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The claudin family of membrane proteins is responsible for the backbone structure and function of tight junctions (TJs), which regulate the paracellular permeability of epithelia. It is thought that each claudin subtype has its own unique function and the combination of expressed subtypes determines the permeability property of each epithelium. However, many issues remain unsolved in regard to claudin functions, including the detailed functional differences between claudin subtypes and the effect of the combinations of specific claudin subtypes on the structure and function of TJs. To address these issues, it would be useful to have a way of reconstituting TJs containing only the claudin subtype(s) of interest in epithelial cells. In this study, we attempted to reconstitute TJs of individual claudin subtypes in TJ-deficient MDCK cells, designated as claudin quinKO cells, which were previously established from MDCK II cells by deleting the genes of claudin-1, -2, -3, -4, and -7. Exogenous expression of each of claudin-1, -2, -3, -4, and -7 in claudin quinKO cells resulted in the reconstitution of functional TJs. These TJs did not contain claudin-12 and -16, which are endogenously expressed in claudin quinKO cells. Furthermore, overexpression of neither claudin-12 nor claudin-16 resulted in the reconstitution of TJs, demonstrating the existence of claudin subtypes lacking TJ-forming activity in epithelial cells. Exogenous expression of the channel-forming claudin-2, -10a, -10b, and -15 reconstituted TJs with reported paracellular channel properties, demonstrating that these claudin subtypes form paracellular channels by themselves without interaction with other subtypes. Thus, the reconstitution of TJs in claudin quinKO cells is advantageous for further investigation of claudin functions. Key words: tight junction, claudin, paracellular permeability, epithelial barrier

Summary: To infer a “live” protein network in single cells, we developed a novel Protein Localization and Modification-based Covariation Network (PLOM-CON) analysis method using a large set of quantitative data on the abundance (quantity), post-translational modification state (quality), and localization/morphological information of target proteins from microscope immunostained images. The generated network exhibited synchronized time-dependent behaviors of the target proteins to visualize how a live protein network develops or changes in cells under specific experimental conditions. As a proof of concept for PLOM-CON analysis, we applied this method to elucidate the role of actin scaffolds, in which actin fibers and signaling molecules accumulate and form membrane-associated protein condensates, in insulin signaling in rat hepatoma cells. We found that the actin scaffold in cells may function as a platform for glycogenesis and protein synthesis upon insulin stimulation.

Tight junctions (TJs) are cellular junctions within the mammalian epithelial cell sheet that function as a physical barrier to molecular transport within the intercellular space. Dysregulation of TJs leads to various diseases. Tricellular TJs (tTJs), specialized structural variants of TJs, are formed by multiple transmembrane proteins (e.g., lipolysis-stimulated lipoprotein receptor [LSR] and tricellulin) within tricellular contacts in the mammalian epithelial cell sheet. However, the mechanism for recruiting LSR and tricellulin to tTJs is largely unknown. Previous studies have identified that tyrphostin 9, the dual inhibitor of Pyk2 (a nonreceptor tyrosine kinase) and receptor tyrosine kinase platelet-derived growth factor receptor (PDGFR), suppresses LSR and tricellulin recruitment to tTJs in EpH4 (a mouse mammary epithelial cell line) cells. In this study, we investigated the effect of Pyk2 inhibition on LSR and tricellulin localization to tTJs. Pyk2 inactivation by its specific inhibitor or repression by RNAi inhibited the localization of LSR and downstream tricellulin to tTJs without changing their expression level in EpH4 cells. Pyk2-dependent changes in subcellular LSR and tricellulin localization were independent of c-Jun N-terminal kinase (JNK) activation and expression. Additionally, Pyk2-dependent LSR phosphorylation at Tyr-237 was required for LSR and tricellulin localization to tTJs and decreased epithelial barrier function. Our findings indicated a novel mechanism by which Pyk2 regulates tTJ assembly and epithelial barrier function in the mammalian epithelial cell sheet.

Brucella species replicate within host cells in the form of endoplasmic reticulum (ER)-derived vacuoles. The mechanisms by which the bacteria are sequestered into such vacuoles and obtain a continuous membrane supply for their replication remain to be elucidated. In the present study, we provided several lines of evidence that demonstrate the mechanism by which B. abortus acquires the ER-derived membrane. First, during Brucella infection, the IRE1 pathway, but not the PERK and ATF6 pathways, of the unfolded protein response (UPR) was activated in a time-dependent manner, and the COPII vesicle components Sar1, Sec23, and Sec24D were upregulated. Second, a marked accretion of ER-derived vacuoles was observed around replicating bacteria using fluorescent microscopy and electron microscopy. Third, we identified a novel host factor, Yip1A, for the activation of the IRE1 pathway in response to both tunicamycin treatment and infection with B. abortus. We found that Yip1A is responsible for the phosphorylation of IRE1 through high-order assembly of Ire1 molecules at ER exit sites (ERES) under the UPR conditions. In Yip1A-knockdown cells, B. abortus failed to generate the ER-derived vacuoles, and remained in endosomal/lysosomal compartments. These results indicate that the activation of the IRE1 pathway and the subsequent formation of ER-derived vacuoles are critical for B. abortus to establish a safe replication niche, and that Yip1A is indispensable for these processes. Furthermore, we showed that the autophagy-related proteins Atg9 and WIPI1, but not DFCP1, were required for the biogenesis of the ER-derived membrane compartments. On the basis of our findings, we propose a model for intracellular Brucella replication that exploits the host UPR and ER-derived vacuole formation machineries, both of which depend on Yip1A-mediated IRE1 activation.

Cell-based assay systems that can serve as cellular models of aberrant function in pathogenic organs would be novel and useful tools for screening drugs and clarifying the molecular mechanisms of various diseases. We constructed model cells that replicated the conditions in diabetic hepatocytes by using the cell resealing technique, which enables the exchange of cytosol. The plasma membrane of HeLa cells was permeabilized with the streptococcal toxin streptolysin O, and cytosol that had been prepared from wild-type or db/db diabetic mice was introduced into the resulting semi-intact cells. By resealing the plasma membrane by exposure to Ca(2+), we created WT or Db model cells, in which the cytosolic conditions replicated those of healthy or diabetic liver. Interestingly, phosphorylation of p38 MAPK was promoted, whereas the level of endosomal phosphatidylinositol-3-phosphate was decreased, in Db cells. We investigated several endocytic pathways in WT and Db cells, and found that retrograde endosome-to-Golgi transport was delayed in a p38 MAPK-dependent manner in Db cells. Furthermore, the degradation pathway of the EGF receptor from endosomes to lysosomes was enhanced in Db cells, and this did not depend on the activation of p38 MAPK. The disease model cell system should become a powerful tool for the detection of aberrant processes in cells under pathogenic conditions and for therapeutic applications.

Summary: Body rocking can either induce sleep or arousal. That is, the vestibular sense influences sleep-wake states. Neuronal interactions between sleep-wake systems and vestibular systems, however, remain unclear. In this study, we found that GABAergic neurons in the lateral part of the medial vestibular nucleus (LMVN), a primary vestibular afferent projection site, control sleep-wake states. Specific inhibition of LMVN GABAergic neurons revealed that the firing of LMVN GABAergic neurons underlies stable wakefulness and smooth transitions from non-rapid-eye-movement (NREM) sleep to rapid eye movement (REM) sleep and that LMVN GABAergic neurons do not affect body balance control in freely moving conditions. Selective axonal tracing of LMVN GABAergic neurons indicated that LMVN GABAergic neurons send axons not only to areas involved in vestibular and oculomotor functions but also to areas regulating sleep-wake states. Our findings suggest that LMVN GABAergic neurons stabilize wakefulness and gate the entry into REM sleep through the use of vestibular information.

Disease-modifying therapies, such as neuroprotective and neurorestorative interventions, are strongly desired for Alzheimer’s disease (AD) treatment. Several studies have suggested that histone deacetylase 2 (HDAC2) inhibition can exhibit disease-modifying effects in AD patients. However, whether HDAC2 inhibition shows neuroprotective and neurorestorative effects under neuropathic conditions, such as amyloid β (Aβ)-elevated states, remains poorly understood. Here, we performed HDAC2-specific knockdown in CA1 pyramidal cells and showed that HDAC2 knockdown increased the length of dendrites and the number of mushroom-like spines of CA1 basal dendrites in APP/PS1 transgenic mouse model. Furthermore, HDAC2 knockdown also ameliorated the deficits in hippocampal CA1 long-term potentiation and memory impairment in contextual fear conditioning tests. Taken together, our results support the notion that specific inhibition of HDAC2 has the potential to slow the disease progression of AD through ameliorating Aβ-induced neuronal impairments.

Increase in the concentration of plasma L-cysteine is closely associated with defective insulin secretion from pancreatic β-cells, which results in type 2 diabetes (T2D). In this study, we investigated the effects of prolonged L-cysteine treatment on glucosestimulated insulin secretion (GSIS) from mouse insulinoma 6 (MIN6) cells and from mouse pancreatic islets, and found that the treatment reversibly inhibited glucose-induced ATP production and resulting GSIS without affecting proinsulin and insulin synthesis. Comprehensive metabolic analyses using capillary electrophoresis time-of-flight mass spectrometry showed that prolonged L-cysteine treatment decreased the levels of pyruvate and its downstream metabolites. In addition, methyl pyruvate, a membrane- permeable form of pyruvate, rescued L-cysteine-induced inhibition of GSIS. Based on these results, we found that both in vitro and in MIN6 cells, L-cysteine specifically inhibited the activity of pyruvate kinase muscle isoform 2 (PKM2), an isoform of pyruvate kinases that catalyze the conversion of phosphoenolpyruvate to pyruvate. L-cysteine also induced PKM2 subunit dissociation (tetramers to dimers/monomers) in cells, which resulted in impaired glucose-induced ATP production for GSIS. DASA-10 (NCGC00181061, a substituted N,N'-diarylsulfonamide), a specific activator for PKM2, restored the tetramer formation and the activity of PKM2, glucoseinduced ATP production, and biphasic insulin secretion in L-cysteine- treated cells. Collectively, our results demonstrate that impaired insulin secretion due to exposure to L-cysteine resulted from its direct binding and inactivation of PKM2 and suggest that PKM2 is a potential therapeutic target for T2D. [ABSTRACT FROM AUTHOR]