Tight junction formation in early Xenopus laevis embryos: identification and ultrastructural characterization of junctional crests and junctional vesicles

How tight junctions (TJ) form during early amphibian embryogenesis is still an open question. We used time-lapse video microscopy, scanning electron microscopy (SEM), TEM and freeze-fracture to gain new insight into TJ biogenesis in early clevages of Xenopus laevis. Video analysis suggests three phases in junction formation between blastomeres. A first "waiting” phase, where new unpigmented lateral membranes are generated. A second "mixing” phase, where the unpigmented lateral membrane is separated from the pigmented apical membrane by an area showing a limited degree of intermingling of cortical pigment. And a third "sealing” phase, characterized by the formation of cingulin-containing boundaries between membrane domains, and their rapid directional adhesion in a zipper-like fashion. By SEM, we characterized these boundaries ("junctional crests”, JC) as arrays of villiform protrusions at the border between old and new membranes. In the 2-cell embryo, JC are deeply located, and thus not visible at the surface, but they become increasingly more superficial as cleavages progress. After adjacent blastomeres have adhered to each other, fractured JC display linear arrays of junctional vesicles (JV) of 1-3μm diameter. TEM analysis shows that JV are symmetrically located near the apposed membranes of adjacent blastomeres, and that the membranes near the JV display focal sites of intimate contact, typical of TJ. Freeze-fracture analysis confirms that intramembrane fibrils, typical of TJ, are present at adhesion sites. We conclude that TJ are formed following the sealing of JC, through the recruitment, sorting and assembly of membrane and cytoplasmic proteins at or near J

ZO Proteins Redundantly Regulate the Transcription Factor DbpA/ZONAB

The localization and activities of DbpA/ZONAB and YAP transcription factors are in part regulated by the density-dependent assembly of epithelial junctions. DbpA activity and cell proliferation are inhibited by exogenous overexpression of the tight junction (TJ) protein ZO-1, leading to a model whereby ZO-1 acts by sequestering DbpA at the TJ. However, mammary epithelial cells and mouse tissues knock-out for ZO-1 do not show increased proliferation, as predicted by this model. To address this discrepancy, we examined the localization and activity of DbpA and YAP in Madin-Darby canine kidney cells depleted either of ZO-1, or one of the related proteins ZO-2 and ZO-3 (ZO proteins), or all three together. Depletion of only one ZO protein had no effect on DbpA localization and activity, whereas depletion of ZO-1 and ZO-2, which is associated with reduced ZO-3 expression, resulted in increased DbpA localization in the cytoplasm. Only depletion of ZO-2 reduced the nuclear import of YAP. Mammary epithelial (Eph4) cells KO for ZO-1 showed junctional DbpA, demonstrating that ZO-1 is not required to sequester DbpA at junctions. However, further depletion of ZO-2 in Eph4 ZO-1KO cells, which do not express ZO-3, caused decreased junctional localization and expression of DbpA, which were rescued by the proteasome inhibitor MG132. In vitro binding assays showed that full-length ZO-1 does not interact with DbpA. These results show that ZO-2 is implicated in regulating the nuclear shuttling of YAP, whereas ZO proteins redundantly control the junctional retention and stability of DbpA, without affecting its shuttling to the nucleus

PLEKHA7 Is an Adherens Junction Protein with a Tissue Distribution and Subcellular Localization Distinct from ZO-1 and E-Cadherin

The pleckstrin-homology-domain-containing protein PLEKHA7 was recently identified as a protein linking the E-cadherin-p120 ctn complex to the microtubule cytoskeleton. Here we characterize the expression, tissue distribution and subcellular localization of PLEKHA7 by immunoblotting, immunofluorescence microscopy, immunoelectron microscopy, and northern blotting in mammalian tissues. Anti-PLEKHA7 antibodies label the junctional regions of cultured kidney epithelial cells by immunofluorescence microscopy, and major polypeptides of Mr ∼135 kDa and ∼145 kDa by immunoblotting of lysates of cells and tissues. Two PLEKHA7 transcripts (∼5.5 kb and ∼6.5 kb) are detected in epithelial tissues. PLEKHA7 is detected at epithelial junctions in sections of kidney, liver, pancreas, intestine, retina, and cornea, and its tissue distribution and subcellular localization are distinct from ZO-1. For example, PLEKHA7 is not detected within kidney glomeruli. Similarly to E-cadherin, p120 ctn, β-catenin and α-catenin, PLEKHA7 is concentrated in the apical junctional belt, but unlike these adherens junction markers, and similarly to afadin, PLEKHA7 is not localized along the lateral region of polarized epithelial cells. Immunoelectron microscopy definitively establishes that PLEKHA7 is localized at the adherens junctions in colonic epithelial cells, at a mean distance of 28 nm from the plasma membrane. In summary, we show that PLEKHA7 is a cytoplasmic component of the epithelial adherens junction belt, with a subcellular localization and tissue distribution that is distinct from that of ZO-1 and most AJ proteins, and we provide the first description of its distribution and localization in several tissues

Intestinal barriers protect against disease

Leaky cell-cell junctions contribute to inflammatory and autoimmune diseases</p