Tight Junction Formation Research Articles

Multipotent vascular stem cells contribute to neurovascular regeneration of peripheral nerve

BackgroundNeurovascular unit restoration is crucial for nerve regeneration, especially in critical gaps of injured peripheral nerve. Multipotent vascular stem cells (MVSCs) harvested from an adult blood vessel are involved in vascular remodeling; however, the therapeutic benefit for nerve regeneration is not clear.MethodsMVSCs were isolated from rats expressing green fluorescence protein (GFP), expanded, mixed with Matrigel matrix, and loaded into the nerve conduits. A nerve autograft or a nerve conduit (with acellular matrigel or MVSCs in matrigel) was used to bridge a transected sciatic nerve (10-mm critical gap) in rats. The functional motor recovery and cell fate in the regenerated nerve were investigated to understand the therapeutic benefit.ResultsMVSCs expressed markers such as Sox 17 and Sox10 and could differentiate into neural cells in vitro. One month following MVSC transplantation, the compound muscle action potential (CMAP) significantly increased as compared to the acellular group. MVSCs facilitated the recruitment of Schwann cell to regenerated axons. The transplanted cells, traced by GFP, differentiated into perineurial cells around the bundles of regenerated myelinated axons. In addition, MVSCs enhanced tight junction formation as a part of the blood-nerve barrier (BNB). Furthermore, MVSCs differentiated into perivascular cells and enhanced microvessel formation within regenerated neurovascular bundles.ConclusionsIn rats with peripheral nerve injuries, the transplantation of MVSCs into the nerve conduits improved the recovery of neuromuscular function; MVSCs differentiated into perineural cells and perivascular cells and enhanced the formation of tight junctions in perineural BNB. This study demonstrates the in vivo therapeutic benefit of adult MVSCs for peripheral nerve regeneration and provides insight into the role of MVSCs in BNB regeneration.

Peptides Derived from the Tight Junction Protein CLDN1 Disrupt the Skin Barrier and Promote Responsiveness to an Epicutaneous Vaccine

Keratinocytes express many pattern recognition receptors that enhance the skin's adaptive immune response to epicutaneous antigens. We have shown that these pattern recognition receptors are expressed below tight junctions (TJ), strongly implicating TJ disruption as a critical step in antigen responsiveness. To disrupt TJs, we designed peptides inspired by the first extracellular loop of the TJ transmembrane protein CLDN1. These peptides transiently disrupted TJs in the human lung epithelial cell line 16HBE and delayed TJ formation in primary human keratinocytes. Building on these observations, we tested whether vaccinating mice with an epicutaneous influenza patch containing TJ-disrupting peptides was an effective strategy to elicit an immunogenic response. Application of a TJ-disrupting peptide patch resulted in barrier disruption as measured by increased transepithelial water loss. We observed a significant increase in antigen-specific antibodies when we applied patches with TJ-disrupting peptide plus antigen (influenza hemagglutinin) in either a patch-prime or a patch-boost model. Collectively, these observations demonstrate that our designed peptides perturb TJs in human lung as well as human and murine skin epithelium, enabling epicutaneous vaccine delivery. We anticipate that this approach could obviate currently used needle-based vaccination methods that require administration by health care workers and biohazard waste removal.

Biomimetic Human Tissue Model for Long-Term Study of Neisseria gonorrhoeae Infection.

Gonorrhea is the second most common sexually transmitted infection in the world and is caused by Gram-negative diplococcus Neisseria gonorrhoeae. Since N. gonorrhoeae is a human-specific pathogen, animal infection models are only of limited use. Therefore, a suitable in vitro cell culture model for studying the complete infection including adhesion, transmigration and transport to deeper tissue layers is required. In the present study, we generated three independent 3D tissue models based on porcine small intestinal submucosa (SIS) scaffold by co-culturing human dermal fibroblasts with human colorectal carcinoma, endometrial epithelial, and male uroepithelial cells. Functional analyses such as transepithelial electrical resistance (TEER) and FITC-dextran assay indicated the high barrier integrity of the created monolayer. The histological, immunohistochemical, and ultra-structural analyses showed that the 3D SIS scaffold-based models closely mimic the main characteristics of the site of gonococcal infection in human host including the epithelial monolayer, the underlying connective tissue, mucus production, tight junction, and microvilli formation. We infected the established 3D tissue models with different N. gonorrhoeae strains and derivatives presenting various phenotypes regarding adhesion and invasion. The results indicated that the disruption of tight junctions and increase in interleukin production in response to the infection is strain and cell type-dependent. In addition, the models supported bacterial survival and proved to be better suitable for studying infection over the course of several days in comparison to commonly used Transwell® models. This was primarily due to increased resilience of the SIS scaffold models to infection in terms of changes in permeability, cell destruction and bacterial transmigration. In summary, the SIS scaffold-based 3D tissue models of human mucosal tissues represent promising tools for investigating N. gonorrhoeae infections under close-to-natural conditions.

The microstructure of laminin-111 compensates for dystroglycan loss in mammary epithelial cells in downstream expression of milk proteins

Laminin-111 (Ln-1), an extracellular matrix (ECM) glycoprotein found in the basement membrane of mammary gland epithelia, is essential for lactation. In mammary epithelial cells (MECs), dystroglycan (Dg) is believed to be necessary for polymerization of laminin-111 into networks., thus we asked whether correct polymerization could compensate for Dg loss. Artificially polymerized laminin-111 and the laminin-glycoprotein mix Matrigel, both formed branching, spread networks with fractal dimensions from 1.7 to 1.8, whereas laminin-111 in neutral buffers formed small aggregates without fractal properties (a fractal dimension of 2). In Dg knockout cells, either polymerized laminin-111 or Matrigel readily attached to the cell surface, whereas aggregated laminin-111 did not. In contrast, polymerized and aggregated laminin-111 bound similarly to Dg knock-ins. Both polymerized laminin-111 and Matrigel promoted cell rounding, clustering, formation of tight junctions, and expression of milk proteins, whereas aggregated Ln-1 did not attach to cells or promote functional differentiation. These findings support that the microstructure of Ln-1 networks in the basement membrane regulates mammary epithelial cell function.

E‐cadherin is downregulated in benign prostatic hyperplasia and required for tight junction formation and permeability barrier in the prostatic epithelial cell monolayer

We previously reported the presence of prostate-specific antigen (PSA) in the stromal compartment of benign prostatic hyperplasia (BPH). Since PSA is expressed exclusively by prostatic luminal epithelial cells, PSA in the BPH stroma suggests increased tissue permeability and the compromise of epithelial barrier integrity. E-cadherin, an important adherens junction component and tight junction regulator, is known to exhibit downregulation in BPH. These observations suggest that the prostate epithelial barrier is disrupted in BPH and E-cadherin downregulation may increase epithelial barrier permeability. The ultra-structure of cellular junctions in BPH specimens was observed using transmission electron microscopy (TEM) and E-cadherin immunostaining analysis was performed on BPH and normal adjacent specimens from BPH patients. In vitro cell line studies using benign prostatic epithelial cell lines were performed to determine the impact of small interfering RNA knockdown of E-cadherin on transepithelial electrical resistance and diffusion of fluorescein isothiocyanate (FITC)-dextran in transwell assays. The number of kiss points in tight junctions was reduced in BPH epithelial cells as compared with the normal adjacent prostate. Immunostaining confirmed E-cadherin downregulation and revealed a discontinuous E-cadherin staining pattern in BPH specimens. E-cadherin knockdown increased monolayer permeability and disrupted tight junction formation without affecting cell density. Our results indicate that tight junctions are compromised in BPH and loss of E-cadherin is potentially an important underlying mechanism, suggesting targeting E-cadherin loss could be a potential approach to prevent or treat BPH.

Cell Adhesion Structures in Epithelial Cells Are Formed in Dynamic and Cooperative Ways.

There are many morphologically distinct membrane structures with different functions at the surface of epithelial cells. Among these, adherens junctions (AJ) and tight junctions (TJ) are responsible for the mechanical linkage of epithelial cells and epithelial barrier function, respectively. In the process of new cell-cell adhesion formation between two epithelial cells, such as after wounding, AJ form first and then TJ form on the apical side of AJ. This process is very complicated because AJ formation triggers drastic changes in the organization of actin cytoskeleton, the activity of Rho family of small GTPases, and the lipid composition of the plasma membrane, all of which are required for subsequent TJ formation. In this review, the authors focus on the relationship between AJ and TJ as a representative example of specialization of plasma membrane regions and introduce recent findings on how AJ formation promotes the subsequent formation of TJ.

The second PDZ domain of scaffold protein Frmpd2 binds to GluN2A of NMDA receptors

The scaffold proteins Frmpd2 is localized at the basolateral membranes of polarized epithelial cells and associated with tight junction formation. In this report, we found that the Frmpd2 is specifically expressed at postsynaptic membrane. By using of co-immunoprecipitation and GST pull-down, Frmpd2 was reported to interact with postsynaptic excitatory N-methyl-d-aspartic acid (NMDA) receptors in vivo and in vitro. In addition, we demonstrated that the second PDZ (PDZ2) domain but not the first or third PDZ domain of Frmpd2 binds to the C-terminus of GluN2A and GluN2B, two subunits of NMDA receptors. By surface plasmon resonance, the affinity of Frmpd2-isolated PDZ2 to GluN2A and GluN2B was identified, which indicates that the interaction of Frmpd2 to GluN2A subunit is more strongly than that to GluN2B subunit. The crystal structure of the PDZ2 domain of the mouse homologue of Frmpd2 was further solved. Some amino acid residues of the PDZ2 structure are supposed to associate with the GluN2A binding. Our study suggests that the scaffold protein Frmpd2 is probably involved in synaptic NMDA receptors-mediated neural excitatory and neurotoxicity in a PDZ2 domain-dependent manner.

Recent Progress in Microfluidic Models of the Blood-Brain Barrier.

The blood-brain barrier (BBB) is a critical physical and chemical barrier that maintains brain homeostasis. Researchers in academia and industry are highly motivated to develop experimental models that can accurately mimic the physiological characteristics of the BBB. Microfluidic systems, which manipulate fluids at the micrometer scale, are ideal tools for simulating the BBB microenvironment. In this review, we summarized the progress in the design and evaluation of microfluidic in vitro BBB models, including advances in chip materials, porous membranes, the use of endothelial cells, the importance of shear stress, the detection specific markers to monitor tight junction formation and integrity, measurements of TEER and permeability. We also pointed out several shortcomings of the current microfluidic models. The purpose of this paper is to let the readers understand the characteristics of different types of model design, and select appropriate design parameters according to the research needs, so as to obtain the best experimental results. We believe that the microfluidics BBB models will play an important role in neuroscience and pharmaceutical research.

Magnetic Human Corneal Endothelial Cell Transplant: Delivery, Retention, and Short-Term Efficacy

PurposeCorneal endothelial dysfunction leads to corneal edema, pain, and vision loss. Adequate animal models are needed to study the safety and efficacy of novel cell therapies as an alternative to corneal transplantation.MethodsPrimary human corneal endothelial cells (HCECs) were isolated from cadaveric donor corneas, expanded in vitro, transduced to express green fluorescent protein (GFP), loaded with superparamagnetic nanoparticles, and injected into the anterior chamber of adult rabbits immediately after endothelial cell or Descemet's membrane stripping. The same volume of balanced salt solution plus (BSS+) was injected in control eyes. We compared different models for inducing corneal edema in rabbits, and examined the ability of transplanted HCECs to reduce corneal edema over time by measuring central corneal thickness and tracking corneal clarity. GFP-positive donor cells were tracked in vivo using optical coherence tomography (OCT) fluorescence angiography module, and the transplanted cells were confirmed by human nuclei immunostaining.ResultsMagnetic HCECs integrated onto the recipient corneas with intact Descemet's membrane, and donor identity was confirmed by GFP expression and immunostaining for human nuclei marker. Donor HCECs formed a monolayer on the posterior corneal surface and expressed HCEC functional markers of tight junction formation. No GFP-positive cells were observed in the trabecular meshwork or on the iris, and intraocular pressure remained stable through the length of the study.ConclusionsOur results demonstrate magnetic cell-based therapy efficiently delivers HCECs to restore corneal transparency without detectable toxicity or adverse effect on intraocular pressure. Magnetic delivery of HCECs may enhance corneal function and should be explored further for human therapies.

Inactivation of CFTR by CRISPR/Cas9 alters transcriptional regulation of inflammatory pathways and other networks

BackgroundIndividuals with cystic fibrosis (CF) experience elevated inflammation in multiple organs, but whether this reflects an inherent feature of CF cells or is a consequence of a pro-inflammatory environment is not clear. MethodUsing CRISPR/Cas9-mediated mutagenesis of CFTR, 17 subclonal cell lines were generated from Caco-2 cells. Clonal lines with functional CFTR (CFTR+) were compared to those without (CFTR−) to directly address the role of CFTR in inflammatory gene regulation. ResultsAll lines maintained CFTR mRNA production and formation of tight junctions. CFTR+ lines displayed short circuit currents in response to forskolin, while the CFTR− lines did not. Baseline expression of cytokines IL6 and CXCL8 (IL8) was not different between the lines regardless of CFTR genotype. All lines responded to TNFα and IL1β by increasing IL6 and CXCL8 mRNA levels, but the CFTR− lines produced more CXCL8 mRNA than the CFTR+ lines. Transcriptomes of 6 CFTR− and 6 CFTR+ lines, before and after stimulation by TNFα, were compared for differential expression as a function of CFTR genotype. While some genes appeared to be differentially expressed simply because of CFTR's absence, others required stimulation for differences to be apparent. ConclusionTogether, these data suggest cells respond to CFTR's absence by modulating transcriptional networks, some of which are only apparent when cells are exposed to different environmental contexts, such as inflammation. With regards to inflammation, these data suggest a model in which CFTR's absence leads to a poised, pro-inflammatory state of cells that is only revealed by stimulation.

Development of a Novel ex vivo Nasal Epithelial Cell Model Supporting Colonization With Human Nasal Microbiota.

The nasal mucosa provides first line defense against inhaled pathogens while creating a unique microenvironment for bacterial communities. Studying the impact of microbiota in the nasal cavity has been difficult due to limitations with current models including explant cultures, primary cells, or neoplastic cell lines. Most notably, none have been shown to support reproducible colonization by bacterial communities from human donors. Therefore, to conduct controlled studies of the human nasal ecosystem, we have developed a novel ex vivo mucosal model that supports bacterial colonization of a cultured host mucosa created by immortalized human nasal epithelial cells (NEC). For this model, immortalized NEC established from 5 male and 5 female donors were cultured with an air-interfaced, apical surface on a porous transwell membrane. NEC were grown from nasal turbinate tissues harvested from willed bodies or from discarded tissue collected during sinonasal procedures. Immortalized cells were evaluated through molecular verification of cell type, histological confirmation of tissue differentiation including formation of tight junctions, NEC multilayer viability, metabolism, physiology and imaging of the luminal surface by scanning electron microscopy. Results showed proper differentiation and multilayer formation at seven to 10 days after air interface that was maintained for up to 3 weeks. The optimized mucosal cultures created an environment necessary to sustain colonization by nasal microbiomes (NMBs) that were collected from healthy volunteers, cryogenically preserved and characterized with customized quantitative polymerase chain reaction (qPCR) arrays. Polymicrobial communities of nasal bacteria associated with healthy and inflamed states were consistently reproduced in matured NEC co-cultures by transplant of NMBs from multiple community types. The cultured NMBs were stable after an initial period of bacterial replication and equilibration. This novel ex vivo culture system is the first model that supports controlled cultivation of NMBs, allowing for lab-based causation studies and further experimentation to explore the complexities of host-microbe and microbe-microbe interactions.

Proteomic Assessment of iTRAQ-Based NaoMaiTong in the Treatment of Ischemic Stroke in Rats.

Background NaoMaiTong (NMT) is widely used in the treatment of cerebral ischemia but the molecular details of its beneficial effects remain poorly characterized. Materials and Methods In this study, we used iTRAQ using 2D LC-MS/MS technology to investigate the cellular mechanisms governing the protective effects of NMT. The transient middle cerebral artery occlusion (MCAO) rat model was established and evaluated. The degree of cerebral ischemia was assessed through scoring for nerve injury symptoms and through the assessment of the areas of cerebral infarction. Brain tissues were subjected to analysis by iTRAQ. High-pH HPLC and RSLC-MS/MS analysis were performed to detect differentially expressed proteins (DEPs) between the treatment groups (Sham, MCAO, and NMT). Bioinformatics were employed for data analysis and DEPs were validated by western blot. Results The results showed that NMT offers protection to the neurological damage caused by MCAO and was found to reduce the areas of cerebral infarction. We detected 3216 DEPs via mass spectrometry. Of these proteins, 21 displayed altered expression following NMT intervention. These included DEPs involved in translation, cell cycle regulation, cellular nitrogen metabolism, and stress responses. Pathway analysis revealed seven key DEPs that were enriched in ribosomal synthesis pathways, tight junction formation, and regulation of the actin cytoskeleton. According to protein-protein interaction analysis, RPL17, Tuba, and Rac1 were affected by NMT treatment, which was validated by western blot analysis. Discussion We therefore identify new pharmacodynamic mechanisms of NMT for the prevention and treatment of ischemic stroke. These DEPs reveal new targets to prevent ischemic stroke induced neuronal damage.

In vitro model for assessing the effect of T helper cell cytokines on the barrier function of alveolar type II epithelial cells

Abstract There are still diseases of global burden for which satisfactory vaccines are unavailable, e.g. tuberculosis. Vaccines against intracellular microbes has relied on the activation of the adaptive T cell response. Our laboratory is interested in investigating whether the innate epithelial host defense could be integrated in a novel approach. In response to airborne pathogens, airway epithelia release antimicrobial peptides like human beta defensin-2 and -3 (HBD2/3), antimicrobial lipids, and chemokines, like IL-8, to attract other immune cells including T helper cells that are integral to the adaptive immune response. We hypothesize that T cell derived cytokines augment the antimicrobial function of airway epithelia and we have developed an in vitro model to generate pilot data. A549 cells, alveolar type II epithelial cells, were grown in air-liquid interface and stimulated with TNF-α & IFN-γ and IL-17 & IL-22, at 5 ng/mL each, for 24 h. IL-8 and HBD2/3 gene expression was quantified by RT-PCR and secretion of antimicrobial peptides was assessed by AU-PAGE and Western blot probing for HBD2/3. Lipid production was assessed with Bodipy and the physical barrier integrity with Phalloidin. IL-8 gene expression was 9-fold elevated when cells were stimulated with TNF-α & IFN-γ. Secretions from stimulated cells appeared to contain more cationic peptides that were not reactive with HBD2/3 polyclonal antibodies. Cellular Bodipy fluorescence was pronounced after IL-17 & IL22 stimulation. Phalloidin staining was consistent with tight junction formation in all samples. This data provides evidence that T helper cells may directly augment epithelial barrier function supporting a novel vaccine design that integrates the epithelial cell response.

In vitro model for predicting acute inhalation toxicity by using a Calu-3 epithelium cytotoxicity assay

IntroductionAs the current methods to predict the inhalation toxicity of chemicals using animal models are limited, alternative methods are required. We present a new in vitro prediction method for acute inhalation toxicity using the Calu-3 epithelial cytotoxicity assay applicable for water-soluble inhalable chemicals. MethodTo confirm the characteristics of the optimal Calu-3 epithelium, tight-junction formation, morphology, and mucus secretion were verified using scanning electron microscopy, transepithelial electrical resistance analysis, and immunofluorescence after growth in an air-liquid interface (ALI). Sixty chemicals, including 38 positive and 22 negative for acute inhalation toxicity, were selected from the European Chemical Agency chemical database. The cell viability of the exposed cells was assessed using an MTT assay to predict the acute inhalation toxicity by calculating the area under the receiver operating characteristic (ROC) curve and accuracy. ResultsWhen cultivated in an ALI, the epithelium was thicker and secreted more mucin than that under submerged cultivation, characteristic of the in vivo respiratory epithelium. The areas under the ROC curve were 0.75 and 0.78 when exposed to chemicals at concentrations of 2.5 and 5%, respectively. The highest accuracy of the methods was 68 and 78% at cut-off values of 85 and 40% cell viability, respectively. DiscussionThe in vitro model was moderately accurate with good prediction. It is replicable because of its advantages, i.e., the use of cultured cells and the simplicity of the method. Overall, the Calu-3 epithelial cytotoxicity assay may be a useful and simple approach to identify substances that cause acute inhalation toxicity.

A novel 3D-printed silk fibroin-based scaffold facilitates tracheal epithelium proliferation in vitro.

The functional epithelial regeneration is important for repairing tracheal defects. However, the potential of 3D-printed SF-based scaffolds for tracheal epithelial regeneration is still unknown. In this study, we developed a novel silk fibroin-based scaffold prepared by 3D printing of silk fibroin/hydroxypropyl methyl cellulose (SF/HPMC) thixotropic hydrogel and evaluated the tracheal epithelium proliferation on this scaffold in vitro. Combined with the freeze-dried technology, the 3D-printed SF/HPMC scaffolds had porous structures in the printed bars. After evaluation of their pore sizes, porosities, water contents and mechanical properties, the scaffolds were co-cultured with the normal human bronchial epithelial cell line (BEAS-2B) for seven days. We detected the BEAS-2B cells proliferation on the scaffolds using a CCK-8 assay, determined their mucin secretion and intercellular tight junction formation by immunofluorescence, as well as observed their cell viability and morphology by live/dead staining and scanning electronic microscopy. The results showed that the SF/HPMC scaffolds had good porosity, water content and mechanical properties. In addition, the BEAS-2B cells proliferated well on SF/HPMC scaffolds, during the seven-day co-culture, with high viability, mucin expression, and intercellular tight junction formation. In summary, these results demonstrated that the BEAS-2B cells could attach and proliferate on the 3D-printed SF/HPMC scaffolds, which were expected to have potential for facilitating tracheal epithelial regeneration.

361 Ephrin-A1 ligand strengthens tight junction function and prevents Th2 cytokine-induced barrier collapse

Atopic dermatitis (AD) is a chronic skin disease affecting all ages at a worldwide prevalence rate up to 20%. AD is largely driven by immune signals initiated by Th2 cytokines including interleukin (IL)-4 and IL-13. However, mounting evidence points to a central role for epidermal barrier dysfunction in the pathogenesis of AD. We have shown that the epidermal tight junction (TJ) network, a critical component of the barrier, depends on the receptor tyrosine kinase EphA2. Therefore, we hypothesize that EphA2 is a positive regulator of TJs in epidermis and can be targeted to improve epidermal barrier function. In 2D keratinocyte cultures, engaging EphA2 with its ligand ephrin-A1 enhances TJ barrier function compared to control (P=0.0001), as measured by trans-epithelial electrical resistance (TEER). This enhancement is accompanied by the immunofluorescent accumulation of TJ proteins, but only small changes in protein and transcript expression. Increased TEER (P<0.05) and TJ formation are also seen in 3D human epidermal equivalents (3D HEE) after ephrin-A1 treatment. These results led us to ask if ephrin-A1 can prevent epidermal barrier defects in in vitro models of AD. Concurrent treatment with IL-4 and IL-13 induces an AD-like phenotype apparent by changes in epidermal morphology. We show that ephrin-A1 treatment in 3D human skin equivalents (3D HSE) restores keratohyalin granules and the stratum corneum which are lost in 3D HSE after cytokine treatment alone. In human explant cultures of truncal skin, IL-4+IL-13 cause epidermal spongiosis and increased epidermal thickness, effects that are abrogated by ephrin-A1. In addition to altering epidermal morphology, we find that IL-4+IL-13 reduce TEER in 3D HEE compared to controls (P<0.05) and obliterate TJs, shown by loss of occludin immunofluorescence. This cytokine-induced catastrophe of TJ function and formation was rescued by co-treatment with ephrin-A1. Taken together, these results suggest that targeting the EphA2 signaling axis not only enhances TJs, but can also restore epidermal barrier function in the face of Th2 cytokine-driven TJ disassembly.

First person – Mathias Cobbaut and Ahmed Elbediwy

ABSTRACT First Person is a series of interviews with the first authors of a selection of papers published in Journal of Cell Science, helping early-career researchers promote themselves alongside their papers. Mathias Cobbaut and Ahmed Elbediwy are co-first authors on ‘The Rho family GEF FARP2 is activated by aPKCι to control tight junction formation and polarity’, published in JCS. Mathias is a postdoc in the lab of Prof. Peter Parker at The Francis Crick Institute, London, investigating protein kinase regulation/biochemistry and the functional role of protein kinase signaling. Ahmed is a lecturer at Kingston University, investigating cancer biology, polarity and signaling.

The Rho family GEF FARP2 is activated by aPKCι to control tight junction formation and polarity.

ABSTRACTThe elaboration of polarity is central to organismal development and to the maintenance of functional epithelia. Among the controls determining polarity are the PAR proteins, PAR6, aPKCι and PAR3, regulating both known and unknown effectors. Here, we identify FARP2 as a ‘RIPR’ motif-dependent partner and substrate of aPKCι that is required for efficient polarisation and junction formation. Binding is conferred by a FERM/FA domain–kinase domain interaction and detachment promoted by aPKCι-dependent phosphorylation. FARP2 is shown to promote GTP loading of Cdc42, which is consistent with it being involved in upstream regulation of the polarising PAR6–aPKCι complex. However, we show that aPKCι acts to promote the localised activity of FARP2 through phosphorylation. We conclude that this aPKCι−FARP2 complex formation acts as a positive feedback control to drive polarisation through aPKCι and other Cdc42 effectors.This article has an associated First Person interview with the first author of the paper.

3D in vitro model for human corneal endothelial cell maturation

Corneal endothelium is a cellular monolayer positioned on the Descemet's membrane at the anterior cornea, and it plays a critical role in maintaining corneal clarity. Our present study examines the feasibility of utilizing our 3-dimensional (3D) corneal stromal construct, which consists of human corneal fibroblasts (HCF) and their self-assembled matrix, to observe the development and maturation of human corneal endothelial cells (HCEndoCs) in a co-culture model. Three-dimensional HCF constructs were created by growing the HCFs on Transwell membranes in Eagles' minimum essential medium (EMEM) + 10% FBS + 0.5 mM Vitamin C (VitC) for about 4 weeks. HCEndoCs, either primary (pHCEndoC) or cell line (HCEndoCL), were either seeded in chamber slides, directly on the Transwell membranes, or on the 3D HCF constructs and cultivated for 5 days or 2 weeks. The HCEndoCs that were seeded directly on the Transwell membranes were exposed indirectly to HCF by culturing the HCF on the plate beneath the membrane. Cultures were examined for morphology and ultrastructure using light and transmission electron microscopy (TEM). In addition, indirect-immunofluorescence microscopy (IF) was used to examine tight junction formation (ZO-1), maturation (ALDH1A1), basement membrane formation (Laminin), cell proliferation (Ki67), cell death (caspase-3), and fibrotic response (CTGF). As expected, both pHCEndoCs and HCEndoCLs formed monolayers on the constructs; however, the morphology of the HCEndoCLs appeared to be similar to that seen in vivo, uniform and closely packed, whereas the pHCEndoCs remained elongated. The IF data showed that laminin localization was present in the HCEndoCs' cytoplasm as cell-cell contact increased, and when they were grown in the 3D co-culture, the beginnings of what appears to be a continuous DM-like structure was observed. In addition, in co-cultures, ALDH1A1-positive HCEndoCs were present, ZO-1 expression localized within the tight junctions, minimal numbers of HCEndoCs were Ki67-or Caspase-3-positive, and CTGF was positive in both the HCEndoCs cytoplasm and the matrix of the co-culture. Also, laminin localization was stimulated in HCEndoCs upon indirect stimuli secreted by HCF. The present data suggests our 3D co-culture model is useful for studying corneal endothelium maturation in vitro since the co-culture promotes new DM-like formation, HCEndoCs develop in vivo-like characteristics, and the fibrotic response is activated. Our current findings are applicable to understanding the implications of corneal endothelial injection therapy, such as if the abnormal DM has to be removed from the patient, the newly injected endothelial cells will seed onto the wound area and deposit a new DM-like membrane. However, caution should be observed and as much of the normal DM should be left intact since removal of the DM can cause a posterior stromal fibrotic response.

Moderately Neutralizing Epitopes in Nonfunctional Regions Dominate the Antibody Response to Plasmodium falciparum EBA-140.

Plasmodium falciparum erythrocyte-binding antigen 140 (EBA-140) plays a role in tight junction formation during parasite invasion of red blood cells and is a potential vaccine candidate for malaria. Individuals in areas where malaria is endemic possess EBA-140-specific antibodies, and individuals with high antibody titers to this protein have a lower rate of reinfection by parasites. The red blood cell binding segment of EBA-140 is comprised of two Duffy-binding-like domains, called F1 and F2, that together create region II. The sialic acid-binding pocket of F1 is essential for binding, whereas the sialic acid-binding pocket in F2 appears dispensable. Here, we show that immunization of mice with the complete region II results in poorly neutralizing antibodies. In contrast, immunization of mice with the functionally relevant F1 domain of region II results in antibodies that confer a 2-fold increase in parasite neutralization compared to that of the F2 domain. Epitope mapping of diverse F1 and F2 monoclonal antibodies revealed that the functionally relevant F1 sialic acid-binding pocket is a privileged site inaccessible to antibodies, that the F2 sialic acid-binding pocket contains a nonneutralizing epitope, and that two additional epitopes reside in F1 on the opposite face from the sialic acid-binding pocket. These studies indicate that focusing the immune response to the functionally important F1 sialic acid binding pocket improves the protective immune response of EBA-140. These results have implications for improving future vaccine designs and emphasize the importance of structural vaccinology for malaria.