Department of Neuroscience and Cell Biology, University of Texas Medical Branch
Akimzhanov, A. M., Boehning, D. Monitoring Dynamic Changes In Mitochondrial Calcium Levels During Apoptosis Using A Genetically Encoded Calcium Sensor. J. Vis. Exp. (50), e2579, doi:10.3791/2579 (2011).
Dynamic changes in intracellular calcium concentration in response to various stimuli regulates many cellular processes such as proliferation, differentiation, and apoptosis1. During apoptosis, calcium accumulation in mitochondria promotes the release of pro-apoptotic factors from the mitochondria into the cytosol2. It is therefore of interest to directly measure mitochondrial calcium in living cells in situ during apoptosis. High-resolution fluorescent imaging of cells loaded with dual-excitation ratiometric and non-ratiometric synthetic calcium indicator dyes has been proven to be a reliable and versatile tool to study various aspects of intracellular calcium signaling. Measuring cytosolic calcium fluxes using these techniques is relatively straightforward. However, measuring intramitochondrial calcium levels in intact cells using synthetic calcium indicators such as rhod-2 and rhod-FF is more challenging. Synthetic indicators targeted to mitochondria have blunted responses to repetitive increases in mitochondrial calcium, and disrupt mitochondrial morphology3. Additionally, synthetic indicators tend to leak out of mitochondria over several hours which makes them unsuitable for long-term experiments. Thus, genetically encoded calcium indicators based upon green fluorescent protein (GFP)4 or aequorin5 targeted to mitochondria have greatly facilitated measurement of mitochondrial calcium dynamics. Here, we describe a simple method for real-time measurement of mitochondrial calcium fluxes in response to different stimuli. The method is based on fluorescence microscopy of 'ratiometric-pericam' which is selectively targeted to mitochondria. Ratiometric pericam is a calcium indicator based on a fusion of circularly permuted yellow fluorescent protein and calmodulin4. Binding of calcium to ratiometric pericam causes a shift of its excitation peak from 415 nm to 494 nm, while the emission spectrum, which peaks around 515 nm, remains unchanged. Ratiometric pericam binds a single calcium ion with a dissociation constant in vitro of ~1.7 μM4. These properties of ratiometric pericam allow the quantification of rapid and long-term changes in mitochondrial calcium concentration. Furthermore, we describe adaptation of this methodology to a standard wide-field calcium imaging microscope with commonly available filter sets. Using two distinct agonists, the purinergic agonist ATP and apoptosis-inducing drug staurosporine, we demonstrate that this method is appropriate for monitoring changes in mitochondrial calcium concentration with a temporal resolution of seconds to hours. Furthermore, we also demonstrate that ratiometric pericam is also useful for measuring mitochondrial fission/fragmentation during apoptosis. Thus, ratiometric pericam is particularly well suited for continuous long-term measurement of mitochondrial calcium dynamics during apoptosis.
1. Pericam-mt Transfection and Cell Preparation.
2. Microscope Setup and Image Acquisition
3. Image Processing and Analysis
4. Representative Results:
Figure 1. (A) Subcellular localization of ratiometric-pericam-mt. This first image shows live HeLa cell expressing ratiometric-pericam-mt. The second image shows fluorescent staining with mitochondrion-selective dye MitoTracker Red CMXRos. The yellow fluorescence in the merged image demonstrates co-localization of ratiometric-pericam-mt and MitoTracker fluorescence. (B) A series of pseudocolor ratio (495:380 nm) images of 4 HeLa cells expressing mt-ratiometric pericam treated with 10 mM ATP. (C) Quantification of changes in mitochondrial calcium levels in the region of interest (ROI) indicated in (B) treated with 10 mM ATP. (D) Series of pseudocolor ratio (495:380 nm) images of a HeLa cell expressing mt-ratiometric pericam treated with 0.5 μM staurosporine. Heterogeneity in the calcium response in individual mitochondria is evident, as well as significant fragmentation of mitochondria by 60 minutes. Some mitochondria have oscillatory increases in calcium (white arrow head), whereas others do not show significant changes in calcium level (yellow arrow head).(E) Quantification of increases in global mitochondrial calcium levels in a single HeLa cell after induction of apoptosis with 0.5 μM staurosporine.
Here we present a very simple method for measuring mitochondrial calcium using mitochondrial-targeted ratiometric pericam. As shown in Figure 1A, using standard widefield optics with no deconvolution it is possible to easily view individual mitochondria in HeLa cells with acceptable signal-to-noise ratio. This is because HeLa cells, like most cells in culture, flatten out significantly when adherent obviating the need for confocal microscopy or other specialized equipment. We have found similar results in Jurkat cells adhered to poly-lysine coated coverslips8. In contrast to methodologies using dyes, it is also possible to non-invasively monitor mitochondrial calcium levels for hours using genetically encoded calcium indicators such as ratiometric pericam (Figure 1E). This is especially important when analyzing mitochondrial calcium during cell death. Furthermore, it is also possible to visualize fission/fragmentation of mitochondria during the apoptotic process. As shown in Figures 1D and E, staurosporine treatment causes a slow increase in calcium in select subpopulations of mitochondria which peaks at 20 minutes. Calcium levels go down again concomitant with mitochondrial fragmentation before going up again 2 hours after treatment. This is consistent with the slow waves in cytosolic calcium induced by staurosporine treatment measured using Fura-29. Thus, important kinetic information can be obtained which is not possible with methods employing static measurements. Although we have presented ratios and not calcium concentrations in Figure 1, it is possible to calibrate the sensor in situ to calculate absolute calcium levels4, 10. One important caveat to consider is that ratiometric pericam is sensitive to pH4, 10. As both cytosolic and mitochondrial pH levels can change dramatically during apoptosis11, this is an important consideration. Titration of pH in isolated mitochondria expressing pericam demonstrate that increasing [H+] increases emission of pericam when excited at 495 nm, with little effect on emission stimulated by excitation at 410 nm, a property which has been exploited to simultaneously measure both mitochondrial calcium and pH12. Thus, selectively monitoring emission with 410 nm excitation provides a means to non-ratiometrically monitor mitochondrial calcium with reduced concern for pH. Finally, the protocol presented here only requires access to a standard epifluorescent microscope with widely available filters, thus making this technique accessible to most laboratories.
No conflicts of interest declared.
We would like to thank Atsushi Miyawaki for providing us with the ratiometric-pericam-mt expression construct. This work was supported by grant GM081685 from the National Institutes of Health (DB).
|Inverted wide-field fluorescent calcium imaging microscope||Nikon Instruments||MEA53310||Any wide-field inverted microscope equipped with 380 nm/495 nm excitation filters and appropriate longpass and emission filter can be easily adapted to use this protocol.|
|Imaging Software||Molecular Devices||Metafluor|
|Attofluor cell chamber||Invitrogen||A-7816||Any imaging chamber for an inverted microscope which allows perfusion would suffice.|
|Lipofectamine 2000||Invitrogen||11668-019||Most transfection reagents/protocols would be appropriate for these studies|
|MitoTracker Red CMXRos||Invitrogen||M7512|