Title Observation of Stress-Induced Autophagy in Fish Cells
Authers Takeshi YABU and Michiaki YAMASHITA
Keywords autophagy, protein degradation, amino acids starvation, heat stress, microtubule-associated protein 1-light chain 3
Citation Bull. Fish. Res. Agen. No.26, 23-28, 2008
Abstract
Starvation is one of important stress conditions that cause various physiological dysfunctions and deteriorate fish quality as a food. Here, we developed a biomarker to detect the state of stress and/or starvation in fish and cultured cells. In particular, we focused on the autophagic pathway that forms autophagic vacuoles, i.e . autophagosomes, in the starved cells by the withdrawal of amino acids from culture medium. The autophagy has been characterized to be an apoptotic pathway induced by stress conditions, such as starvation, heat shock and hypoxia, followed by intracellular protein degradation (bulk degradation) differs from the ubiquitin-proteasome system. We used fish cultured cells as a model to evaluate the influence and the degree of biochemical changes by heat stress and amino acids starvation. A microtubule binding protein that localized in autophagosome membranes can be used as a molecular marker for induction of autophagy. We established stable transformants of ZE cell line introduced with a fusion protein of green fluorescent protein( GFP) and microtubule-associated protein 1-light chain 3( MAP1- LC3) to detect fluorescent autophagosome associated with the GFP-MAP1-LC3-fusion protein. The induction of autophagy was examined under amino acids starvation and heat stress conditions. The high temperature and amino acids withdrawal in the ZE cells induced autophagy in time-dependent manner by counting the number of fluorescent particles localized with GFP-LC3 fusion protein in the cell. The autophagy induced by the amino acids withdrawal was suppressed in the presence of phosphatidylinositol 3 kinase inhibtor, 3-methyladenine, in the culture medium and by overexperssion of the bcl-2 gene. A phosphatidylinositol 3 kinase, target of rapamycin (TOR), mediated autophagic signaling pathway that executes the starvation induced-autophagy. Therefore, we proposed a new biological model of autophagic signaling pathway induced under heat stress and amino acid starvation conditions in fish cultured cells.
URI http://www.fra.affrc.go.jp/bulletin/bull/bull26/yabu2.pdf