Characterization of the strain-rate-dependent mechanical response of single cell-cell junctions.

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.

Accumulation of Neurofascin at Nodes of Ranvier Is Regulated by a Paranodal Switch.

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.

Physics of lumen growth.

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.

Phosphorylation hotspot in the C-terminal domain of occludin regulates the dynamics of epithelial junctional complexes.

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.

Mechanochemical Coupling and Junctional Forces during Collective Cell Migration.

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.

Surface Tension Controls the Hydraulic Fracture of Adhesive Interfaces Bridged by Molecular Bonds.

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.

Paired octamer rings of retinoschisin suggest a junctional model for cell-cell adhesion in the retina.

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.

Advanced Methods for the Investigation of Cell Contact Dynamics in Endothelial Cells Using Florescence-Based Live Cell Imaging.

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.

Organization of Non-centrosomal Microtubules in Epithelial Cells.

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.

Endoplasmic reticulum-plasma membrane junctions: structure, function and dynamics.

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.

The CellBorderTracker, a novel tool to quantitatively analyze spatiotemporal endothelial junction dynamics at the subcellular level.

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.

Localization of cystic fibrosis transmembrane conductance regulator signaling complexes in human salivary gland striated duct cells.

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.

A novel protein complex, Mesh-Ssk, is required for septate junction formation in the Drosophila midgut.

Septate junctions (SJs) are specialized intercellular junctions that restrict the free diffusion of solutes through the paracellular route in invertebrate epithelia. In arthropods, two morphologically different types of SJs have been reported: pleated SJs and smooth SJs (sSJs), which are found in ectodermally and endodermally derived epithelia, respectively. However, the molecular and functional differences between these SJ types have not been fully elucidated. Here, we report that a novel sSJ-specific component, a single-pass transmembrane protein, which we term 'Mesh' (encoded by CG31004), is highly concentrated in Drosophila sSJs. Compromised mesh expression causes defects in the organization of sSJs, in the localizations of other sSJ proteins, and in the barrier function of the midgut. Ectopic expression of Mesh in cultured cells induces cell-cell adhesion. Mesh forms a complex with Ssk, another sSJ-specific protein, and these proteins are mutually interdependent for their localization. Thus, a novel protein complex comprising Mesh and Ssk has an important role in sSJ formation and in intestinal barrier function in Drosophila.

Afadin-nectin forces its way to the front.

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.

The transcription factor grainyhead-like 2 regulates the molecular composition of the epithelial apical junctional complex.

Differentiation of epithelial cells and morphogenesis of epithelial tubes or layers is closely linked with the establishment and remodeling of the apical junctional complex, which includes adherens junctions and tight junctions. Little is known about the transcriptional control of apical junctional complex components. Here, we show that the transcription factor grainyhead-like 2 (Grhl2), an epithelium-specific mammalian homolog of Drosophila Grainyhead, is essential for adequate expression of the adherens junction gene E-cadherin and the tight junction gene claudin 4 (Cldn4) in several types of epithelia, including gut endoderm, surface ectoderm and otic epithelium. We have generated Grhl2 mutant mice to demonstrate defective molecular composition of the apical junctional complex in these compartments that coincides with the occurrence of anterior and posterior neural tube defects. Mechanistically, we show that Grhl2 specifically associates with cis-regulatory elements localized at the Cldn4 core promoter and within intron 2 of the E-cadherin gene. Cldn4 promoter activity in epithelial cells is crucially dependent on the availability of Grhl2 and on the integrity of the Grhl2-associated cis-regulatory element. At the E-cadherin locus, the intronic Grhl2-associated cis-regulatory region contacts the promoter via chromatin looping, while loss of Grhl2 leads to a specific decrease of activating histone marks at the E-cadherin promoter. Together, our data provide evidence that Grhl2 acts as a target gene-associated transcriptional activator of apical junctional complex components and, thereby, crucially participates in epithelial differentiation.

The Na-K-ATPase α1ß1 heterodimer as a cell adhesion molecule in epithelia.

The ion gradients generated by the Na-K-ATPase play a critical role in epithelia by driving transepithelial transport of various solutes. The efficiency of this Na-K-ATPase-driven vectorial transport depends on the integrity of epithelial junctions that maintain polar distribution of membrane transporters, including the basolateral sodium pump, and restrict paracellular diffusion of solutes. The review summarizes the data showing that, in addition to pumping ions, the Na-K-ATPase located at the sites of cell-cell junction acts as a cell adhesion molecule by interacting with the Na-K-ATPase of the adjacent cell in the intercellular space accompanied by anchoring to the cytoskeleton in the cytoplasm. The review also discusses the experimental evidence on the importance of a specific amino acid region in the extracellular domain of the Na-K-ATPase ß(1) subunit for the Na-K-ATPase trans-dimerization and intercellular adhesion. Furthermore, a possible role of N-glycans linked to the Na-K-ATPase ß(1) subunit in regulation of epithelial junctions by modulating ß(1)-ß(1) interactions is discussed.

Control of Gliotactin localization and levels by tyrosine phosphorylation and endocytosis is necessary for survival of polarized epithelia.

The tricellular junction (TCJ) forms at the convergence of bicellular junctions from three adjacent cells in polarized epithelia and is necessary for maintaining the transepithelial barrier. In the fruitfly Drosophila, the TCJ is generated at the meeting point of bicellular septate junctions. Gliotactin was the first identified component of the TCJ and is necessary for TCJ and septate junction development. Gliotactin is a member of the neuroligin family and associates with the PDZ protein discs large. Beyond this interaction, little is known about the mechanisms underlying Gliotactin localization and function at the TCJ. In this study, we show that Gliotactin is phosphorylated at conserved tyrosine residues, a process necessary for endocytosis and targeting to late endosomes and lysosomes for degradation. Regulation of Gliotactin levels through phosphorylation and endocytosis is necessary as overexpression results in displacement of Gliotactin away from the TCJ throughout the septate junction domain. Excessive Gliotactin in polarized epithelia leads to delamination, paired with subsequent migration, and apoptosis. The apoptosis and the resulting compensatory proliferation resulting from high levels of Gliotactin are mediated by the Drosophila JNK pathway. Therefore, Gliotactin levels within the cell membrane are regulated to ensure correct protein localization and cell survival.

A new diagnostic test for arrhythmogenic right ventricular cardiomyopathy.

BACKGROUND: The diagnosis of arrhythmogenic right ventricular cardiomyopathy (ARVC) can be challenging because the clinical presentation is highly variable and genetic penetrance is often low. METHODS: To determine whether a change in the distribution of desmosomal proteins can be used as a sensitive and specific diagnostic test for ARVC, we performed immunohistochemical analysis of human myocardial samples. RESULTS: We first tested myocardium from 11 subjects with ARVC; of these samples, 8 had desmosomal gene mutations. We also tested myocardium obtained at autopsy from 10 subjects with no clinical or pathological evidence of heart disease as control samples. All ARVC samples but no control samples showed a marked reduction in immunoreactive signal levels for plakoglobin (also known as gamma-catenin), a protein that links adhesion molecules at the intercalated disk to the cytoskeleton. Other desmosomal proteins showed variable changes, but signal levels for the nondesmosomal adhesion molecule N-cadherin were normal in all subjects with ARVC. To determine whether a diminished plakoglobin signal level was specific for ARVC, we analyzed myocardium from 15 subjects with hypertrophic, dilated, or ischemic cardiomyopathies. In every sample, levels of N-cadherin and plakoglobin signals at junctions were indistinguishable from those in control samples. Finally, we performed blinded immunohistochemical analysis of heart-biopsy samples from the Johns Hopkins ARVC registry. We made the correct diagnosis in 10 of 11 subjects with definite ARVC on the basis of clinical criteria and correctly ruled out ARVC in 10 of 11 subjects without ARVC, for a sensitivity of 91%, a specificity of 82%, a positive predictive value of 83%, and a negative predictive value of 90%. The plakoglobin signal level was reduced diffusely in ARVC samples, including those obtained in the left ventricle and the interventricular septum. CONCLUSIONS: Routine immunohistochemical analysis of a conventional endomyocardial-biopsy sample appears to be a highly sensitive and specific diagnostic test for ARVC.

Stepwise polarisation of the Drosophila follicular epithelium.

The function of epithelial tissues is dependent on their polarised architecture, and loss of cell polarity is a hallmark of various diseases. Here we analyse cell polarisation in the follicular epithelium of Drosophila, an epithelium that arises by a mesenchymal-epithelial transition. Although many epithelia are formed by mesenchymal precursors, it is unclear how they polarise. Here we show how lateral, apical, and adherens junction proteins act stepwise to establish polarity in the follicular epithelium. Polarisation starts with the formation of adherens junctions, whose positioning is controlled by combined activities of Par-3, beta-catenin, and Discs large. Subsequently, Par-6 and aPKC localise to the apical membrane in a Par-3-dependent manner. Apical membrane specification continues by the accumulation of the Crumbs complex, which is controlled by Par-3, Par-6, and aPKC. Thus, our data elucidate the genetic mechanisms leading to the stepwise polarisation of an epithelium with a mesenchymal origin.