Publikationsdatenbank

Heterologous expression of claudin-5 in Xenopus laevis oocytes (2019)

Art

Poster

Autoren

Brunner, N. (WE 2)

Amasheh, S. (WE 2)

Kongress

98th meeting of the German Physiological Society

Ulm, 30.09. – 02.10.2019

Quelle

Acta physiologica Scandinavica

Bandzählung: 227

Heftzählung: S719

Seiten: 197

ISSN: 0001-6772

Sprache

Englisch

Verweise

URL (Volltext): https://onlinelibrary.wiley.com/doi/epdf/10.1111/apha.13385

Kontakt

Institut für Veterinär-Physiologie

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

Abstract / Zusammenfassung

Outline:
The tight junction protein claudin-5 features barrier-tightening properties, and represents a major component of the blood brain barrier (1). We used the established heterologous expression system of Xenopus oocytes (2) to investigate the contribution of claudin-5 to the contact area of clustered oocytes and to evaluate the isolated effect of claudin-5 to cell-cell-interaction.

Methods:
Oocytes were harvested from 4 adult female African claw frogs via surgical laparoscopy and injected with 1 ng cRNA encoding for human claudin-5, or RNase-free water as controls, respectively. After 3 days, oocytes were devitellinized and clustered in pairs of claudin-5-expressing and control oocytes as follows: cldn5-cldn5 (n=33), cldn5-ctrl (n=20), and ctrl-ctrl (n=34) respectively. Width of contact was measured after 1h, 24h and 48h after clustering via bright field microscopy and the area of contact was calculated by using the circle equation (A = π × (measured width/2)2). Additionally, membrane protein fraction from injected oocytes were obtained and used for Western blot analysis, and oocytes were prepared for immunohistochemical staining.

Results:
After injection of cRNA, Western blots of the membrane fraction revealed claudin-5- specific signals, whereas the water injected controls were negative. In accordance with these results, immunohistochemical staining revealed specific signals for claudin-5 in the plasma membrane. The contact area showed a time-dependent increase over time in all tested combinations. Statistical testing revealed no significant differences between cldn5-cldn5, cldn5- ctrl, and ctrl-ctrl (Mann-Whitney-U, p >0.05).

Conclusion:
In our study, for the first time, we were able to expand the heterologous Xenopus laevis oocyte expression system for the analysis of tight junction proteins to claudin-5. The injection of claudin-5 cRNA resulted in the integration of the protein into the plasma membrane, but the contact area did not change in contrast to oocytes coexpressing claudin-1,-2 and -3, as reported recently (2).