of the cytoplasmic dynein complex, Dynlt3, is required for efficient melanosome transport, acidity and transfer. In Mus musculus melanocytes with decreased levels of Dynlt3, pigmented melanosomes undergo a more directional motion, leading to their peripheral location in the cell. Stage IV melanosomes are more acidic, but still heavily pigmented, resulting in a less efficient melanosome transfer. Finally, the level of Dynlt3 is dependent on ß-catenin activity, revealing a function of the Wnt/ß-catenin signalling pathway during melanocyte and skin pigmentation, by coupling the transport, positioning and acidity of melanosomes required for their transfer.
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Dineínas/genética , Melanocitos/metabolismo , Melanosomas/fisiología , Animales , Dineínas/metabolismo , Masculino , Ratones , Ratones Endogámicos C57BL , Pigmentación de la PielRESUMEN
Autosomal Dominant Polycystic Kidney Disease (ADPKD) is a major renal pathology provoked by the deletion of PKD1 or PKD2 genes leading to local renal tubule dilation followed by the formation of numerous cysts, ending up with renal failure in adulthood. In vivo, renal tubules are tightly packed, so that dilating tubules and expanding cysts may have mechanical influence on adjacent tubules. To decipher the role of this coupling between adjacent tubules, we developed a kidney-on-chip reproducing parallel networks of tightly packed tubes. This original microdevice is composed of cylindrical hollow tubes of physiological dimensions, parallel and closely packed with 100-200 µm spacing, embedded in a collagen I matrix. These multitubular systems were properly colonized by different types of renal cells with long-term survival, up to 2 months. While no significant tube dilation over time was observed with Madin-Darby Canine Kidney (MDCK) cells, wild-type mouse proximal tubule (PCT) cells, or with PCT Pkd1 +/- cells (with only one functional Pkd1 allele), we observed a typical 1.5-fold increase in tube diameter with isogenic PCT Pkd1 -/- cells, an ADPKD cellular model. This tube dilation was associated with an increased cell proliferation, as well as a decrease in F-actin stress fibers density along the tube axis. With this kidney-on-chip model, we also observed that for larger tube spacing, PCT Pkd1 -/- tube deformations were not spatially correlated with adjacent tubes whereas for shorter spacing, tube deformations were increased between adjacent tubes. Our device reveals the interplay between tightly packed renal tubes, constituting a pioneering tool well-adapted to further study kidney pathophysiology.
RESUMEN
Transient receptor potential (TRP) channels form a family of polymodal cation channels gated by thermal, mechanical, or chemical stimuli, with many of them involved in the control of proliferation, apoptosis, or cell cycle. From an evolutionary point of view, TRP family is characterized by high conservation of duplicated genes originating from whole-genome duplication at the onset of vertebrates. The conservation of such "ohnolog" genes is theoretically linked to an increased probability of generating phenotypes deleterious for the organism upon gene mutation. We aimed to test experimentally the hypothesis that TRP mutations, in particular gain-of-function, could be involved in the generation of deleterious phenotypes involved in cancer, such as gain of invasiveness. Indeed, a number of TRP channels have been linked to cancer progression, and exhibit changes in expression levels in various types of cancers. However, TRP mutations in cancer have been poorly documented. We focused on 2 TRPV family members, TRPV4 and TRPV6, and studied the effect of putative gain-of-function mutations on invasiveness properties. TRPV channels have a C-terminal calmodulin-binding domain (CaMBD) that has important functions for regulating protein function, through different mechanisms depending on the channel (channel inactivation/potentiation, cytoskeleton regulation). We studied the effect of mutations mimicking constitutive phosphorylation in TRPV4 and TRPV6 CaMBDs: TRPV4 S823D, S824D and T813D, TRPV6 S691D, S692D and T702. We found that most of these mutants induced a strong gain of invasiveness of colon adenocarcinoma SW480 cells, both for TRPV4 and TRPV6. While increased invasion with TRPV6 S692D and T702D mutants was correlated to increased mutant channel activity, it was not the case for TRPV4 mutants, suggesting different mechanisms with the same global effect of gain in deleterious phenotype. This highlights the potential importance to search for TRP mutations involved in cancer.
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Adenocarcinoma/metabolismo , Neoplasias del Colon/metabolismo , Mutación/genética , Canales Catiónicos TRPV/genética , Canales Catiónicos TRPV/metabolismo , Adenocarcinoma/genética , Línea Celular Tumoral , Neoplasias del Colon/genética , Electrofisiología , Mutación con Ganancia de Función , Humanos , Unión ProteicaRESUMEN
Cells are able to develop various types of membrane protrusions that modulate their adhesive, migratory, or functional properties. However, their ability to form basal protrusions, particularly in the context of epithelial sheets, is not widely characterized. The authors built hexagonal lattices to probe systematically the microtopography-induced formation of epithelial cell protrusions. Lattices of hexagons of various sizes (from 1.5 to 19 µm) and 5-10 µm height were generated by two-photon photopolymerization in NOA61 or poly(ethylene glycol) diacrylate derivatives. The authors found that cells generated numerous, extensive, and deep basal protrusions for hexagons inferior to cell size (3-10 µm) while maintaining a continuous epithelial layer above structures. They characterized the kinetics of protrusion formation depending on scaffold geometry and size. The reported formation of extensive protrusions in 3D microtopography could be beneficial to develop new biomaterials with increased adhesive properties or to improve tissue engineering.
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Adhesión Celular , Membrana Celular/metabolismo , Extensiones de la Superficie Celular/ultraestructura , Células Epiteliales/fisiología , Células Epiteliales/ultraestructura , Propiedades de Superficie , Animales , Perros , Imagenología Tridimensional , Células de Riñón Canino Madin Darby , Microscopía Confocal , Microscopía FluorescenteRESUMEN
Multicellular tubes are structures ubiquitously found during development and in adult organisms. Their topologies (diameter, direction or branching), together with their mechanical characteristics, play fundamental roles in organ function and in the emergence of pathologies. In tubes of micrometric range diameters, typically found in the vascular system, renal tubules or excretory ducts, cells are submitted to a strong curvature and confinement effects in addition to flow. Then, small tubes with change in diameter are submitted to a local gradient of shear stress and curvature, which may lead to complex mechanotransduction responses along tubes, and may be involved in the onset or propagation of cystic or obstructive pathologies. We describe here a simple method to build a microfluidic device that integrates cylindrical channels with changes in diameter that mimic in vivo tube geometries. This microfabrication approach is based on molding of etched tungsten wires, which can achieve on a flexible way any change in diameter in a polydimethylsiloxane (PDMS) microdevice. The interest of this biomimetic multitube system has been evidenced by reproducing renal tubules on chip. In particular, renal cell lines were successfully seeded and grown in PDMS circular tubes with a transition between 80 µm and 50 µm diameters. Thanks to this biomimetic platform, the effect of the tube curvature has been investigated especially regarding cell morphology and orientation. The effect of shear stress on confluent cells has also been assessed simultaneously in both parts of tubes. It is thus possible to study interconnected cell response to differential constraints which is of central importance when mimicking tubes present in the organism.
RESUMEN
We report here a simple yet robust transient compartmentalization system for microfluidic platforms. Cylindrical microfilaments made of commercially available fishing lines are embedded in a microfluidic chamber and employed as removable walls, dividing the chamber into several compartments. These partitions allow tight sealing for hours, and can be removed at any time by longitudinal sliding with minimal hydrodynamic perturbation. This allows the easy implementation of various functions, previously impossible or requiring more complex instrumentation. In this study, we demonstrate the applications of our strategy, firstly to trigger chemical diffusion, then to make surface co-coating or cell co-culture on a two-dimensional substrate, and finally to form multiple cell-laden hydrogel compartments for three-dimensional cell co-culture in a microfluidic device. This technology provides easy and low-cost solutions, without the use of pneumatic valves or external equipment, for constructing well-controlled microenvironments for biochemical and cellular assays.
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Citoesqueleto de Actina/metabolismo , Técnicas de Cultivo de Célula/instrumentación , Dispositivos Laboratorio en un Chip , Animales , Hidrogeles/química , Ratones , Neuroglía/citología , Neuronas/citologíaRESUMEN
In the natural and technological world, multi-agent systems strongly depend on how the interactions are ruled between their individual components, and the proper control of time-scales and synchronization is a key issue. This certainly applies to living tissues when multicellular assemblies such as epithelial cells achieve complex morphogenetic processes. In epithelia, because cells are known to individually generate actomyosin contractile stress, each individual intercellular adhesive junction line is subjected to the opposed stresses independently generated by its two partner cells. Contact lines should thus move unless their two partner cells mechanically match. The geometric homeostasis of mature epithelia observed at short enough time-scale thus raises the problem to understand how cells, if considered as noisy individual actuators, do adapt across individual intercellular contacts to locally balance their time-average contractile stress. Structural components of adherens junctions, cytoskeleton (F-actin) and homophilic bonds (E-cadherin) are quickly renewed at steady-state. These turnovers, if they depend on forces exerted at contacts, may play a key role in the mechanical adaptation of epithelia. Here we focus on E-cadherin as a force transducer, and we study the local regulation and the mechanosensitivity of its turnover in junctions. We show that E-cadherin turnover rates match remarkably well on either side of mature intercellular contacts, despite the fact that they exhibit large fluctuations in time and variations from one junction to another. Using local mechanical and biochemical perturbations, we find faster turnover rates with increased tension, and asymmetric rates at unbalanced junctions. Together, the observations that E-cadherin turnover, and its local symmetry or asymmetry at each side of the junction, are mechanosensitive, support the hypothesis that E-cadherin turnover could be involved in mechanical homeostasis of epithelia.
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Uniones Adherentes/metabolismo , Cadherinas/metabolismo , Animales , Cadherinas/genética , Citoesqueleto/metabolismo , Perros , Endocitosis , Recuperación de Fluorescencia tras Fotoblanqueo , Células de Riñón Canino Madin Darby , Microscopía Fluorescente , Proteínas Recombinantes de Fusión/biosíntesis , Proteínas Recombinantes de Fusión/genética , Factores de TiempoRESUMEN
Homeostasis of adherens junctions is achieved through complex regulatory mechanisms. The junctions are highly dynamic in contact establishment, in remodeling events during development, and during processes involving a loss of adhesion like epithelial-mesenchyme transition. It appeared recently that they are also dynamically renewed in mature, steady-state adhesions. Indeed, maintenance of a steady state must be integrated into a tight control of force equilibrium between a cell and its neighbors. Therefore, it appears that E-cadherin dynamics allows to respond constantly to various biochemical and mechanical stimuli and to regulate the movement and shape of junctions in active remodeling processes. E-cadherin dynamics is mediated through several mechanisms (diffusion, trafficking) in function of the biological system. In mature junctions, membrane E-cadherin is quickly renewed by endocytosis in many cell types. E-cadherin endocytosis shows a complex regulation, depending on small G proteins, ubiquitination, cleavage events, actomyosin cytoskeleton, and other trans molecules in adherens junctions. It is modulated by growth factor stimulations and physical factors. Consequently, E-cadherin endocytosis tightly controls a number of functional processes: cell movements, junction maintenance, cell sorting, and polarity. Misregulated E-cadherin endocytosis is involved in many diseases, from cancerous processes to organogenesis defects.
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Cadherinas/metabolismo , Endocitosis , Uniones Adherentes/metabolismo , Animales , Polaridad Celular , Humanos , Integrinas/metabolismo , Modelos BiológicosRESUMEN
The analysis of experimental random walks aims at identifying the process(es) that generate(s) them. It is in general a difficult task, because statistical dispersion within an experimental set of random walks is a complex combination of the stochastic nature of the generating process, and the possibility to have more than one simple process. In this paper, we study by numerical simulations how the statistical distribution of various geometric descriptors such as the second, third and fourth order moments of two-dimensional random walks depends on the stochastic process that generates that set. From these observations, we derive a method to classify complex sets of random walks, and resolve the generating process(es) by the systematic comparison of experimental moment distributions with those numerically obtained for candidate processes. In particular, various processes such as Brownian diffusion combined with convection, noise, confinement, anisotropy, or intermittency, can be resolved by using high order moment distributions. In addition, finite-size effects are observed that are useful for treating short random walks. As an illustration, we describe how the present method can be used to study the motile behavior of epithelial microvilli. The present work should be of interest in biology for all possible types of single particle tracking experiments.
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Biometría , Movimiento/fisiología , Animales , Anisotropía , Simulación por Computador , Difusión , Células LLC-PK1 , Matemática , Microvellosidades/fisiología , Modelos Biológicos , Procesos Estocásticos , PorcinosRESUMEN
The molecular structure of the brush-border of enterocytes has been investigated since the 1980s, but the dynamics of this highly specialized subcellular domain have been difficult to study due to its small size. To perform a detailed analysis of the dynamics of cytoskeleton proteins in this domain, we developed two-photon fluorescence recovery after photobleaching and a theoretical framework for data analysis. With this method, fast dynamics of proteins in the microvilli of the brush border of epithelial intestinal cells can be measured on the millisecond timescale in volumes smaller than 1 microm3. Two major proteins of the cytoskeleton of the microvilli, actin and myosin 1a (Myo1a; formerly named brush border myosin I), are mobile in the brush-border of Caco-2 cells, an enterocyte-like cellular model. However, the mobility of actin is very different from that of Myo1a and they appear to be unrelated (diffusion coefficient of 15 microm2 s(-1) with a mobile fraction of 60% for actin, and 4 microm2 s(-1) with a mobile fraction of 90% for Myo1a). Furthermore, we show for the first time, in vivo, that the dynamics of Myo1a in microvilli reflect its motor activity.
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Actinas/fisiología , Proteínas de Unión a Calmodulina/fisiología , Citoesqueleto/fisiología , Enterocitos/fisiología , Recuperación de Fluorescencia tras Fotoblanqueo/métodos , Microscopía de Fluorescencia por Excitación Multifotónica/métodos , Proteínas Motoras Moleculares/fisiología , Actinas/ultraestructura , Células CACO-2 , Proteínas de Unión a Calmodulina/ultraestructura , Movimiento Celular/fisiología , Simulación por Computador , Citoesqueleto/ultraestructura , Enterocitos/ultraestructura , Humanos , Microvellosidades/fisiología , Microvellosidades/ultraestructura , Modelos Biológicos , Proteínas Motoras Moleculares/ultraestructura , Cadenas Pesadas de Miosina , Miosina Tipo IRESUMEN
Ezrin plays a key role in coupling signal transduction to cortical cell organization. This actin-membrane linker undergoes a series of conformational changes that modulate its interactions with various partners and its localization in membrane or cytosolic pools. Its mobility and exchange rates within and between these two pools were assessed by two-photon fluorescence recovery after photobleaching in epithelial cell microvilli. Analysis of ezrin mutants with an altered actin-binding site revealed three ezrin membrane states of different mobilities and exchange properties, reflecting sequential association with membrane components and F-actin in the context of a fast overall turnover.