Publikationsdatenbank

Analysis of ZO-1 in claudin-expressing Xenopus laevis oocytes (2022)

Art

Poster

Autoren

Brunner, N. (WE 2)

Stein, L. (WE 2)

Amasheh, S. (WE 2)

Kongress

Tagung der Fachgruppe Physiologie und Biochemie der Deutschen Veterinärmedizinischen Gesellschaft (DVG)

Gießen, 24. – 26.06.2022

Quelle

Abstractheft der Tagung der Fachgruppe Physiologie und Biochemie der Deutschen Veterinärmedizinischen Gesellschaft (DVG)

— S. 53

Sprache

Englisch

Kontakt

Institut für Veterinär-Physiologie

Oertzenweg 19 b
14163 Berlin
+49 30 838 62600
physiologie@vetmed.fu-berlin.de

Abstract / Zusammenfassung

Outline
In the past years we established Xenopus laevis oocytes for the heterologous expression and analysis of major tight junction proteins, namely claudins (1,2). In this study, the claudin-expression was set into context of the cells’ capacity to express the scaffolding protein Zona occludens 1 (ZO-1). We
were able to detect endogenous ZO-1 expression in protein and mRNA analyses of unfertilized Xenopus laevis oocytes expressing human claudin-1 to -5 and further validate the amphibian cell model for the analysis of claudin interactions.
Methods
Oocytes from mature Xenopus laevis were surgically removed and injected with cRNA encoding for human claudins, or RNase-free water as controls. Cell membrane fractions were used for western blotting. Using our established protocols (2) for the paired oocyte assay, we induced adhering contact areas by pairwise clustering. After incubation for 24 h, oocytes were fixed and prepared for immunohistochemical analysis of claudins and ZO-1. Additionally, qPCR experiments were performed. dCT values were analyzed by one-way analysis of variance (ANOVA).
Results
Western blots revealed claudin expression in accordance with the injected cRNAs, and ZO-1 was detectable. Immunohistochemical stainings revealed ZO-1 signals located in the submembraneous space that appear as a submembraneous belt underneath the oocyte plasma membrane. As expected, claudin signals were located to the plasma membrane. In mRNA analysis, ZO-1 mRNA levels of the distinct claudin-expressing oocytes and water-injected controls were equal.
Conclusion
In this study we utilized protein analysis to gain a comprehensive understanding of the expression, localization and interaction of heterologously expressed claudins with ZO-1 in the Xenopus laevis oocytes. Claudin-scaffold interactions were reflected by partial colocalization of the two binding
partners. The limitation of merged signals indicated, that claudins and ZO-1 are only periodically associated, which might reflect a dynamic coupling of TJ proteins with ZO-1.