RESUMEN
Force transmission at cell-cell junctions critically regulates embryogenesis, tissue homeostasis, and diseases including cancer. The cadherin-catenin linkage has been considered the keystone of junctional force transmission, but new findings challenge this paradigm, arguing instead that the nectin-afadin linkage plays the more important role in mature junctions in the intestinal epithelium.
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Uniones Intercelulares , Proteínas de Microfilamentos , Nectinas , Cadherinas/metabolismo , Cateninas/metabolismo , Proteínas de Microfilamentos/metabolismo , Nectinas/metabolismo , Uniones Intercelulares/química , HumanosRESUMEN
Cell-cell adhesions are often subjected to mechanical strains of different rates and magnitudes in normal tissue function. However, the rate-dependent mechanical behavior of individual cell-cell adhesions has not been fully characterized due to the lack of proper experimental techniques and therefore remains elusive. This is particularly true under large strain conditions, which may potentially lead to cell-cell adhesion dissociation and ultimately tissue fracture. In this study, we designed and fabricated a single-cell adhesion micro tensile tester (SCAµTT) using two-photon polymerization and performed displacement-controlled tensile tests of individual pairs of adherent epithelial cells with a mature cell-cell adhesion. Straining the cytoskeleton-cell adhesion complex system reveals a passive shear-thinning viscoelastic behavior and a rate-dependent active stress-relaxation mechanism mediated by cytoskeleton growth. Under low strain rates, stress relaxation mediated by the cytoskeleton can effectively relax junctional stress buildup and prevent adhesion bond rupture. Cadherin bond dissociation also exhibits rate-dependent strengthening, in which increased strain rate results in elevated stress levels at which cadherin bonds fail. This bond dissociation becomes a synchronized catastrophic event that leads to junction fracture at high strain rates. Even at high strain rates, a single cell-cell junction displays a remarkable tensile strength to sustain a strain as much as 200% before complete junction rupture. Collectively, the platform and the biophysical understandings in this study are expected to build a foundation for the mechanistic investigation of the adaptive viscoelasticity of the cell-cell junction.
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Uniones Intercelulares/metabolismo , Estrés Mecánico , Cadherinas/metabolismo , Adhesión Celular , Línea Celular Tumoral , Citoesqueleto/metabolismo , Elasticidad , Humanos , Uniones Intercelulares/química , ViscosidadRESUMEN
The paranodal junctions flank mature nodes of Ranvier and provide a barrier between ion channels at the nodes and juxtaparanodes. These junctions also promote node assembly and maintenance by mechanisms that are poorly understood. Here, we examine their role in the accumulation of NF186, a key adhesion molecule of PNS and CNS nodes. We previously showed that NF186 is initially targeted/accumulates via its ectodomain to forming PNS (hemi)nodes by diffusion trapping, whereas it is later targeted to mature nodes by a transport-dependent mechanism mediated by its cytoplasmic segment. To address the role of the paranodes in this switch, we compared accumulation of NF186 ectodomain and cytoplasmic domain constructs in WT versus paranode defective (i.e., Caspr-null) mice. Both pathways are affected in the paranodal mutants. In the PNS of Caspr-null mice, diffusion trapping mediated by the NF186 ectodomain aberrantly persists into adulthood, whereas the cytoplasmic domain/transport-dependent targeting is impaired. In contrast, accumulation of NF186 at CNS nodes does not undergo a switch; it is predominantly targeted to both forming and mature CNS nodes via its cytoplasmic domain and requires intact paranodes. Fluorescence recovery after photobleaching analysis indicates that the paranodes provide a membrane diffusion barrier that normally precludes diffusion of NF186 to nodes. Linkage of paranodal proteins to the underlying cytoskeleton likely contributes to this diffusion barrier based on 4.1B and ßII spectrin expression in Caspr-null mice. Together, these results implicate the paranodes as membrane diffusion barriers that regulate targeting to nodes and highlight differences in the assembly of PNS and CNS nodes.SIGNIFICANCE STATEMENT Nodes of Ranvier are essential for effective saltatory conduction along myelinated axons. A major question is how the various axonal proteins that comprise the multimeric nodal complex accumulate at this site. Here we examine how targeting of NF186, a key nodal adhesion molecule, is regulated by the flanking paranodal junctions. We show that the transition from diffusion-trapping to transport-dependent accumulation of NF186 requires the paranodal junctions. We also demonstrate that these junctions are a barrier to diffusion of axonal proteins into the node and highlight differences in PNS and CNS node assembly. These results provide new insights into the mechanism of node assembly and the pathophysiology of neurologic disorders in which impaired paranodal function contributes to clinical disability.
Asunto(s)
Moléculas de Adhesión Celular/metabolismo , Ganglios Espinales/metabolismo , Factores de Crecimiento Nervioso/metabolismo , Nódulos de Ranvier/metabolismo , Animales , Moléculas de Adhesión Celular/análisis , Células Cultivadas , Femenino , Ganglios Espinales/química , Ganglios Espinales/citología , Uniones Intercelulares/química , Uniones Intercelulares/metabolismo , Masculino , Ratones , Ratones Noqueados , Ratones Transgénicos , Factores de Crecimiento Nervioso/análisis , Nódulos de Ranvier/químicaRESUMEN
Epithelial and endothelial monolayers are multicellular sheets that form barriers between the 'outside' and 'inside' of tissues. Cell-cell junctions, made by adherens junctions, tight junctions and desmosomes, hold together these monolayers. They form intercellular contacts by binding their receptor counterparts on neighboring cells and anchoring these structures intracellularly to the cytoskeleton. During tissue development, maintenance and pathogenesis, monolayers encounter a range of mechanical forces from the cells themselves and from external systemic forces, such as blood pressure or tissue stiffness. The molecular landscape of cell-cell junctions is diverse, containing transmembrane proteins that form intercellular bonds and a variety of cytoplasmic proteins that remodel the junctional connection to the cytoskeleton. Many junction-associated proteins participate in mechanotransduction cascades to confer mechanical cues into cellular responses that allow monolayers to maintain their structural integrity. We will discuss force-dependent junctional molecular events and their role in cell-cell contact organization and remodeling.
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Uniones Adherentes/química , Uniones Intercelulares/química , Fenómenos Mecánicos , Proteínas de la Membrana/química , Uniones Adherentes/genética , Presión Sanguínea/genética , Citoesqueleto/química , Desmosomas/química , Desmosomas/genética , Células Endoteliales/química , Células Epiteliales/química , Humanos , Uniones Intercelulares/genética , Proteínas de la Membrana/genética , Uniones Estrechas/química , Uniones Estrechas/genéticaRESUMEN
Under physiological and pathological conditions, mechanical forces generated from cells themselves or transmitted from extracellular matrix (ECM) through focal adhesions (FAs) and adherens junctions (AJs) are known to play a significant role in regulating various cell behaviors. Substantial progresses have been made in the field of mechanobiology towards novel methods to understand how cells are able to sense and adapt to these mechanical forces over the years. To address these issues, this review will discuss recent advancements of traction force microscopy (TFM), intracellular force microscopy (IFM), and monolayer stress microscopy (MSM) to measure multiple aspects of cellular forces exerted by cells at cell-ECM and cell-cell junctional intracellular interfaces. We will also highlight how these methods can elucidate the roles of mechanical forces at interfaces of cell-cell/cell-ECM in regulating various cellular functions. [BMB Reports 2020; 53(2): 74-81].
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Matriz Extracelular/fisiología , Mecanotransducción Celular/fisiología , Microscopía de Fuerza Atómica/métodos , Biopolímeros , Adhesión Celular/fisiología , Matriz Extracelular/química , Adhesiones Focales/química , Adhesiones Focales/fisiología , Hidrogeles , Uniones Intercelulares/química , Uniones Intercelulares/fisiología , Estrés Mecánico , TracciónRESUMEN
Biological function requires cell-cell adhesions to tune their cohesiveness; for instance, during the opening of new fluid-filled cavities under hydraulic pressure. To understand the physical mechanisms supporting this adaptability, we develop a stochastic model for the hydraulic fracture of adhesive interfaces bridged by molecular bonds. We find that surface tension strongly enhances the stability of these interfaces by controlling flaw sensitivity, lifetime, and optimal architecture in terms of bond clustering. We also show that bond mobility embrittles adhesions and changes the mechanism of decohesion. Our study provides a mechanistic background to understand the biological regulation of cell-cell cohesion and fracture.
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Adhesión Celular/fisiología , Uniones Intercelulares/química , Uniones Intercelulares/fisiología , Modelos Biológicos , Membrana Celular/química , Membrana Celular/fisiología , Simulación por Computador , Procesos Estocásticos , Tensión SuperficialRESUMEN
Cell migration, a fundamental physiological process in which cells sense and move through their surrounding physical environment, plays a critical role in development and tissue formation, as well as pathological processes, such as cancer metastasis and wound healing. During cell migration, dynamics are governed by the bidirectional interplay between cell-generated mechanical forces and the activity of Rho GTPases, a family of small GTP-binding proteins that regulate actin cytoskeleton assembly and cellular contractility. These interactions are inherently more complex during the collective migration of mechanically coupled cells because of the additional regulation of cell-cell junctional forces. In this study, we adapted a recent minimal modeling framework to simulate the interactions between mechanochemical signaling in individual cells and interactions with cell-cell junctional forces during collective cell migration. We find that migration of individual cells depends on the feedback between mechanical tension and Rho GTPase activity in a biphasic manner. During collective cell migration, waves of Rho GTPase activity mediate mechanical contraction/extension and thus synchronization throughout the tissue. Further, cell-cell junctional forces exhibit distinct spatial patterns during collective cell migration, with larger forces near the leading edge. Larger junctional force magnitudes are associated with faster collective cell migration and larger tissue size. Simulations of heterogeneous tissue migration exhibit a complex dependence on the properties of both leading and trailing cells. Computational predictions demonstrate that collective cell migration depends on both the emergent dynamics and interactions between cellular-level Rho GTPase activity and contractility and multicellular-level junctional forces.
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Movimiento Celular , Uniones Intercelulares/metabolismo , Mecanotransducción Celular , Modelos Teóricos , Citoesqueleto de Actina/metabolismo , Animales , Retroalimentación Fisiológica , Uniones Intercelulares/química , Estrés Mecánico , Proteínas de Unión al GTP rho/metabolismoRESUMEN
Research on nanoparticle (NP)-cell interactions has been extensively carried out in dilute cell cultures, where NPs are heavily internalized by cells. However, it is not known whether the findings from cell culture studies are still true in tissues where cells are tightly packed. Here, we show experimentally and theoretically that when cells are tightly packed, cellular uptake is strongly hindered. When simultaneously encountering two adjacent cells as is often the case in tissues, adhesion, bending and protrusion of at least two membranes from these cells generate complicated energy contributions that cause trapping of NPs at cell junctions with impeded uptake.
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Uniones Intercelulares/metabolismo , Nanopartículas/metabolismo , Células A549 , Endocitosis , Ácido Fólico/química , Oro/química , Células HeLa , Humanos , Interacciones Hidrofóbicas e Hidrofílicas , Uniones Intercelulares/química , Nanopartículas del Metal/química , Modelos Teóricos , Nanopartículas/química , TermodinámicaRESUMEN
P120 catenin (p120) is a non-redundant master regulatory protein of cadherin-based cell-cell junctions, intracellular signaling, and tissue homeostasis and repair. Alternative splicing can generate p120 isoforms 1 and 3 (p120-1 and p120-3), which are implicated in non-overlapping functions by differential expression regulation and unique interactions in different cell types, with often predominant expression of p120-1 in mesenchymal cells, and p120-3 generally prevalent in epithelial cells. However, the lack of specific p120-3 protein detection has precluded analysis of their relative abundance in tissues. Here, we have developed a p120-3 isoform-specific antibody and analyzed the p120-3 localization relative to p120-1 in human tissues. p120-3 but not p120-1 is highly expressed in cell-cell junctions of simple gastrointestinal epithelia such as colon and stomach, and the acini of salivary glands and the pancreas. Conversely, the basal layer of the epidermis and hair follicles expressed p120-1 with reduced p120-3, whereas most other epithelia co-expressed p120-3 and p120-1, including bronchial epithelia and mammary luminal epithelial cells. These data provide an inventory of tissue-specific p120 isoform expression and suggest a link between p120 isoform expression and epithelial differentiation.
Asunto(s)
Cateninas/análisis , Epitelio/química , Isoformas de Proteínas/análisis , Transcriptoma , Humanos , Inmunoensayo , Uniones Intercelulares/química , Catenina deltaRESUMEN
Endothelial cells of the vascular system are dynamic cells whose molecular adaptability is decisive for the adjustment of homeostasis and organ perfusion. Advanced microscopic techniques, automation processing, and image analysis software was shown to improve the understanding of vascular biology. In this work, we describe advanced methods that allow investigating the dynamics of endothelial cell contacts. The development of viral vectors has contributed significantly to the genetic manipulation of endothelial cells. We used the Gibson assembly as a quick and cheap cloning system for introducing sequences into the lentiviral-based pFUGW vector. Furthermore, classical fluorescence tags such as mCherry and EGFP were compared with self-labeling tags such as Halo and SNAP for their suitability to study junction dynamics in cultured endothelium, and found the self-labeling tags as useful tools. Using such combinations, we found maintained cell junction integrity during shear stress-induced junction remodeling using VE-cadherin-EGFP. Remodeling was accompanied by VE-cadherin plaque formation, indicating that this process is mediated by the for-mation of the actin-driven junction-associated intermittent lamellipodia, JAIL. The combined methods including the Gibson assembly, lentiviral mediated gene transfer, spinning disk-based live cell imaging, and software for quantification allow analyses of the endothelial cell junction dynamics under static and under shear stress conditions.
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Clonación Molecular/métodos , Células Endoteliales/fisiología , Células Endoteliales/ultraestructura , Colorantes Fluorescentes , Uniones Intercelulares/fisiología , Animales , Anticuerpos , Anticuerpos Monoclonales , Cadherinas/análisis , Cadherinas/genética , Expresión Génica , Vectores Genéticos , Cabras/inmunología , Proteínas Fluorescentes Verdes/genética , Células Endoteliales de la Vena Umbilical Humana , Humanos , Immunoblotting , Uniones Intercelulares/química , Ratones , Conejos/inmunología , beta Catenina/análisis , gamma Catenina/análisisRESUMEN
Sertoli cells of the mammalian seminiferous epithelium form unique subcellular actin-related structures at intercellular junctions. The appearance of these so called "tubulobulbar complexes" (TBCs) precedes both sperm release at the apex of the epithelium and the movement of early spermatogenic cells out of the spermatogonial stem cell niche at the base of the epithelium. TBCs are considered to be part of the mechanism of junction endocytosis by Sertoli cells. The structures contain junction proteins and morphologically identifiable junctions, and are associated with markers of endocytosis. Here we review the current state of knowledge about the structure and function of TBCs. As the complexes form, they morphologically resemble and have the molecular signature of clathrin-coated pits with extremely long necks. As they mature, the actin filament networks around the "necks" of the structures progressively disassemble and the membrane cores expand or swell into distinct "bulbs". These bulbs acquire extensive membrane contact sites with associated cisternae of endoplasmic reticulum. Eventually the bulbs undergo scission and continue through endosomal compartments of the Sertoli cells. The morphology and composition of TBC indicates to us that the structures likely evolved from the basic clathrin-mediated endocytosis mechanism common to cells generally, and along the way they incorporated unique features to accommodate the cyclic turnover of massive and "intact" intercellular junctions that occurs during spermatogenesis. Anat Rec, 301:2080-2085, 2018. © 2018 Wiley Periodicals, Inc.
Asunto(s)
Actinas/metabolismo , Clatrina/metabolismo , Endocitosis/fisiología , Uniones Intercelulares/metabolismo , Testículo/metabolismo , Actinas/análisis , Animales , Clatrina/análisis , Humanos , Uniones Intercelulares/química , Masculino , Epitelio Seminífero/química , Epitelio Seminífero/citología , Epitelio Seminífero/metabolismo , Células de Sertoli/química , Células de Sertoli/metabolismo , Testículo/química , Testículo/citologíaRESUMEN
We model the dynamics of formation of intercellular secretory lumens. Using conservation laws, we quantitatively study the balance between paracellular leaks and the build-up of osmotic pressure in the lumen. Our model predicts a critical pumping threshold to expand stable lumens. Consistently with experimental observations in bile canaliculi, the model also describes a transition between a monotonous and oscillatory regime during luminogenesis as a function of ion and water transport parameters. We finally discuss the possible importance of regulation of paracellular leaks in intercellular tubulogenesis.
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Hepatocitos/citología , Hepatocitos/fisiología , Uniones Intercelulares/química , Uniones Intercelulares/fisiología , Modelos Teóricos , Ósmosis , Animales , Células Cultivadas , RatasRESUMEN
The apical junctional complex (AJC), which includes tight junctions (TJs) and adherens junctions (AJs), determines the epithelial polarity, cell-cell adhesion and permeability barrier. An intriguing characteristic of a TJ is the dynamic nature of its multiprotein complex. Occludin is the most mobile TJ protein, but its significance in TJ dynamics is poorly understood. On the basis of phosphorylation sites, we distinguished a sequence in the C-terminal domain of occludin as a regulatory motif (ORM). Deletion of ORM and expression of a deletion mutant of occludin in renal and intestinal epithelia reduced the mobility of occludin at the TJs. ORM deletion attenuated Ca2+ depletion, osmotic stress and hydrogen peroxide-induced disruption of TJs, AJs and the cytoskeleton. The double point mutations T403A/T404A, but not T403D/T404D, in occludin mimicked the effects of ORM deletion on occludin mobility and AJC disruption by Ca2+ depletion. Both Y398A/Y402A and Y398D/Y402D double point mutations partially blocked AJC disruption. Expression of a deletion mutant of occludin attenuated collective cell migration in the renal and intestinal epithelia. Overall, this study reveals the role of ORM and its phosphorylation in occludin mobility, AJC dynamics and epithelial cell migration.
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Uniones Adherentes/química , Ocludina/química , Fosfoproteínas/química , Uniones Estrechas/química , Uniones Adherentes/genética , Animales , Calcio/metabolismo , Movimiento Celular/genética , Polaridad Celular/genética , Citoesqueleto/química , Citoesqueleto/genética , Perros , Células Epiteliales/química , Recuperación de Fluorescencia tras Fotoblanqueo , Proteínas Fluorescentes Verdes/química , Proteínas Fluorescentes Verdes/genética , Humanos , Uniones Intercelulares/química , Uniones Intercelulares/genética , Células de Riñón Canino Madin Darby , Ocludina/genética , Fosfoproteínas/genética , Fosforilación/genética , Mutación Puntual/genética , Dominios Proteicos/genética , Uniones Estrechas/genéticaRESUMEN
Fluorescence microscopy of FRET-based biosensors allow nanoscale interactions to be probed in living cells. This paper describes a novel approach to spectrally resolved fluorescence microscopy, termed sensorFRET, that enables quantitative measurement of FRET efficiency. This approach is an improvement on existing methods (FLIM, sRET, luxFRET, pFRET), as it does not require single fluorophore standards to be measured with every experiment and the acquisition is intensity independent, allowing the laser power to be optimized for varying levels of fluorophore expression. Additionally, it was found that all spectral based methods, including sensorFRET, fail at specific fluorophore-excitation wavelength combinations. These combinations can be determined a priori using sensorFRET, whereas other methods would give no indication of inaccuracies. This method was thoroughly validated and compared to existing methods using simulated spectra, Fluorescein and TAMRA dye mixtures as a zero FRET control, and Cerulean-Venus FRET standards as positive FRET controls. Simulations also provided a means of quantifying the uncertainty in each measurement by relating the fit residual of noisy spectra to the standard deviation of the measured FRET efficiency. As an example application, Teal-Venus force sensitive biosensors integrated into E-cadherin were used to resolve piconewton scale forces along different parts of an individual cell junction.
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Técnicas Biosensibles/métodos , Transferencia Resonante de Energía de Fluorescencia/métodos , Microscopía Fluorescente/métodos , Cadherinas/química , Fluoresceína/química , Humanos , Uniones Intercelulares/química , Rodaminas/químicaRESUMEN
Polarized epithelial cells contain a characteristic array of microtubules in which non-centrosomal microtubules are aligned along the apical-to-basal axis of the cell with their minus ends oriented towards the apical pole. Although this unique orientation of microtubules was discovered in the late 1980s, how this orientation is established remains unresolved partly because of limited information about molecular factors that regulate the minus ends of non-centrosomal microtubules. Recent studies, however, identified novel minus end-associated proteins, revealing mechanisms by which the polarized arrays of microtubules are established in epithelial cells. These studies have also demonstrated the importance of apico-basally orientated microtubules in intra-structural organization of cells. This review focuses on recent progress of our understanding of the molecular basis for epithelium-specific microtubule assembly and function.
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Epitelio/metabolismo , Microtúbulos/metabolismo , Animales , Epitelio/química , Uniones Intercelulares/química , Uniones Intercelulares/metabolismo , Proteínas Asociadas a Microtúbulos/metabolismo , Microtúbulos/química , Modelos MolecularesRESUMEN
Retinoschisin (RS1) is involved in cell-cell junctions in the retina, but is unique among known cell-adhesion proteins in that it is a soluble secreted protein. Loss-of-function mutations in RS1 lead to early vision impairment in young males, called X-linked retinoschisis. The disease is characterized by separation of inner retinal layers and disruption of synaptic signaling. Using cryo-electron microscopy, we report the structure at 4.1 Å, revealing double octamer rings not observed before. Each subunit is composed of a discoidin domain and a small N-terminal (RS1) domain. The RS1 domains occupy the centers of the rings, but are not required for ring formation and are less clearly defined, suggesting mobility. We determined the structure of the discoidin rings, consistent with known intramolecular and intermolecular disulfides. The interfaces internal to and between rings feature residues implicated in X-linked retinoschisis, indicating the importance of correct assembly. Based on this structure, we propose that RS1 couples neighboring membranes together through octamer-octamer contacts, perhaps modulated by interactions with other membrane components.
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Moléculas de Adhesión Celular/química , Moléculas de Adhesión Celular/ultraestructura , Adhesión Celular , Proteínas del Ojo/química , Proteínas del Ojo/ultraestructura , Uniones Intercelulares/ultraestructura , Retina/química , Retina/ultraestructura , Secuencia de Aminoácidos , Animales , Simulación por Computador , Dimerización , Uniones Intercelulares/química , Ratones , Modelos Biológicos , Modelos Moleculares , Complejos Multiproteicos/química , Complejos Multiproteicos/ultraestructura , Conformación ProteicaRESUMEN
Endoplasmic reticulum (ER)-plasma membrane (PM) junctions are contact sites between the ER and the PM; the distance between the two organelles in the junctions is below 40 nm and the membranes are connected by protein tethers. A number of molecular tools and technical approaches have been recently developed to visualise, modify and characterise properties of ER-PM junctions. The junctions serve as the platforms for lipid exchange between the organelles and for cell signalling, notably Ca(2+) and cAMP signalling. Vice versa, signalling events regulate the development and properties of the junctions. Two Ca(2+) -dependent mechanisms of de novo formation of ER-PM junctions have been recently described and characterised. The junction-forming proteins and lipids are currently the focus of vigorous investigation. Junctions can be relatively short-lived and simple structures, forming and dissolving on the time scale of a few minutes. However, complex, sophisticated and multifunctional ER-PM junctions, capable of attracting numerous protein residents and other cellular organelles, have been described in some cell types. The road from simplicity to complexity, i.e. the transformation from simple 'nascent' ER-PM junctions to advanced stable multiorganellar complexes, is likely to become an attractive research avenue for current and future junctologists. Another area of considerable research interest is the downstream cellular processes that can be activated by specific local signalling events in the ER-PM junctions. Studies of the cell physiology and indeed pathophysiology of ER-PM junctions have already produced some surprising discoveries, likely to expand with advances in our understanding of these remarkable organellar contact sites.
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Membrana Celular/química , Membrana Celular/fisiología , Retículo Endoplásmico/química , Retículo Endoplásmico/fisiología , Uniones Intercelulares/química , Uniones Intercelulares/fisiología , Animales , HumanosRESUMEN
Endothelial junctions are dynamic structures organized by multi-protein complexes that control monolayer integrity, homeostasis, inflammation, cell migration and angiogenesis. Newly developed methods for both the genetic manipulation of endothelium and microscopy permit time-lapse recordings of fluorescent proteins over long periods of time. Quantitative data analyses require automated methods. We developed a software package, the CellBorderTracker, allowing quantitative analysis of fluorescent-tagged cell junction protein dynamics in time-lapse sequences. The CellBorderTracker consists of the CellBorderExtractor that segments cells and identifies cell boundaries and mapping tools for data extraction. The tool is illustrated by analyzing fluorescent-tagged VE-cadherin the backbone of adherence junctions in endothelium. VE-cadherin displays high dynamics that is forced by junction-associated intermittent lamellipodia (JAIL) that are actin driven and WASP/ARP2/3 complex controlled. The manual segmentation and the automatic one agree to 90 %, a value that indicates high reliability. Based on segmentations, different maps were generated allowing more detailed data extraction. This includes the quantification of protein distribution pattern, the generation of regions of interest, junction displacements, cell shape changes, migration velocities and the visualization of junction dynamics over many hours. Furthermore, we demonstrate an advanced kymograph, the J-kymograph that steadily follows irregular cell junction dynamics in time-lapse sequences for individual junctions at the subcellular level. By using the CellBorderTracker, we demonstrate that VE-cadherin dynamics is quickly arrested upon thrombin stimulation, a phenomenon that was largely due to transient inhibition of JAIL and display a very heterogeneous subcellular and divers VE-cadherin dynamics during intercellular gap formation and resealing.
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Cadherinas/análisis , Endotelio Vascular/citología , Uniones Intercelulares/metabolismo , Programas Informáticos , Animales , Cadherinas/metabolismo , Células Cultivadas , Drosophila , Endotelio Vascular/metabolismo , Fluorescencia , Técnica del Anticuerpo Fluorescente , Humanos , Uniones Intercelulares/químicaRESUMEN
The cystic fibrosis transmembrane conductance regulator (CFTR) is a cyclic AMP-dependent protein kinase (PKA)-regulated Cl(-) channel, crucial for epithelial cell regulation of salt and water transport. Previous studies showed that ezrin, an actin binding and A-kinase anchoring protein (AKAP), facilitates association of PKA with CFTR. We used immunohistochemistry and immunogold transmission electron microscopy to localize CFTR, ezrin, and PKA type II regulatory (RII) and catalytic (C) subunits in striated duct cells of human parotid and submandibular glands. Immunohistochemistry localized the four proteins mainly to the apical membrane and the apical cytoplasm of striated duct cells. In acinar cells, ezrin localized to the luminal membrane, and PKA RII subunits were present in secretory granules, as previously described. Immunogold labeling showed that CFTR and PKA RII and C subunits were localized to the luminal membrane and associated with apical granules and vesicles of striated duct cells. Ezrin was present along the luminal membrane, on microvilli and along the junctional complexes between cells. Double labeling showed specific protein associations with apical granules and vesicles and along the luminal membrane. Ezrin, CFTR, and PKA RII and C subunits are co-localized in striated duct cells, suggesting the presence of signaling complexes that serve to regulate CFTR activity.