Visualization of Endoplasmic Reticulum and Mitochondria in Aurantiochytrium limacinum by the Expression of EGFP with Cell Organelle-Specific Targeting/Retaining Signals
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Thraustochytrids are single cell marine eukaryotes that produce large amounts of polyunsaturated fatty acids such as docosahexaenoic acid. In the present study, we report the visualization of endoplasmic reticulum (ER) and mitochondria in a type strain of the thraustochytrid, Aurantiochytrium limacinum ATCC MYA-1381, using the enhanced green fluorescent protein (EGFP) with specific targeting/retaining signals. We expressed the egfp gene with ER targeting/retaining signals from A. limacinum calreticulin or BiP/GRP78 in the thraustochytrid, resulting in the distribution of EGFP signals at the perinuclear region and near lipid droplets. ER-Tracker™ Red, an authentic fluorescent probe for the visualization of ER in mammalian cells, also stained the same region. We observed small lipid droplets generated from the visualized ER in the early growth phase of cell culture. Expression of the egfp gene with the mitochondria targeting signal from A. limacinum cytochrome c oxidase resulted in the localization of EGFP near the plasma membrane. The distribution of EGFP signals coincided with that of MitoTracker® Red CMXRos, which is used to visualize mitochondria in eukaryotes. The ER and mitochondria of A. limacinum were visualized for the first time by EGFP with thraustochytrid cell organelle-specific targeting/retaining signals. These results will contribute to classification of the intracellular localization of proteins expressed in ER and mitochondria as well as analyses of these cell organelles in thraustochytrids.
KeywordsThraustochytrids Aurantiochytrium limacinum Endoplasmic reticulum Mitochondrion Cell organelle Green fluorescent protein Lipid droplet Targeting signal
We thank Dr. Masahiro Hayashi at the University of Miyazaki (Japan) for providing A. limacinum mh0186. We also thank Dr. Daisuke Honda, Konan University (Japan), for his helpful comments on thraustochytrids. We are indebted to Ms. Yuki Okugawa, the Center for Advanced Instrumental and Educational Supports, Faculty of Agriculture, Kyushu University, for the confocal microscope (Leica TCS SP8 STED) analysis. This work was supported in part by the Science and Technology Research Promotion Program for Agriculture, Forestry, Fisheries and Food Industry, Japan.
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Conflict of Interest
The authors declare that they have no conflict of interest.
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