Hyperosmolality Induces Nuclear Translocation of Osmotic Stress Transcription Factor 1 in Oreochromis Mossambicus Cells

Abstract

Euryhaline fish tolerate a wide range of environmental salinity by employing molecular mechanisms for coping with the associated osmotic stress. We have previously shown that osmotic stress transcription factor 1 (OSTF1) is part of these mechanisms. OSTF1 is transiently and rapidly upregulated in gill epithelial cells of tilapia (Oreochromis mossambicus) exposed to hyperosmolality. Hyperosmotic induction of OSTF1 was shown in tilapia gills, a response that was reproduced in the tilapia OmB cell brain fibroblast cell line (OmB). OSTF1 shares the signature sequence of the TSC‐22 family predicting that it is a transcription factor. If in fact OSTF1 is a transcription factor, we hypothesize it will localize to the nucleus during hyperosmotic stress. Using molecular cloning, OSTF1 was tagged with enhanced green fluorescent protein (EGFP) at either the C‐ or N‐terminus. Using fluorescent microscopy we showed that the fusion proteins were retained in the cytosol under iso‐osmotic conditions. To evaluate potential nuclear translocation of OSTF1 during hyperosmotic stress, we subjected OmB cells expressing the OSTF1:EGFP fusion protein to hyperosmotic media and imaged at time intervals from 5 minutes to 4 hours using a Leica Dmi8 microscope. At four hours and 650 mOsmol/kg, subcellular localization quantified by LASX image analysis (Leica) demonstrated that OSTF1:EGFP was mostly localized to the nucleus. This result supports our hypothesis that OSTF1 is indeed an osmotically inducible transcription factor. Current work evaluates the persistency of nuclear localization of OSTF1 during sustained hyperosmotic stress and isosmotic recovery.Support or Funding InformationThis study was funded by a grant from NSF (IOS‐1656371).