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Functioning of Fluorescent Proteins in Aggregates in Anthozoa Species and in Recombinant Artificial Models

Despite great advances in practical applications of fluorescent proteins (FPs), their natural function is poorly understood. FPs display complex spatio-temporal expression patterns in living Anthozoa coral polyps. Here we applied confocal microscopy, specifically, the fluorescence recovery after pho...

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Autores principales: Povarova, Natalia V., Petri, Natalia D., Blokhina, Anna E., Bogdanov, Alexey M., Gurskaya, Nadya G., Lukyanov, Konstantin A.
Formato: Online Artículo Texto
Lenguaje:English
Publicado: MDPI 2017
Materias:
Acceso en línea:https://www.ncbi.nlm.nih.gov/pmc/articles/PMC5535993/
https://www.ncbi.nlm.nih.gov/pubmed/28704934
http://dx.doi.org/10.3390/ijms18071503
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author Povarova, Natalia V.
Petri, Natalia D.
Blokhina, Anna E.
Bogdanov, Alexey M.
Gurskaya, Nadya G.
Lukyanov, Konstantin A.
author_facet Povarova, Natalia V.
Petri, Natalia D.
Blokhina, Anna E.
Bogdanov, Alexey M.
Gurskaya, Nadya G.
Lukyanov, Konstantin A.
author_sort Povarova, Natalia V.
collection PubMed
description Despite great advances in practical applications of fluorescent proteins (FPs), their natural function is poorly understood. FPs display complex spatio-temporal expression patterns in living Anthozoa coral polyps. Here we applied confocal microscopy, specifically, the fluorescence recovery after photobleaching (FRAP) technique to analyze intracellular localization and mobility of endogenous FPs in live tissues. We observed three distinct types of protein distributions in living tissues. One type of distribution, characteristic for Anemonia, Discosoma and Zoanthus, is free, highly mobile cytoplasmic localization. Another pattern is seen in FPs localized to numerous intracellular vesicles, observed in Clavularia. The third most intriguing type of intracellular localization is with respect to the spindle-shaped aggregates and lozenge crystals several micrometers in size observed in Zoanthus samples. No protein mobility within those structures was detected by FRAP. This finding encouraged us to develop artificial aggregating FPs. We constructed “trio-FPs” consisting of three tandem copies of tetrameric FPs and demonstrated that they form multiple bright foci upon expression in mammalian cells. High brightness of the aggregates is advantageous for early detection of weak promoter activities. Simultaneously, larger aggregates can induce significant cytostatic and cytotoxic effects and thus such tags are not suitable for long-term and high-level expression.
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spelling pubmed-55359932017-08-04 Functioning of Fluorescent Proteins in Aggregates in Anthozoa Species and in Recombinant Artificial Models Povarova, Natalia V. Petri, Natalia D. Blokhina, Anna E. Bogdanov, Alexey M. Gurskaya, Nadya G. Lukyanov, Konstantin A. Int J Mol Sci Article Despite great advances in practical applications of fluorescent proteins (FPs), their natural function is poorly understood. FPs display complex spatio-temporal expression patterns in living Anthozoa coral polyps. Here we applied confocal microscopy, specifically, the fluorescence recovery after photobleaching (FRAP) technique to analyze intracellular localization and mobility of endogenous FPs in live tissues. We observed three distinct types of protein distributions in living tissues. One type of distribution, characteristic for Anemonia, Discosoma and Zoanthus, is free, highly mobile cytoplasmic localization. Another pattern is seen in FPs localized to numerous intracellular vesicles, observed in Clavularia. The third most intriguing type of intracellular localization is with respect to the spindle-shaped aggregates and lozenge crystals several micrometers in size observed in Zoanthus samples. No protein mobility within those structures was detected by FRAP. This finding encouraged us to develop artificial aggregating FPs. We constructed “trio-FPs” consisting of three tandem copies of tetrameric FPs and demonstrated that they form multiple bright foci upon expression in mammalian cells. High brightness of the aggregates is advantageous for early detection of weak promoter activities. Simultaneously, larger aggregates can induce significant cytostatic and cytotoxic effects and thus such tags are not suitable for long-term and high-level expression. MDPI 2017-07-12 /pmc/articles/PMC5535993/ /pubmed/28704934 http://dx.doi.org/10.3390/ijms18071503 Text en © 2017 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (http://creativecommons.org/licenses/by/4.0/).
spellingShingle Article
Povarova, Natalia V.
Petri, Natalia D.
Blokhina, Anna E.
Bogdanov, Alexey M.
Gurskaya, Nadya G.
Lukyanov, Konstantin A.
Functioning of Fluorescent Proteins in Aggregates in Anthozoa Species and in Recombinant Artificial Models
title Functioning of Fluorescent Proteins in Aggregates in Anthozoa Species and in Recombinant Artificial Models
title_full Functioning of Fluorescent Proteins in Aggregates in Anthozoa Species and in Recombinant Artificial Models
title_fullStr Functioning of Fluorescent Proteins in Aggregates in Anthozoa Species and in Recombinant Artificial Models
title_full_unstemmed Functioning of Fluorescent Proteins in Aggregates in Anthozoa Species and in Recombinant Artificial Models
title_short Functioning of Fluorescent Proteins in Aggregates in Anthozoa Species and in Recombinant Artificial Models
title_sort functioning of fluorescent proteins in aggregates in anthozoa species and in recombinant artificial models
topic Article
url https://www.ncbi.nlm.nih.gov/pmc/articles/PMC5535993/
https://www.ncbi.nlm.nih.gov/pubmed/28704934
http://dx.doi.org/10.3390/ijms18071503
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