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Marine Biotechnology

, Volume 20, Issue 2, pp 182–192 | Cite as

Visualization of Endoplasmic Reticulum and Mitochondria in Aurantiochytrium limacinum by the Expression of EGFP with Cell Organelle-Specific Targeting/Retaining Signals

  • Nozomu Okino
  • Hiroyoshi Wakisaka
  • Yohei Ishibashi
  • Makoto Ito
Original Article
  • 266 Downloads

Abstract

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.

Keywords

Thraustochytrids Aurantiochytrium limacinum Endoplasmic reticulum Mitochondrion Cell organelle Green fluorescent protein Lipid droplet Targeting signal 

Notes

Acknowledgments

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.

Compliance with Ethical Standards

Conflict of Interest

The authors declare that they have no conflict of interest.

References

  1. Bolte S, Talbot C, Boutte Y, Catrice O, Read ND, Satiat-Jeunemaitre B (2004) FM-dyes as experimental probes for dissecting vesicle trafficking in living plant cells. J Microsc 214(2):159–173CrossRefPubMedGoogle Scholar
  2. Breslow JL (2006) n-3 fatty acids and cardiovascular disease. Am J Clin Nutr 83(6 Suppl):S1477–S1482CrossRefGoogle Scholar
  3. Calder PC (2006) n-3 polyunsaturated fatty acids, inflammation, and inflammatory diseases. Am J Clin Nutr 83(6 Suppl):S1505–S1519CrossRefGoogle Scholar
  4. Cubitt AB, Heim R, Adams SR, Boyd AE, Gross LA, Tsien RY (1995) Understanding, improving and using green fluorescent proteins. Trends Biochem Sci 20(11):448–455CrossRefPubMedGoogle Scholar
  5. Emanuelsson O, Nielsen H, Brunak S, von Heijne G (2000) Predicting subcellular localization of proteins based on their N-terminal amino acid sequence. J Mol Biol 300(4):1005–1016CrossRefPubMedGoogle Scholar
  6. Finco AMO, Mamani LDG, Carvalho JC, de Melo Pereira GV, Thomaz-Soccol V, Soccol CR (2017) Technological trends and market perspectives for production of microbial oils rich in omega-3. Crit Rev Biotechnol 37(5):656–671CrossRefPubMedGoogle Scholar
  7. Fujimoto T, Ohsaki Y, Cheng J, Suzuki M, Shinohara Y (2008) Lipid droplets: a classic organelle with new outfits. Histochem Cell Biol 130(2):263–279CrossRefPubMedPubMedCentralGoogle Scholar
  8. Gerdes H-H, Kaether C (1996) Green fluorescent protein: applications in cell biology. FEBS Lett 389(1):44–47CrossRefPubMedGoogle Scholar
  9. Gocze PM, Freeman DA (1994) Factors underlying the variability of lipid droplet fluorescence in MA-10 leydig tumor cells. Cytometry 17(2):151–158CrossRefPubMedGoogle Scholar
  10. Greenspan P, Mayer EP, Fowler SD (1985) Nile red: a selective fluorescent stain for intracellular lipid droplets. J Cell Biol 100(3):965–973CrossRefPubMedGoogle Scholar
  11. Innis SM (2008) Dietary omega 3 fatty acids and the developing brain. Brain Res 1237:35–43CrossRefPubMedGoogle Scholar
  12. Janssen CI, Kiliaan AJ (2014) Long-chain polyunsaturated fatty acids (LCPUFA) from genesis to senescence: the influence of LCPUFA on neural development, aging, and neurodegeneration. Prog Lipid Res 53:1–17CrossRefPubMedGoogle Scholar
  13. Katayama H, Yamamoto A, Mizushima N, Yoshimori T, Miyawaki A (2008) GFP-like proteins stably accumulate in lysosomes. Cell Struct Funct 33:1–12CrossRefPubMedGoogle Scholar
  14. Kaya K, Nakazawa A, Matsuura H, Honda D, Inouye I, Watanabe MM (2011) Thraustochytrid Aurantiochytrium sp. 18W-13a accummulates high amounts of squalene. Biosci Biotechnol Biochem 75(11):2246–2248CrossRefPubMedGoogle Scholar
  15. Kim K, Jung Kim E, Ryu BG, Park S, Choi YE, Yang JW (2013) A novel fed-batch process based on the biology of Aurantiochytrium sp. KRS101 for the production of biodiesel and docosahexaenoic acid. Bioresour Technol 135:269–274CrossRefPubMedGoogle Scholar
  16. Klionsky DJ, Herman PK, Emr SD (1990) The fungal vacuole: composition, function, and biogenesis. Microbiol Rev 54(3):266–292PubMedPubMedCentralGoogle Scholar
  17. Köhler RH, Zipfel WR, Webb WW, Hanson MR (1997) The green fluorescent protein as a marker to visualize plant mitochondria in vivo. Plant J 11(3):613–621CrossRefPubMedGoogle Scholar
  18. Lenihan-Geels G, Bishop KS, Ferguson LR (2013) Alternative sources of omega-3 fats: can we find a sustainable substitute for fish? Nutrients 5(4):1301–1315CrossRefPubMedPubMedCentralGoogle Scholar
  19. Lewis TE, Nichols PD, McMeekin TA (1999) The biotechnological potential of thraustochytrids. Mar Biotechnol (NY) 1(6):580–587CrossRefGoogle Scholar
  20. Li SC, Kane PM (2009) The yeast lysosome-like vacuole: endpoint and crossroads. Biochim Biophys Acta 1793:650–663CrossRefPubMedGoogle Scholar
  21. Lydon MJ, Keeler KD, Thomas DB (1980) Vital DNA staining and cell sorting by flow microfluorometry. J Cell Physiol 102(2):175–181CrossRefPubMedGoogle Scholar
  22. Martins DA, Custodio L, Barreira L, Pereira H, Ben-Hamadou R, Varela J, Abu-Salah KM (2013) Alternative sources of n-3 long-chain polyunsaturated fatty acids in marine microalgae. Mar Drugs 11(7):2259–2281CrossRefPubMedPubMedCentralGoogle Scholar
  23. Matsuda T, Sakaguchi K, Hamaguchi R, Kobayashi T, Abe E, Hama Y, Hayashi M, Honda D, Okita Y, Sugimoto S, Okino N, Ito M (2012) Analysis of delta12-fatty acid desaturase function revealed that two distinct pathways are active for the synthesis of PUFAs in T. aureum ATCC 34304. J Lipid Res 53(6):1210–1222CrossRefPubMedPubMedCentralGoogle Scholar
  24. Morita E, Kumon Y, Nakahara T, Kagiwada S, Noguchi T (2006) Docosahexaenoic acid production and lipid-body formation in Schizochytrium limacinum SR21. Mar Biotechnol (NY) 8(3):319–327CrossRefGoogle Scholar
  25. Nagano N, Taoka Y, Honda D, Hayashi M (2013) Effect of trace elements on growth of marine eukaryotes, tharaustochytrids. J Biosci Bioeng 116(3):337–339CrossRefPubMedGoogle Scholar
  26. Nakai K, Horton P (1999) PSORT: a program for detecting sorting signals in proteins and predicting their subcellular localization. Trends Biochem Sci 24(1):34–35CrossRefPubMedGoogle Scholar
  27. Nilsson T, Warren G (1994) Retention and retrieval in the endoplasmic reticulum and the Golgi apparatus. Curr Opin Cell Biol 6(4):517–521CrossRefPubMedGoogle Scholar
  28. Ohara J, Sakaguchi K, Okita Y, Okino N, Ito M (2013) Two fatty acid elongases possessing C18-delta6/C18-delta9/C20-delta5 or C16-delta9 elongase activity in Thraustochytrium sp. ATCC 26185. Mar Biotechnol (NY) 15(4):476–486CrossRefGoogle Scholar
  29. Raghukumar S (2008) Thraustochytrid marine protists: production of PUFAs and other emerging technologies. Mar Biotechnol (NY) 10(6):631–640CrossRefGoogle Scholar
  30. Rizzuto R, Brini M, Pizzo P, Murgia M, Pozzan T (1995) Chimeric green fluorescent protein as a tool for visualizing subcellular organelles in living cells. Curr Biol 5(6):635–642CrossRefPubMedGoogle Scholar
  31. Sakaguchi K, Matsuda T, Kobayashi T, Ohara Ji, Hamaguchi R, Abe E, Nagano N, Hayashi M, Ueda M, Honda D, Okita Y, Taoka Y, Sugimoto S, Okino N, Ito M (2012) Versatile transformation system that is applicable to both multiple transgene expression and gene targeting for Thraustochytrids. Appl Environ Microbiol 78(9):3193–3202CrossRefPubMedPubMedCentralGoogle Scholar
  32. Scheuring D, Schöller M, Kleine-Vehn J, Löfke C (2015) Vacuolar staining methods in plant cells. In: Estevez JM (ed) Plant cell expansion: methods and protocols. Springer New York, New York, NY, pp 83–92Google Scholar
  33. Seibel NM, Eljouni J, Nalaskowski MM, Hampe W (2007) Nuclear localization of enhanced green fluorescent protein homomultimers. Anal Biochem 368(1):95–99CrossRefPubMedGoogle Scholar
  34. Thiam AR, Foret L (2016) The physics of lipid droplet nucleation, growth and budding. Biochim Biophys Acta 1861(8):715–722CrossRefPubMedGoogle Scholar
  35. Velmurugan N, Sathishkumar Y, Yim SS, Lee YS, Park MS, Yang JW, Jeong KJ (2014) Study of cellular development and intracellular lipid bodies accumulation in the thraustochytrid Aurantiochytrium sp. KRS101. Bioresour Technol 161:149–154CrossRefPubMedGoogle Scholar
  36. Vida TA, Emr SD (1995) A new vital stain for visualizing vacuolar membrane dynamics and endocytosis in yeast. J Cell Biol 128(5):779–792CrossRefPubMedGoogle Scholar

Copyright information

© Springer Science+Business Media, LLC, part of Springer Nature 2018

Authors and Affiliations

  1. 1.Department of Bioscience and Biotechnology, Graduate School of Bioresource and Bioenvironmental SciencesKyushu UniversityFukuokaJapan

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