Immunocytochemical studies of the interactions of cadherins and catenins in the early Xenopus embryo

Abstract

Linkage of cadherins to the cytoskeleton is crucial for their adhesive function. Since alpha- and beta-catenin play a key role in this linkage, these proteins are possible targets for processes that control cell-cell adhesion. To achieve a better understanding of the regulation of cell-cell adhesion in embryonic morphogenesis, we used immunohistology to investigate how in Xenopus blastomeres catenins respond to disturbances in the expression of maternal cadherins. Overexpression of myc-tagged maternal cadherin leads to a proportionate increase of the level of beta-catenin. The two proteins colocalize in the endoplasmic reticulum, in cytoplasmic vesicles, and along the cell membrane, indicating that the beta-catenin binds to overexpressed cadherin early in its passage to the plasma membrane. Expression of cadherin is essential for the stable presence of beta-catenin, as depletion from maternal cadherin mRNA leads to a complete loss of beta-catenin from the blastomeres. alpha-Catenin behaves differently. Overexpression of cadherin leaves the amount and localization of alpha-catenin largely unaffected, and additional cadherin inserts itself into the membrane without a proportionate rise in the level of membrane-bound alpha-catenin. However, cadherin mRNA depletion leads to a redistribution of alpha-catenin from the membrane to the cytoplasm. Thus, cadherin is required to localize alpha-catenin to the membrane, but the amount of alpha-catenin along the membrane seems to be restricted to a certain level which cannot be exceeded. The relevance of these observations for the regulation of cadherin-mediated cell adhesion in the Xenopus embryo is discussed. Additionally, we demonstrate that plakoglobin, like beta-catenin an armadillo repeat protein, shows neither accumulation after overexpression nor colocalization with the overexpressed cadherin.

Cadherin-dependent differential cell adhesion in Xenopus causes cell sorting in vitro, but not in the embryo

“…S2A-A0). Classic cadherins bind to b-catenin, and as expected (Schneider et al, 1993;Fagotto and Gumbiner, 1994;Kurth et al, 1999), membrane b-catenin paralleled C-cadherin expression (supplementary material Fig. S2E-G0), indicating that other cadherins do not compensate for changes in C-cadherin levels.…”

Oct-3/4 regulates stem cell identity and cell fate decisions by modulating Wnt/β-catenin signalling

“…The pCS2‐Oct‐3/4 was constructed by cloning the full‐length Oct‐3/4 cDNA into the ClaI site of pCS2. The other expression plasmids were pEVRF‐Oct‐3/4, Oct‐3/4‐ΔN, Oct‐3/4‐ΔC (Ben‐Shushan et al , 1998); pOct1 (Tanaka and Herr, 1990); pOct‐2 (Clerc et al , 1988); pOct‐6 (Meijer et al , 1992); Flag‐β‐catenin, Flag‐DP, Flag‐D32N, Flag‐S33, Flag‐Axin1, Myc‐Axin1 and GFP (Amit et al , 2002); pCS2‐xWnt‐8 (Kelly et al , 1995); pCS2‐xSiamois (Lemaire et al , 1995); pCS2‐x β‐catenin (Kurth et al , 1999) and TOP‐Flash and FOP‐Flash (Korinek et al , 1997). The pLKO.1 lentivral vector‐expressing shRNA against β‐catenin was bought from Openbiosystems (Cat RMM4534).…”

A cell cycle arrest is necessary for bottle cell formation in the early Xenopus gastrula: Integrating cell shape change, local mitotic control and mesodermal patterning

“…Double immunofluorescent whole-mount labelling of embryos was performed as described previously (Kurth et al, 1999;Kurth, 2003) using the following primary antibodies: P14L (polyclonal rabbit antibody against b-catenin; Schneider et al, 1993;, and a mouse monoclonal antinucleoplasmin antibody (b7-1A9, Wedlich et al, 1985). As secondary antibodies Cy3-coupled goat-anti-rabbit IgGs (Dianova) and Alexa 488-coupled goat-anti-mouse IgGs (Molecular Probes) were applied.…”