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Generation of Red-Shifted Cameleons for Imaging Ca²⁺ Dynamics of the Endoplasmic Reticulum.

Waldeck-Weiermair M, Bischof H, Blass S, Deak AT, Klec C, Graier T, Roller C, Rost R, Eroglu E, Gottschalk B, Hofmann NA, Graier WF, Malli R - Sensors (Basel) (2015)

Bottom Line: Cameleons are sophisticated genetically encoded fluorescent probes that allow quantifying cellular Ca2+ signals.The probes are based on Förster resonance energy transfer (FRET) between terminally located fluorescent proteins (FPs), which move together upon binding of Ca2+ to the central calmodulin myosin light chain kinase M13 domain.However, ER targeted GFP/RFP-based probes were suitable to sense ER Ca2+ in a reliable manner.

View Article: PubMed Central - PubMed

Affiliation: Institute of Molecular Biology and Biochemistry, Centre of Molecular Medicine, Medical University of Graz, Harrachgasse 21, 8010 Graz, Austria. markus.weiermair@medunigraz.at.

ABSTRACT
Cameleons are sophisticated genetically encoded fluorescent probes that allow quantifying cellular Ca2+ signals. The probes are based on Förster resonance energy transfer (FRET) between terminally located fluorescent proteins (FPs), which move together upon binding of Ca2+ to the central calmodulin myosin light chain kinase M13 domain. Most of the available cameleons consist of cyan and yellow FPs (CFP and YFP) as the FRET pair. However, red-shifted versions with green and orange or red FPs (GFP, OFP, RFP) have some advantages such as less phototoxicity and minimal spectral overlay with autofluorescence of cells and fura-2, a prominent chemical Ca2+ indicator. While GFP/OFP- or GFP/RFP-based cameleons have been successfully used to study cytosolic and mitochondrial Ca2+ signals, red-shifted cameleons to visualize Ca2+ dynamics of the endoplasmic reticulum (ER) have not been developed so far. In this study, we generated and tested several ER targeted red-shifted cameleons. Our results show that GFP/OFP-based cameleons due to miss-targeting and their high Ca2+ binding affinity are inappropriate to record ER Ca2+ signals. However, ER targeted GFP/RFP-based probes were suitable to sense ER Ca2+ in a reliable manner. With this study we increased the palette of cameleons for visualizing Ca2+ dynamics within the main intracellular Ca2+ store.

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Related in: MedlinePlus

Column statistics of maximal ER Ca2+-release in HeLa cells transfected with individual GFP/RFP-based cameleons. Bars representing FRET change upon treatment with 100 µM histamine and 15 µM BHQ detected by D1ERRG-Cam1 (pink bar, n = 13), D1ERRG-Cam2 (purple bar, n = 10), D1ERGR (light grey bar, n = 4), D1ERTG (violet bar, n = 7), D1ERRC (light green bar, n = 10), D1ERCR (grey bar, n = 4), D1ERGmR2 (black bar, n = 11), D1ERmR2G (dark red bar, n = 6), D1ERmR2C (blue bar, n = 11) or D1ERCmR2 (red bar, n = 7). * P < 0.05 for D1ERCmR2 vs. all other indicators.
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sensors-15-13052-f003: Column statistics of maximal ER Ca2+-release in HeLa cells transfected with individual GFP/RFP-based cameleons. Bars representing FRET change upon treatment with 100 µM histamine and 15 µM BHQ detected by D1ERRG-Cam1 (pink bar, n = 13), D1ERRG-Cam2 (purple bar, n = 10), D1ERGR (light grey bar, n = 4), D1ERTG (violet bar, n = 7), D1ERRC (light green bar, n = 10), D1ERCR (grey bar, n = 4), D1ERGmR2 (black bar, n = 11), D1ERmR2G (dark red bar, n = 6), D1ERmR2C (blue bar, n = 11) or D1ERCmR2 (red bar, n = 7). * P < 0.05 for D1ERCmR2 vs. all other indicators.

Mentions: Under consideration of the fluorescent properties of various FPs, we selected several green FRET-donor and red FRET-acceptor proteins for the generation of novel functional red-shifted ER targeted cameleons (Table S2). For this purpose, we also included the green Clover and the red mRuby FP that had been recently used as an optimized FRET pair for the development of red-shifted genetically encoded probes for kinase activities and the membrane potential [41] Moreover the N- or C-terminal orientation of the FRET-donor and -acceptor FPs within ER targeted cameleons was considered as a relevant determinant of the sensors’ properties (ER localization and Ca2+ sensitivity). In total, we generated and tested 10 different ER targeted GFP/RFP-based cameleons that differ either in the FP-FRET pair or the orientation of FPs within the construct (C- or N-terminal location, Table 1). To test the properties of the novel red-shifted ER targeted cameleons, HeLa cells expressing the probes were imaged. During imaging at different excitation wavelengths ranging from 400 to 490 nm, ER Ca2+ levels were manipulated by cell treatment with digitonin/ionomycin in the presence and absence of Ca2+ in the experimental buffer (Figure 2A,B). Out of these spectral scans, the signal to noise ratio (SNR) for each sensor at each excitation wavelength was calculated to evaluate the best working red-shifted cameleon in the ER under these conditions (Table 2). All the GFP/RFP-based sensors were suitable to detect changes of [Ca2+]ER (Figure 2). D1ERTG (Table 1 and Figure 2A), which was built of the bright tandem dimer Tomato as the FRET acceptor and EGFP as the FRET donor at the C-terminal end, appeared with the highest basal FRET ratio (Figure 2A), while the SNR was rather low (Table 2). Interestingly, the orientation of the FPs within the EGFP/tagRFP-consisting cameleons influenced the SNR significantly. If the tagRFP FRET acceptor was on the N-terminus of the ER targeted cameleon, better signals could be obtained (Figure 2A and Table 2). However, the cameleon with Clover on its N-terminal end and mRuby2 on its C-terminal end, named D1ERCmR2, showed the best performance with the highest SNR (Table 1 and Table 2; Figure 2B). This finding is in line with the original report about the usage of the Clover/mRuby FRET pair in genetically encoded probes [41] These are reporters of kinase activity (Camuiα-CR, AKAR-CR), small GTPase activity (Raichu-CR) and transmembrane voltage (VSFP-CR) [41]. Within all these indicators, the standard FRET pair CFP/YFP has been replaced by Clover as donor at the N-terminal end and by mRuby2 as FRET acceptor at the C-terminal end. Moreover, Clover and mRuby2 were characterized as the brightest green and red FPs exhibiting the highest Förster radius of any ratiometric FRET pair to date. We also tested if the orientation of Clover and mRuby in ER targeted cameleons impact the performance of the probe. In this case, the exchange of the FPs resulting in the construct D1ERmR2C (with mRuby2 on the N-terminus and Clover on the C-terminus) dramatically worsened the respective signal (Figure 2B; compare red curve with blue curve and respective SNR values in Table 2). Similar results were also found in intact HeLa cells that were treated for several minutes with histamine and the reversible sarcoplasmic/endoplasmic reticulum calcium ATPase (SERCA) inhibitor 2,5-di-tert-butylhydroquinone (BHQ) to transiently deplete the ER Ca2+ content (Figure 3). In line with the data obtained from the spectral scans, D1ERCmR2 showed a significantly higher delta FRET ratio signal in comparison with all other ER targeted probes in intact cells under these conditions (Figure 3). These findings indicate that in GFP/RFP-based cameleons that contain D1 as the Ca2+ sensing domain keep the high Kd of approximately 200 µM and are, hence, suitable to monitor Ca2+ dynamics of the ER. However, the origin and orientation of FPs forming the FRET pair within ER targeted cameleons have a huge impact on FRET ratio signals in response to ER Ca2+ mobilization.


Generation of Red-Shifted Cameleons for Imaging Ca²⁺ Dynamics of the Endoplasmic Reticulum.

Waldeck-Weiermair M, Bischof H, Blass S, Deak AT, Klec C, Graier T, Roller C, Rost R, Eroglu E, Gottschalk B, Hofmann NA, Graier WF, Malli R - Sensors (Basel) (2015)

Column statistics of maximal ER Ca2+-release in HeLa cells transfected with individual GFP/RFP-based cameleons. Bars representing FRET change upon treatment with 100 µM histamine and 15 µM BHQ detected by D1ERRG-Cam1 (pink bar, n = 13), D1ERRG-Cam2 (purple bar, n = 10), D1ERGR (light grey bar, n = 4), D1ERTG (violet bar, n = 7), D1ERRC (light green bar, n = 10), D1ERCR (grey bar, n = 4), D1ERGmR2 (black bar, n = 11), D1ERmR2G (dark red bar, n = 6), D1ERmR2C (blue bar, n = 11) or D1ERCmR2 (red bar, n = 7). * P < 0.05 for D1ERCmR2 vs. all other indicators.
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4507692&req=5

sensors-15-13052-f003: Column statistics of maximal ER Ca2+-release in HeLa cells transfected with individual GFP/RFP-based cameleons. Bars representing FRET change upon treatment with 100 µM histamine and 15 µM BHQ detected by D1ERRG-Cam1 (pink bar, n = 13), D1ERRG-Cam2 (purple bar, n = 10), D1ERGR (light grey bar, n = 4), D1ERTG (violet bar, n = 7), D1ERRC (light green bar, n = 10), D1ERCR (grey bar, n = 4), D1ERGmR2 (black bar, n = 11), D1ERmR2G (dark red bar, n = 6), D1ERmR2C (blue bar, n = 11) or D1ERCmR2 (red bar, n = 7). * P < 0.05 for D1ERCmR2 vs. all other indicators.
Mentions: Under consideration of the fluorescent properties of various FPs, we selected several green FRET-donor and red FRET-acceptor proteins for the generation of novel functional red-shifted ER targeted cameleons (Table S2). For this purpose, we also included the green Clover and the red mRuby FP that had been recently used as an optimized FRET pair for the development of red-shifted genetically encoded probes for kinase activities and the membrane potential [41] Moreover the N- or C-terminal orientation of the FRET-donor and -acceptor FPs within ER targeted cameleons was considered as a relevant determinant of the sensors’ properties (ER localization and Ca2+ sensitivity). In total, we generated and tested 10 different ER targeted GFP/RFP-based cameleons that differ either in the FP-FRET pair or the orientation of FPs within the construct (C- or N-terminal location, Table 1). To test the properties of the novel red-shifted ER targeted cameleons, HeLa cells expressing the probes were imaged. During imaging at different excitation wavelengths ranging from 400 to 490 nm, ER Ca2+ levels were manipulated by cell treatment with digitonin/ionomycin in the presence and absence of Ca2+ in the experimental buffer (Figure 2A,B). Out of these spectral scans, the signal to noise ratio (SNR) for each sensor at each excitation wavelength was calculated to evaluate the best working red-shifted cameleon in the ER under these conditions (Table 2). All the GFP/RFP-based sensors were suitable to detect changes of [Ca2+]ER (Figure 2). D1ERTG (Table 1 and Figure 2A), which was built of the bright tandem dimer Tomato as the FRET acceptor and EGFP as the FRET donor at the C-terminal end, appeared with the highest basal FRET ratio (Figure 2A), while the SNR was rather low (Table 2). Interestingly, the orientation of the FPs within the EGFP/tagRFP-consisting cameleons influenced the SNR significantly. If the tagRFP FRET acceptor was on the N-terminus of the ER targeted cameleon, better signals could be obtained (Figure 2A and Table 2). However, the cameleon with Clover on its N-terminal end and mRuby2 on its C-terminal end, named D1ERCmR2, showed the best performance with the highest SNR (Table 1 and Table 2; Figure 2B). This finding is in line with the original report about the usage of the Clover/mRuby FRET pair in genetically encoded probes [41] These are reporters of kinase activity (Camuiα-CR, AKAR-CR), small GTPase activity (Raichu-CR) and transmembrane voltage (VSFP-CR) [41]. Within all these indicators, the standard FRET pair CFP/YFP has been replaced by Clover as donor at the N-terminal end and by mRuby2 as FRET acceptor at the C-terminal end. Moreover, Clover and mRuby2 were characterized as the brightest green and red FPs exhibiting the highest Förster radius of any ratiometric FRET pair to date. We also tested if the orientation of Clover and mRuby in ER targeted cameleons impact the performance of the probe. In this case, the exchange of the FPs resulting in the construct D1ERmR2C (with mRuby2 on the N-terminus and Clover on the C-terminus) dramatically worsened the respective signal (Figure 2B; compare red curve with blue curve and respective SNR values in Table 2). Similar results were also found in intact HeLa cells that were treated for several minutes with histamine and the reversible sarcoplasmic/endoplasmic reticulum calcium ATPase (SERCA) inhibitor 2,5-di-tert-butylhydroquinone (BHQ) to transiently deplete the ER Ca2+ content (Figure 3). In line with the data obtained from the spectral scans, D1ERCmR2 showed a significantly higher delta FRET ratio signal in comparison with all other ER targeted probes in intact cells under these conditions (Figure 3). These findings indicate that in GFP/RFP-based cameleons that contain D1 as the Ca2+ sensing domain keep the high Kd of approximately 200 µM and are, hence, suitable to monitor Ca2+ dynamics of the ER. However, the origin and orientation of FPs forming the FRET pair within ER targeted cameleons have a huge impact on FRET ratio signals in response to ER Ca2+ mobilization.

Bottom Line: Cameleons are sophisticated genetically encoded fluorescent probes that allow quantifying cellular Ca2+ signals.The probes are based on Förster resonance energy transfer (FRET) between terminally located fluorescent proteins (FPs), which move together upon binding of Ca2+ to the central calmodulin myosin light chain kinase M13 domain.However, ER targeted GFP/RFP-based probes were suitable to sense ER Ca2+ in a reliable manner.

View Article: PubMed Central - PubMed

Affiliation: Institute of Molecular Biology and Biochemistry, Centre of Molecular Medicine, Medical University of Graz, Harrachgasse 21, 8010 Graz, Austria. markus.weiermair@medunigraz.at.

ABSTRACT
Cameleons are sophisticated genetically encoded fluorescent probes that allow quantifying cellular Ca2+ signals. The probes are based on Förster resonance energy transfer (FRET) between terminally located fluorescent proteins (FPs), which move together upon binding of Ca2+ to the central calmodulin myosin light chain kinase M13 domain. Most of the available cameleons consist of cyan and yellow FPs (CFP and YFP) as the FRET pair. However, red-shifted versions with green and orange or red FPs (GFP, OFP, RFP) have some advantages such as less phototoxicity and minimal spectral overlay with autofluorescence of cells and fura-2, a prominent chemical Ca2+ indicator. While GFP/OFP- or GFP/RFP-based cameleons have been successfully used to study cytosolic and mitochondrial Ca2+ signals, red-shifted cameleons to visualize Ca2+ dynamics of the endoplasmic reticulum (ER) have not been developed so far. In this study, we generated and tested several ER targeted red-shifted cameleons. Our results show that GFP/OFP-based cameleons due to miss-targeting and their high Ca2+ binding affinity are inappropriate to record ER Ca2+ signals. However, ER targeted GFP/RFP-based probes were suitable to sense ER Ca2+ in a reliable manner. With this study we increased the palette of cameleons for visualizing Ca2+ dynamics within the main intracellular Ca2+ store.

Show MeSH
Related in: MedlinePlus