A super-ecliptic, pHluorin-mKate2, tandem fluorescent protein-tagged human LC3 for the monitoring of mammalian autophagy.

Tandem fluorescent protein-tagged LC3s that were comprised of a protein tag that emits green fluorescence (e.g., EGFP or mWasabi) fused with another tag that emits red fluorescence (e.g. mCherry or TagRFP) were used for monitoring the maturation step of mammalian autophagosomes. A critical point for...

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Autores principales:	Isei Tanida, Takashi Ueno, Yasuo Uchiyama
Formato:	article
Lenguaje:	EN
Publicado:	Public Library of Science (PLoS) 2014
Materias:	Medicine R Science Q
Acceso en línea:	https://doaj.org/article/60ca3ad51f154a69bffb931223577b1e
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spelling	oai:doaj.org-article:60ca3ad51f154a69bffb931223577b1e2021-11-25T05:55:25ZA super-ecliptic, pHluorin-mKate2, tandem fluorescent protein-tagged human LC3 for the monitoring of mammalian autophagy.1932-620310.1371/journal.pone.0110600https://doaj.org/article/60ca3ad51f154a69bffb931223577b1e2014-01-01T00:00:00Zhttps://doi.org/10.1371/journal.pone.0110600https://doaj.org/toc/1932-6203Tandem fluorescent protein-tagged LC3s that were comprised of a protein tag that emits green fluorescence (e.g., EGFP or mWasabi) fused with another tag that emits red fluorescence (e.g. mCherry or TagRFP) were used for monitoring the maturation step of mammalian autophagosomes. A critical point for this tandem fluorescent-tagged LC3 was the sensitivity of green fluorescence at an acidic pH. EGFP and mWasabi continue to emit a weak, but significant, fluorescence at a pH of approximately 6. To overcome this issue, we focused on super-ecliptic pHluorin, which is a more pH-sensitive GFP variation. The green fluorescence of EGFP and mWasabi in the cells was still observed at weakly acidic levels (pH 6.0-6.5). In contrast, the fluorescence of pHluorin was more significantly quenched at pH 6.5, and was almost completely abolished at pH 5.5-6.0, indicating that pHluorin is more suitable for use in a tandem fluorescent protein-tag for monitoring autophagy. A pHluorin-mKate2 tandem fluorescence protein showed pH-sensitive green fluorescence and pH-resistant far-red fluorescence. We therefore generated expression plasmids for pHluorin-mKate2-tagged human LC3 (PK-hLC3), which could be used as a modifier for LC3-lipidation. The green and far-red fluorescent puncta of PK-hLC3 were increased under starvation conditions. Puncta that were green-negative, but far-red positive, were increased when autolysosomes accumulated, but few puncta of the mutant PK-hLC3ΔG that lacked the carboxyl terminal Gly essential for autophagy were observed in the cells under the same conditions. These results indicated that the PK-hLC3 were more appropriate for the pH-sensitive monitoring of the maturation step of autophagosomes.Isei TanidaTakashi UenoYasuo UchiyamaPublic Library of Science (PLoS)articleMedicineRScienceQENPLoS ONE, Vol 9, Iss 10, p e110600 (2014)
institution	DOAJ
collection	DOAJ
language	EN
topic	Medicine R Science Q
spellingShingle	Medicine R Science Q Isei Tanida Takashi Ueno Yasuo Uchiyama A super-ecliptic, pHluorin-mKate2, tandem fluorescent protein-tagged human LC3 for the monitoring of mammalian autophagy.
description	Tandem fluorescent protein-tagged LC3s that were comprised of a protein tag that emits green fluorescence (e.g., EGFP or mWasabi) fused with another tag that emits red fluorescence (e.g. mCherry or TagRFP) were used for monitoring the maturation step of mammalian autophagosomes. A critical point for this tandem fluorescent-tagged LC3 was the sensitivity of green fluorescence at an acidic pH. EGFP and mWasabi continue to emit a weak, but significant, fluorescence at a pH of approximately 6. To overcome this issue, we focused on super-ecliptic pHluorin, which is a more pH-sensitive GFP variation. The green fluorescence of EGFP and mWasabi in the cells was still observed at weakly acidic levels (pH 6.0-6.5). In contrast, the fluorescence of pHluorin was more significantly quenched at pH 6.5, and was almost completely abolished at pH 5.5-6.0, indicating that pHluorin is more suitable for use in a tandem fluorescent protein-tag for monitoring autophagy. A pHluorin-mKate2 tandem fluorescence protein showed pH-sensitive green fluorescence and pH-resistant far-red fluorescence. We therefore generated expression plasmids for pHluorin-mKate2-tagged human LC3 (PK-hLC3), which could be used as a modifier for LC3-lipidation. The green and far-red fluorescent puncta of PK-hLC3 were increased under starvation conditions. Puncta that were green-negative, but far-red positive, were increased when autolysosomes accumulated, but few puncta of the mutant PK-hLC3ΔG that lacked the carboxyl terminal Gly essential for autophagy were observed in the cells under the same conditions. These results indicated that the PK-hLC3 were more appropriate for the pH-sensitive monitoring of the maturation step of autophagosomes.
format	article
author	Isei Tanida Takashi Ueno Yasuo Uchiyama
author_facet	Isei Tanida Takashi Ueno Yasuo Uchiyama
author_sort	Isei Tanida
title	A super-ecliptic, pHluorin-mKate2, tandem fluorescent protein-tagged human LC3 for the monitoring of mammalian autophagy.
title_short	A super-ecliptic, pHluorin-mKate2, tandem fluorescent protein-tagged human LC3 for the monitoring of mammalian autophagy.
title_full	A super-ecliptic, pHluorin-mKate2, tandem fluorescent protein-tagged human LC3 for the monitoring of mammalian autophagy.
title_fullStr	A super-ecliptic, pHluorin-mKate2, tandem fluorescent protein-tagged human LC3 for the monitoring of mammalian autophagy.
title_full_unstemmed	A super-ecliptic, pHluorin-mKate2, tandem fluorescent protein-tagged human LC3 for the monitoring of mammalian autophagy.
title_sort	super-ecliptic, phluorin-mkate2, tandem fluorescent protein-tagged human lc3 for the monitoring of mammalian autophagy.
publisher	Public Library of Science (PLoS)
publishDate	2014
url	https://doaj.org/article/60ca3ad51f154a69bffb931223577b1e
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A super-ecliptic, pHluorin-mKate2, tandem fluorescent protein-tagged human LC3 for the monitoring of mammalian autophagy.

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