The Role of ZO-2 in Modulating JAM-A and γ-Actin Junctional Recruitment, Apical Membrane and Tight Junction Tension, and Cell Response to Substrate Stiffness and Topography.

This work analyzes the role of the tight junction (TJ) protein ZO-2 on mechanosensation. We found that the lack of ZO-2 reduced apical membrane rigidity measured with atomic force microscopy, inhibited the association of γ-actin and JAM-A to the cell border, and instead facilitated p114RhoGEF and afadin accumulation at the junction, leading to an enhanced mechanical tension at the TJ measured by FRET, with a ZO-1 tension probe, and increased tricellular TJ tension. Simultaneously, adherens junction tension measured with an E-cadherin probe was unaltered. The stability of JAM-A and ZO-2 binding was assessed by a collaborative in silico study. The absence of ZO-2 also impacted the cell response to the substrate, as monolayers plated in 20 kPa hydrogels developed holes not seen in parental cultures and displayed a retarded elongation and formation of cell aggregates. The absence of ZO-2 was sufficient to induce YAP and Snail nuclear accumulation in cells cultured over glass, but when ZO-2 KD cells were plated in nanostructured ridge arrays, they displayed an increased abundance of nuclear Snail and conspicuous internalization of claudin-4. These results indicate that the absence of ZO-2 also impairs the response of cells to substrate stiffness and exacerbates transformation triggered by substrate topography.

ZO-2 favors Hippo signaling, and its re-expression in the steatotic liver by AMPK restores junctional sealing.

ZO-2 is a peripheral tight junction (TJ) protein whose silencing in renal epithelia induces cell hypertrophy. Here, we found that in ZO-2 KD MDCK cells, in compensatory renal hypertrophy triggered in rats by a unilateral nephrectomy and in liver steatosis of obese Zucker (OZ) rats, ZO-2 silencing is accompanied by the diminished activity of LATS, a kinase of the Hippo pathway, and the nuclear concentration of YAP, the final effector of this signaling route. ZO-2 appears to function as a scaffold for the Hippo pathway as it associates to LATS1. ZO-2 silencing in hypertrophic tissue is due to a diminished abundance of ZO-2 mRNA, and the Sp1 transcription factor is critical for ZO-2 transcription in renal cells. Treatment of OZ rats with metformin, an activator of AMPK that blocks JNK activity, augments ZO-2 and claudin-1 expression in the liver, reduces the paracellular permeability of hepatocytes, and serum bile acid content. Our results suggest that ZO-2 silencing is a common feature of hypertrophy, and that ZO-2 is a positive regulator of the Hippo pathway that regulates cell size. Moreover, our observations highlight the importance of AMPK, JNK, and ZO-2 as therapeutic targets for blood-bile barrier dysfunction.

Zonula occludens 2 and Cell-Cell Contacts Are Required for Normal Nuclear Shape in Epithelia.

MAGUK protein ZO-2 is present at tight junctions (TJs) and nuclei. In MDCK ZO-2 knockdown (KD) cells, nuclei exhibit an irregular shape with lobules and indentations. This condition correlates with an increase in DNA double strand breaks, however cells are not senescent and instead become resistant to UV-induced senescence. The irregular nuclear shape is also observed in isolated cells and in those without TJs, due to the lack of extracellular calcium. The aberrant nuclear shape of ZO-2 KD cells is not accompanied by a reduced expression of lamins A/C and B and lamin B receptors. Instead, it involves a decrease in constitutive and facultative heterochromatin, and microtubule instability that is restored with docetaxel. ZO-2 KD cells over-express SUN-1 that crosses the inner nuclear membrane and connects the nucleoskeleton of lamin A to nesprins, which traverse the outer nuclear membrane. Nesprins-3 and -4 that indirectly bind on their cytoplasmic face to vimentin and microtubules, respectively, are also over-expressed in ZO-2 KD cells, whereas vimentin is depleted. SUN-1 and lamin B1 co-immunoprecipitate with ZO-2, and SUN-1 associates to ZO-2 in a pull-down assay. Our results suggest that ZO-2 forms a complex with SUN-1 and lamin B1 at the inner nuclear membrane, and that ZO-2 and cell-cell contacts are required for a normal nuclear shape.

Whole-cell bioconversion of naringenin to high added value hydroxylated compounds using Yarrowia lipolytica 2.2ab in surface and liquid cultures.

The bioconversion process of bioactive naringenin by whole-cells of Yarrowia lipolytica 2.2ab for the production of increased value-added compounds is successfully achieved in surface and liquid cultures. This approach is an alternative to the commercial production of these bioactive compounds from vegetable sources, which are limited due to their low concentrations and the complexity of the purification processes. The experimentation rendered seven value-added compounds in both surface and liquid bioconversion cultures. Some of the compounds produced have not been previously reported as products from the bioconversion processes, such as the case of ampelopsin. Biosynthetic pathways were suggested for the naringenin bioconversion using whole-cells of Y. lipolytica 2.2ab. Finally, the extracts obtained from the naringenin bioconversion in liquid cultures showed higher percentage of inhibition of DPPH· and ABTS· radicals up to 32.88 and 2.08 times, respectively, compared to commercial naringenin.

ZO-2 Is a Master Regulator of Gene Expression, Cell Proliferation, Cytoarchitecture, and Cell Size.

ZO-2 is a cytoplasmic protein of tight junctions (TJs). Here, we describe ZO-2 involvement in the formation of the apical junctional complex during early development and in TJ biogenesis in epithelial cultured cells. ZO-2 acts as a scaffold for the polymerization of claudins at TJs and plays a unique role in the blood-testis barrier, as well as at TJs of the human liver and the inner ear. ZO-2 movement between the cytoplasm and nucleus is regulated by nuclear localization and exportation signals and post-translation modifications, while ZO-2 arrival at the cell border is triggered by activation of calcium sensing receptors and corresponding downstream signaling. Depending on its location, ZO-2 associates with junctional proteins and the actomyosin cytoskeleton or a variety of nuclear proteins, playing a role as a transcriptional repressor that leads to inhibition of cell proliferation and transformation. ZO-2 regulates cell architecture through modulation of Rho proteins and its absence induces hypertrophy due to inactivation of the Hippo pathway and activation of mTOR and S6K. The interaction of ZO-2 with viral oncoproteins and kinases and its silencing in diverse carcinomas reinforce the view of ZO-2 as a tumor regulator protein.

Relationship between G proteins coupled receptors and tight junctions.

Tight junctions (TJs) are sites of cell-cell adhesion, constituted by a cytoplasmic plaque of molecules linked to integral proteins that form a network of strands around epithelial and endothelial cells at the uppermost portion of the lateral membrane. TJs maintain plasma membrane polarity and form channels and barriers that regulate the transit of ions and molecules through the paracellular pathway. This structure that regulates traffic between the external milieu and the organism is affected in numerous pathological conditions and constitutes an important target for therapeutic intervention. Here, we describe how a wide array of G protein-coupled receptors that are activated by diverse stimuli including light, ions, hormones, peptides, lipids, nucleotides and proteases, signal through heterotrimeric G proteins, arrestins and kinases to regulate TJs present in the blood-brain barrier, the blood-retinal barrier, renal tubular cells, keratinocytes, lung and colon, and the slit diaphragm of the glomerulus.

Catsper1 promoter is bidirectional and regulates the expression of a novel lncRNA.

The Catsper1 gene, whose expression is restricted to male germ cells, has great importance in reproductive biology because of its function in sperm motility and fertilization. We previously reported that the promoter of this gene has transcriptional activity in either direction in a heterologous system. In the present study, we found that the Catsper1 promoter has in vitro transcriptional activity in either orientation in GC-1 spg mouse spermatogonial cells. The results also showed that this promoter regulates the expression of a new divergent Catsper1 gene named Catsper1au (Catsper1 antisense upstream transcript). Catsper1au is expressed in adult male mouse testis and liver tissues but not in female mouse liver or ovary tissues. In the testis, Catsper1au is expressed in embryos at 11.5 days post-coitum and from newborns to adults. This gene is also expressed in 1- to 3-week postnatal hearts and in 1-week to adult stage livers. The analysis of the 1402 bp whole genome sequence revealed that Catsper1au is an intronless and polyadenylated lncRNA, located in the nuclei of Sertoli and spermatogenic cells from adult testis. These data indicate that Catsper1au is divergently expressed from the Catsper1 promoter and could regulate gene expression during spermatogenesis.