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

Functional Dicer Is Necessary for Appropriate Specification of Radial Glia during Early Development of Mouse Telencephalon

Early telencephalic development involves transformation of neuroepithelial stem cells into radial glia, which are themselves neuronal progenitors, around the time when the tissue begins to generate postmitotic neurons. To achieve this transformation, radial precursors express a specific combination of proteins. We investigate the hypothesis that micro RNAs regulate the ability of the early telencephalic progenitors to establish radial glia. We ablate functional Dicer, which is required for the generation of mature micro RNAs, by conditionally mutating the Dicer1 gene in the early embryonic telencephalon and analyse the molecular specification of radial glia as well as their progeny, namely postmitotic neurons and basal progenitors. Conditional mutation of Dicer1 from the telencephalon at around embryonic day 8 does not prevent morphological development of radial glia, but their expression of Nestin, Sox9, and ErbB2 is abnormally low. The population of basal progenitors, which are generated by the radial glia, is disorganised and expanded in Dicer1-/- dorsal telencephalon. While the proportion of cells expressing markers of postmitotic neurons is unchanged, their laminar organisation in the telencephalic wall is disrupted suggesting a defect in radial glial guided migration. We found that the laminar disruption could not be accounted for by a reduction of the population of Cajal Retzius neurons. Together, our data suggest novel roles for micro RNAs during early development of progenitor cells in the embryonic telencephalon

The junctional proteins cingulin and paracingulin modulate the expression of tight junction protein genes through GATA-4.

The cytoplamic junctional proteins cingulin and paracingulin have been implicated in the regulation of gene expression in different cultured cell models. In renal epithelial MDCK cells, depletion of either protein results in a Rho-dependent increase in the expression of claudin-2. Here we examined MDCK cell clones depleted of both cingulin and paracingulin (double-KD cells), and we found that unexpectedly the expression of claudin-2, and also the expression of ZO-3 and claudin-3, were decreased, while RhoA activity was still higher than in control cells. The decreased expression of claudin-2 and other TJ proteins in double-KD cells correlated with reduced levels of the transcription factor GATA-4, and was rescued by overexpression of GATA-4, but not by inhibiting RhoA activity. These results indicate that in MDCK cells GATA-4 is required for the expression of claudin-2 and other TJ proteins, and that maintenance of GATA-4 expression requires either cingulin or paracingulin. These results and previous studies suggest a model whereby cingulin and paracingulin redundantly control the expression of specific TJ proteins through distinct GATA-4- and RhoA-dependent mechanisms, and that in the absence of sufficient levels of GATA-4 the RhoA-mediated upregulation of claudin-2 is inhibited

Histone Deacetylase Inhibitors Up-Regulate the Expression of Tight Junction Proteins

Histone deacetylase (HDAC) inhibitors promote cell maturation, differentiation, and apoptosis through changes in gene expression. Differentiated epithelial cells are characterized by apical tight junctions (TJ), which play a role in cell-cell adhesion, polarity, and the permeability barrier function of epithelia. The relationship between cellular differentiation and expression of TJ-associated proteins is not known. Here, we investigated whether HDAC inhibitors affect the expression of TJ proteins in cultured cells by immunoblotting, immunofluorescence, and quantitative real-time, reverse transcription-PCR. We find that the HDAC inhibitor sodium butyrate significantly up-regulates the protein levels of cingulin, ZO-1, and ZO-2 in Rat-1 fibroblasts, cingulin in COS-7 cells, and cingulin and occludin in HeLa cells. Levels of mRNA for cingulin, ZO-1, and ZO-2 are also increased in sodium butyrate–treated Rat-1 fibroblasts. Up-regulation of cingulin is reversible and dose dependent and requires de novo protein synthesis and protein kinase activity, because it is inhibited by cycloheximide and by the protein kinase inhibitor H-7. Up-regulation of TJ proteins by sodium butyrate is linked to the ability of sodium butyrate to inhibit HDAC activity, because suberoylanilide hydroxamic acid, a HDAC inhibitor of a different structural class, also up-regulates cingulin, ZO-1, and ZO-2 expression in Rat-1 fibroblasts. These results indicate that cellular differentiation correlates with kinase-dependent up-regulation of the expression of specific TJ proteins.</p

Increased RhoA activity does not account for the phenotype of double-KD cells.

<p>(<b>A</b>) RhoA activation assay (GST pulldown assay using the rhotekin-binding-domain, see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0055873#pone.0055873-Guillemot3" target="_blank">[19]</a>), showing that RhoA activity is increased at confluence in double-KD cells, similarly to both CGN and CGNL1 single-KD cells. (<b>B</b>) Immunoblot and (<b>C</b>) immunofluorescence analysis of the expression and localization of GEF-H1 in the indicated clonal cell lines. Protein levels of GEF-H1 are not altered in double-KD cells, but the junctional localization of GEF-H1 in confluent cells is decreased in double-KD cells, similar to single-KD cells. Bar, 10 µ m. (<b>D</b>) Histogram displaying cell proliferation (BrdU-incorporating cells) and cell density (cell numbers after 7 days growth at confluence) in wild-type, control, CGN and paracingulin single-KD cell clones, and cingulin/paracingulin double-KD cell clones, these latter either in the absence or in the presence of the ROCK kinase inhibitor Y27632 (50 µM). *p<0.05, compared with WT cells (taken as 100%). (<b>E</b>) Immunoblotting and (<b>F</b>) qRT-PCR analysis of the mRNA and protein levels of claudin-2, ZO-3, GATA-4 and claudin-3 in WT, paracingulin single-KD, and cingulin/paracingulin double-KD cells, without or with the exogenous expression of dominant-negative RhoA (RhoAN19). In (F) data are also shown for double-KD clones expressing only the vector (pcDNA). *p<0.05, compared with WT cells (taken as 100%).</p

Down-regulation of claudin-2, ZO-3, and claudin-3 in cingulin/paracingulin double knockdown MDCK cells.

<p>(<b>A</b>) Histogram showing relative mRNA levels, determined by qRT-PCR, for the indicated transcripts, in wild-type MDCK cells (WT), in a MDCK cell clone expressing a control shRNA (Control), in a cingulin single-KD cell clone (CGN(-)), in a paracingulin single-KD cell clone (CGNL1(-)), and in a double-KD cell clone (CGN(-)/CGNL1(-)). The relative mRNA levels were calculated as the ratio of the mRNA in experimental samples versus WT cells (taken as 100%). *p<0.05, compared with mRNA of WT cells. (<b>B</b>) Immunoblotting analysis of TJ proteins in WT cells, in a clone expressing a control shRNA (Control), in two independent (a and b) paracingulin single-KD cell clones (CGNL1(-)), and in two independent (A and B) double-KD cell clones (CGN(-)/CGNL1(-)). (<b>C</b>) Immunofluorescence localization of CGNL1, CGN, claudin-2, ZO-3, ZO-1 and occludin in WT, control, CGN(-), CGNL1(-) and CGN(-)/CGNL1(-) cells. (<b>D</b>) Immunoblotting analysis and (<b>E</b>) immunofluorescence localization of α-catenin (α-cat), β-catenin (β-cat), and E-cadherin (E-cad) in wild-type (WT) MDCK cells, in a MDCK cell clone expressing a control shRNA (Control), and in a double-KD (CGN(-)/CGNL1(-)) cell clone. Bar (C) and (E) = 10 µm. Protein loadings in (B) and (D) were normalized by immunoblotting samples with anti-β-tubulin antibodies (shown in (B) only). The behaviour of additional double-KD clonal lines was very similar to the representative one shown here. (<b>F</b>) qRT-PCR and (<b>G</b>) immunoblotting analysis of WT, paracingulin single-KD (CGNL1(-)) and cingulin/paracingulin double-KD (CGN(-)/CGNL1(-)) cells with or without additional expression of the tetracycline repressor (TetR), which blocks the expression of shRNA. The relative mRNA levels were calculated as the ratio of the mRNA in experimental samples to that in WT cells (taken as 100%). *p<0.05, compared with WT cells. Protein loadings in (G) were normalized by immunoblotting with anti-β-tubulin antibodies.</p

Hypothetical schematic model for the regulation of claudin-2 expression in MDCK cells.

<p>GATA-4 is required for optimal transcription of claudin-2 (and other TJ proteins, not shown for simplicity) (double arrow in scheme). Cingulin and paracingulin redundantly control the mRNA and protein expression of GATA-4, and independently contribute to the down-regulation of active RhoA in confluent cells. Active RhoA increases claudin-2 expression, either through GATA-4, and/or other, unknown mechanisms (question marks), but is ineffective when GATA-4 is down-regulated.</p

Decreased GATA-4 levels cause reduced expression of claudin-2, ZO-3 and claudin-3 in double-KD cells.

<p>(<b>A</b>) qRT-PCR analysis of the mRNA levels of GATA-4 in the different MDCK cell clones. *p<0.05, compared with WT cells (taken as 100%). (<b>B</b>) Immunoblot analysis of GATA-4 protein levels in wild-type MDCK cells (WT), in a MDCK cell clone expressing a control shRNA (Control), in two independent (a and b) paracingulin single-KD cell clones (CGNL1-), and in two independent (A and B) double-KD cell clones (CGN(-)/CGNL1(-)). Protein loadings were normalized by immunoblotting with anti-β-tubulin antibodies. (<b>C</b>) Immunoblot analysis of protein levels of GATA-4 in WT cells, paracingulin single-KD cells, and cingulin/paracingulin double-KD MDCK cell clones, either in the absence or in the presence (+TetR) of exogenously expressed tetracycline repressor. The decreased expression of GATA-4 is rescued by expression of TetR. (<b>D</b>) and (<b>F</b>) Histograms showing mRNA levels (determined by qRT-PCR) of canine GATA-4 (cGATA-4), mouse+canine GATA-4 (mcGATA-4), claudin-2, ZO-3 and claudin-3 either in double-KD MDCK cells (E) or in WT cells (G), without (white columns) or with (grey columns) the exogenous expression of mouse GATA-4 (m-GATA-4). Mouse GATA-4 was used for the rescue because a canine GATA-4 cDNA was not available, and oligos used for GATA-4 RT-PCR were either canine-, or mouse+canine-specific. (<b>E</b>) Immunoblotting analysis of lysates of the same cell lines shown in (D) and (F), using antibodies against claudin-2, ZO-3, claudin-3, and GATA-4 (mouse-specific antibodies, which do not cross-react with canine GATA-4). Expression of exogenous GATA-4 in double-KD cells rescues mRNA and protein levels of claudin-2, ZO-3, claudin-3, while exogenous expression of mouse GATA-4 in WT cells has little or no effect on the levels of endogenous mRNA and protein.</p

Paracingulin recruits CAMSAP3 to tight junctions and regulates microtubule and polarized epithelial cell organization

Paracingulin (CGNL1) is recruited to tight junctions (TJs) by ZO-1 and to adherens junctions (AJs) by PLEKHA7. PLEKHA7 has been reported to bind to the microtubule minus-end-binding protein CAMSAP3, to tether microtubules to the AJs. Here, we show that knockout (KO) of CGNL1, but not of PLEKHA7, results in the loss of junctional CAMSAP3 and its redistribution into a cytoplasmic pool both in cultured epithelial cells in vitro and mouse intestinal epithelium in vivo. In agreement, GST pulldown analyses show that CGNL1, but not PLEKHA7, interacts strongly with CAMSAP3, and the interaction is mediated by their respective coiled-coil regions. Ultrastructure expansion microscopy shows that CAMSAP3-capped microtubules are tethered to junctions by the ZO-1-associated pool of CGNL1. The KO of CGNL1 results in disorganized cytoplasmic microtubules and irregular nuclei alignment in mouse intestinal epithelial cells, altered cyst morphogenesis in cultured kidney epithelial cells, and disrupted planar apical microtubules in mammary epithelial cells. Together, these results uncover new functions of CGNL1 in recruiting CAMSAP3 to junctions and regulating microtubule cytoskeleton organization and epithelial cell architecture.</p

El pilar de fusta de L’Assut. Conservació i restauració d’una biga de fusta arqueològica d’origen terrestre En

En el següent article es descriuen els treballs de conservació i restauració d’un pilar de fusta provinent del jaciment ibèric de L’Assut a Tivenys (Baix Ebre, Tarragona), que es van dur a terme al laboratori de tercer curs de l’especialitat de Conservació i Restauració d’Arqueologia de l’ESCRBCC durant el curs 2011-2012 sota la supervisió de la professora Júlia Chinchilla Sánchez. Malauradament, trobar objectes de fusta en les excavacions arqueològiques és molt poc comú, sobretot en aquest tipus de jaciments. És per això, que aquesta peça es pot considerar un cas molt interessant i singular. Aquest article recull tota la problemàtica que presentava la fusta i els tractaments que es van aplicar per frenar els seus factors de degradació i garantir la seva preservació, tant per ser estudiada com exposada. Tot el treball s’ha realitzat sota els criteris de conservació i restauració de béns arqueològics, es van prioritzar els treballs de consolidació i estabilització davant dels de reintegració, i es van realitzar només aquelles intervencions necessàries per procurar la conservació de la peça