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A structure-function based screening scrutinizes the p120ctn domains required for cystic EB formation.

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posted on 24.08.2016, 17:30 by Tim Pieters, Steven Goossens, Lieven Haenebalcke, Vanessa Andries, Agata Stryjewska, Riet De Rycke, Kelly Lemeire, Tino Hochepied, Danny Huylebroeck, Geert Berx, Marc P. Stemmler, Dagmar Wirth, Jody J. Haigh, Jolanda van Hengel, Frans van Roy

(A) Morphology (H&E-staining, top panels), ultrastructure (TEM, middle panels) and immunohistochemistry for E-cadherin (bottom panels) of control and p120ctn-null DIV30 EBs. Boxed area in H&E pictures is magnified to visualize the single-layered endodermal layer (arrow). The dashed line in the TEM micrographs separates the endodermal cells (arrows) from the inner EB cells (asterisk). Scale bars: 10 μm. (B) Breeding strategy and mESC isolation procedure to obtain p120ctn-/- mESCs that harbor a recombination-mediated cassette exchange (RMCE)-compatible anti-Luc (AL) allele in the ROSA26 (R26) locus. (C) Micrographs showing DIV30 control and p120ctn-null EBs. Scale bars: 200 μm. (D) Schematic representation of coding potential of key rescue constructs that were introduced into the R26 locus of p120ctn-/-;ALtg/+ mESCs. See Table 1 for the complete list of rescue constructs. p120ctn isoforms contain a central armadillo domain consisting of nine armadillo repeats (grey boxes), one out of four alternative start codons (arrows), and possibly sequences encoded by alternatively used exons A and C (black boxes), and including or excluding two Rho GTPase binding domains (RBDs). p120ctn isoforms 1A, 3A and 4A use the first, third and fourth translation initiation site, respectively. The position of mutations of important AA and the artificial addition of a membrane-targeting motif (CAAX) are shown. E-cadherin (Ecadh) consists of a signal peptide (S), a pro-domain (PRO), four extracellular cadherin (EC) repeats, a membrane-proximal extracellular domain (MPED), a transmembrane domain (T), a juxtamembrane domain (JMD) and a β-catenin-binding domain (CBD). (E-H) Micrographs of DIV30 EBs after introduction of various rescue constructs by RMCE of mESCs, as indicated by the names on top of the images. Scale bars: 200 μm.