Spatiotemporal correlations between cytosolic and mitochondrial Ca(2+) signals using a novel red-shifted mitochondrial targeted cameleon.

The transfer of Ca(2+) from the cytosol into the lumen of mitochondria is a crucial process that impacts cell signaling in multiple ways. Cytosolic Ca(2+) ([Ca(2+)](cyto)) can be excellently quantified with the ratiometric Ca(2+) probe fura-2, while genetically encoded Förster resonance energy trans...

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Autores principales:	Markus Waldeck-Weiermair, Muhammad Rizwan Alam, Muhammad Jadoon Khan, Andras T Deak, Neelanjan Vishnu, Felix Karsten, Hiromi Imamura, Wolfgang F Graier, Roland Malli
Formato:	article
Lenguaje:	EN
Publicado:	Public Library of Science (PLoS) 2012
Materias:	Medicine R Science Q
Acceso en línea:	https://doaj.org/article/a325585103634d5f88d729a960956215
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spelling	oai:doaj.org-article:a325585103634d5f88d729a9609562152021-11-18T07:04:28ZSpatiotemporal correlations between cytosolic and mitochondrial Ca(2+) signals using a novel red-shifted mitochondrial targeted cameleon.1932-620310.1371/journal.pone.0045917https://doaj.org/article/a325585103634d5f88d729a9609562152012-01-01T00:00:00Zhttps://www.ncbi.nlm.nih.gov/pmc/articles/pmid/23029314/pdf/?tool=EBIhttps://doaj.org/toc/1932-6203The transfer of Ca(2+) from the cytosol into the lumen of mitochondria is a crucial process that impacts cell signaling in multiple ways. Cytosolic Ca(2+) ([Ca(2+)](cyto)) can be excellently quantified with the ratiometric Ca(2+) probe fura-2, while genetically encoded Förster resonance energy transfer (FRET)-based fluorescent Ca(2+) sensors, the cameleons, are efficiently used to specifically measure Ca(2+) within organelles. However, because of a significant overlap of the fura-2 emission with the spectra of the cyan and yellow fluorescent protein of most of the existing cameleons, the measurement of fura-2 and cameleons within one given cell is a complex task. In this study, we introduce a novel approach to simultaneously assess [Ca(2+)](cyto) and mitochondrial Ca(2+) ([Ca(2+)](mito)) signals at the single cell level. In order to eliminate the spectral overlap we developed a novel red-shifted cameleon, D1GO-Cam, in which the green and orange fluorescent proteins were used as the FRET pair. This ratiometric Ca(2+) probe could be successfully targeted to mitochondria and was suitable to be used simultaneously with fura-2 to correlate [Ca(2+)](cyto) and [Ca(2+)](mito) within same individual cells. Our data indicate that depending on the kinetics of [Ca(2+)](cyto) rises there is a significant lag between onset of [Ca(2+)](cyto) and [Ca(2+)](mito) signals, pointing to a certain threshold of [Ca(2+)](cyto) necessary to activate mitochondrial Ca(2+) uptake. The temporal correlation between [Ca(2+)](mito) and [Ca(2+)](cyto) as well as the efficiency of the transfer of Ca(2+) from the cytosol into mitochondria varies between different cell types. Moreover, slow mitochondrial Ca(2+) extrusion and a desensitization of mitochondrial Ca(2+) uptake cause a clear difference in patterns of mitochondrial and cytosolic Ca(2+) oscillations of pancreatic beta-cells in response to D-glucose.Markus Waldeck-WeiermairMuhammad Rizwan AlamMuhammad Jadoon KhanAndras T DeakNeelanjan VishnuFelix KarstenHiromi ImamuraWolfgang F GraierRoland MalliPublic Library of Science (PLoS)articleMedicineRScienceQENPLoS ONE, Vol 7, Iss 9, p e45917 (2012)
institution	DOAJ
collection	DOAJ
language	EN
topic	Medicine R Science Q
spellingShingle	Medicine R Science Q Markus Waldeck-Weiermair Muhammad Rizwan Alam Muhammad Jadoon Khan Andras T Deak Neelanjan Vishnu Felix Karsten Hiromi Imamura Wolfgang F Graier Roland Malli Spatiotemporal correlations between cytosolic and mitochondrial Ca(2+) signals using a novel red-shifted mitochondrial targeted cameleon.
description	The transfer of Ca(2+) from the cytosol into the lumen of mitochondria is a crucial process that impacts cell signaling in multiple ways. Cytosolic Ca(2+) ([Ca(2+)](cyto)) can be excellently quantified with the ratiometric Ca(2+) probe fura-2, while genetically encoded Förster resonance energy transfer (FRET)-based fluorescent Ca(2+) sensors, the cameleons, are efficiently used to specifically measure Ca(2+) within organelles. However, because of a significant overlap of the fura-2 emission with the spectra of the cyan and yellow fluorescent protein of most of the existing cameleons, the measurement of fura-2 and cameleons within one given cell is a complex task. In this study, we introduce a novel approach to simultaneously assess [Ca(2+)](cyto) and mitochondrial Ca(2+) ([Ca(2+)](mito)) signals at the single cell level. In order to eliminate the spectral overlap we developed a novel red-shifted cameleon, D1GO-Cam, in which the green and orange fluorescent proteins were used as the FRET pair. This ratiometric Ca(2+) probe could be successfully targeted to mitochondria and was suitable to be used simultaneously with fura-2 to correlate [Ca(2+)](cyto) and [Ca(2+)](mito) within same individual cells. Our data indicate that depending on the kinetics of [Ca(2+)](cyto) rises there is a significant lag between onset of [Ca(2+)](cyto) and [Ca(2+)](mito) signals, pointing to a certain threshold of [Ca(2+)](cyto) necessary to activate mitochondrial Ca(2+) uptake. The temporal correlation between [Ca(2+)](mito) and [Ca(2+)](cyto) as well as the efficiency of the transfer of Ca(2+) from the cytosol into mitochondria varies between different cell types. Moreover, slow mitochondrial Ca(2+) extrusion and a desensitization of mitochondrial Ca(2+) uptake cause a clear difference in patterns of mitochondrial and cytosolic Ca(2+) oscillations of pancreatic beta-cells in response to D-glucose.
format	article
author	Markus Waldeck-Weiermair Muhammad Rizwan Alam Muhammad Jadoon Khan Andras T Deak Neelanjan Vishnu Felix Karsten Hiromi Imamura Wolfgang F Graier Roland Malli
author_facet	Markus Waldeck-Weiermair Muhammad Rizwan Alam Muhammad Jadoon Khan Andras T Deak Neelanjan Vishnu Felix Karsten Hiromi Imamura Wolfgang F Graier Roland Malli
author_sort	Markus Waldeck-Weiermair
title	Spatiotemporal correlations between cytosolic and mitochondrial Ca(2+) signals using a novel red-shifted mitochondrial targeted cameleon.
title_short	Spatiotemporal correlations between cytosolic and mitochondrial Ca(2+) signals using a novel red-shifted mitochondrial targeted cameleon.
title_full	Spatiotemporal correlations between cytosolic and mitochondrial Ca(2+) signals using a novel red-shifted mitochondrial targeted cameleon.
title_fullStr	Spatiotemporal correlations between cytosolic and mitochondrial Ca(2+) signals using a novel red-shifted mitochondrial targeted cameleon.
title_full_unstemmed	Spatiotemporal correlations between cytosolic and mitochondrial Ca(2+) signals using a novel red-shifted mitochondrial targeted cameleon.
title_sort	spatiotemporal correlations between cytosolic and mitochondrial ca(2+) signals using a novel red-shifted mitochondrial targeted cameleon.
publisher	Public Library of Science (PLoS)
publishDate	2012
url	https://doaj.org/article/a325585103634d5f88d729a960956215
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Spatiotemporal correlations between cytosolic and mitochondrial Ca(2+) signals using a novel red-shifted mitochondrial targeted cameleon.

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