Dataset Information

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

ABSTRACT: 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.

SUBMITTER: Waldeck-Weiermair M

PROVIDER: S-EPMC3448721 | biostudies-literature | 2012

REPOSITORIES: biostudies-literature

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

Waldeck-Weiermair Markus M Alam Muhammad Rizwan MR Khan Muhammad Jadoon MJ Deak Andras T AT Vishnu Neelanjan N Karsten Felix F Imamura Hiromi H Graier Wolfgang F WF Malli Roland R

PloS one 20120921 9

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 ...[more]

PMID: 23029314

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Project description:Store-operated Ca²+ entry (SOCE) has been associated with two types of channels: CRAC channels that require Orai1 and STIM1 and SOC channels that involve TRPC1, Orai1, and STIM1. While TRPC1 significantly contributes to SOCE and SOC channel activity, abrogation of Orai1 function eliminates SOCE and activation of TRPC1. The critical role of Orai1 in activation of TRPC1-SOC channels following Ca²+ store depletion has not yet been established. Herein we report that TRPC1 and Orai1 are components of distinct channels. We show that TRPC1/Orai1/STIM1-dependent I(SOC), activated in response to Ca²+ store depletion, is composed of TRPC1/STIM1-mediated non-selective cation current and Orai1/STIM1-mediated I(CRAC); the latter is detected when TRPC1 function is suppressed by expression of shTRPC1 or a STIM1 mutant that lacks TRPC1 gating, STIM1(???EE???). In addition to gating TRPC1 and Orai1, STIM1 mediates the recruitment and association of the channels within ER/PM junctional domains, a critical step in TRPC1 activation. Importantly, we show that Ca²+ entry via Orai1 triggers plasma membrane insertion of TRPC1, which is prevented by blocking SOCE with 1 µM Gd³+, removal of extracellular Ca²+, knockdown of Orai1, or expression of dominant negative mutant Orai1 lacking a functional pore, Orai1-E106Q. In cells expressing another pore mutant of Orai1, Orai1-E106D, TRPC1 trafficking is supported in Ca²+-containing, but not Ca²+-free, medium. Consistent with this, I(CRAC) is activated in cells pretreated with thapsigargin in Ca²+-free medium while I(SOC) is activated in cells pretreated in Ca²+-containing medium. Significantly, TRPC1 function is required for sustained K(Ca) activity and contributes to NF?B activation while Orai1 is sufficient for NFAT activation. Together, these findings reveal an as-yet unidentified function for Orai1 that explains the critical requirement of the channel in the activation of TRPC1 following Ca²+ store depletion. We suggest that coordinated regulation of the surface expression of TRPC1 by Orai1 and gating by STIM1 provides a mechanism for rapidly modulating and maintaining SOCE-generated Ca²+ signals. By recruiting ion channels and other signaling pathways, Orai1 and STIM1 concertedly impact a variety of critical cell functions that are initiated by SOCE.

Dataset Information

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

Publications

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

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