Mitochondria can utilize the electrochemical potential across their inner membrane (ΔΨm) to sequester calcium (Ca2+), allowing them to shape cytosolic Ca2+ signaling within the cell. We describe a method for simultaneously measuring mitochondria Ca2+ uptake and ΔΨm in live cells using fluorescent dyes and confocal microscopy.
Apart from their essential role in generating ATP, mitochondria also act as local calcium (Ca2+) buffers to tightly regulate intracellular Ca2+ concentration. To do this, mitochondria utilize the electrochemical potential across their inner membrane (ΔΨm) to sequester Ca2+. The influx of Ca2+ into the mitochondria stimulates three rate-limiting dehydrogenases of the citric acid cycle, increasing electron transfer through the oxidative phosphorylation (OXPHOS) complexes. This stimulation maintains ΔΨm, which is temporarily dissipated as the positive calcium ions cross the mitochondrial inner membrane into the mitochondrial matrix.
We describe here a method for simultaneously measuring mitochondria Ca2+ uptake and ΔΨm in live cells using confocal microscopy. By permeabilizing the cells, mitochondrial Ca2+ can be measured using the fluorescent Ca2+ indicator Fluo-4, AM, with measurement of ΔΨm using the fluorescent dye tetramethylrhodamine, methyl ester, perchlorate (TMRM). The benefit of this system is that there is very little spectral overlap between the fluorescent dyes, allowing accurate measurement of mitochondrial Ca2+ and ΔΨm simultaneously. Using the sequential addition of Ca2+ aliquots, mitochondrial Ca2+ uptake can be monitored, and the concentration at which Ca2+ induces mitochondrial membrane permeability transition and the loss of ΔΨm determined.
Mitochondria play an important role in regulating intracellular Ca2+ concentration by acting as local Ca2+ buffers1. Ca2+ enters the mitochondria via the Ca2+ uniporter, a process driven by the electrochemical gradient that exists across the mitochondrial inner membrane (ΔΨm)2. Once inside the mitochondrial matrix, Ca2+ can activate oxidative phosphorylation by stimulating three rate-limiting dehydrogenases of the citric acid cycle3. This stimulation maintains ΔΨm, which is temporarily dissipated as the positive calcium ions cross the mitochondrial inner membrane into the mitochondrial matrix. If the Ca2+ concentration within the mitochondria becomes very high, mitochondrial permeability transition can be initiated, resulting in the dissipation of ΔΨm, the cessation of oxidative phosphorylation and the induction of cell death signaling pathways4.
The important role that mitochondria play in the spatial buffering of cellular calcium makes the accurate monitoring of mitochondrial calcium critical. Various methods have been established to monitor mitochondrial calcium, including the use of rhodamine based dyes. One such dye, Rhod-2, AM, is quite effective at partitioning to the mitochondria to measure mitochondrial Ca2+ levels5,6. However, care must be used as some dye will accumulate in other organelles, such as liposomes, or remain in the cell cytosol. Nevertheless, downstream analyses can be employed to distinguish these signals from those from the mitochondria7.
Another technique to monitor mitochondrial calcium utilizes fluorescent reporter constructs8. The benefit of these genetically encoded probes is that they can be specifically targeted to the mitochondria by using endogenous N-terminal peptides, for example the N-terminal targeting signal of human COX subunit VIII. This system has been employed to generate a mitochondrial-targeted aequorin probe which has proved extremely useful for investigating mitochondrial calcium signaling9. The main drawback of these genetically encoded probes is that they need to be introduced into the cells by transient expression (which is not feasible for certain cell types and can produce variable results) or by creating stable expression systems (which is time consuming).
To circumvent the problems outlined above, we have developed a new protocol to measure mitochondrial Ca2+ and ΔΨm simultaneously. This protocol is based on a previously described method that adds exogenous calcium to permeabilized cells10. Our protocol has three main advantages over other methods: firstly, we use Fluo-4, AM and TMRM to monitor mitochondrial Ca2+ and ΔΨm, two dyes that have very distinct spectral properties; secondly, the cells are permeabilized so that the Fluo-4 signal is only detecting mitochondrial Ca2+ and not Ca2+ localized to other organelles or the cell cytosol; and thirdly, the use of Fluo-4 to detect mitochondrial Ca2+ allows for fast and simple cell staining, negating any cell transfection or transformation issues that exist if using genetically encoded probes.
1. Preparation of Cells
2. Buffers for TMRM and Fluo-4 Imaging
3. Staining of Cells with TMRM and Fluo-4, AM
4. Cell Imaging
5. Image Analysis
6. Calculating the Final Free Ca2+ Ion Concentration [Ca2+]
We have used this protocol to examine the effects of an MT-ND5 mutation on the ability of 143B cell mitochondria to buffer increases in calcium12. In the example shown here, control 143B cells were loaded with TMRM and Fluo-4, AM before permeabilization with digitonin. After 5 min of imaging, eight sequential additions of a 1:100 dilution of 40 mM exogenous CaCl2 were made, with the final free Ca2+ ion concentration [Ca2+] calculated.
Following each CaCl2 addition, the TMRM signal decreases as the influx of Ca2+ ions into the mitochondria dissipates the mitochondrial membrane potential ΔΨm (Figure 1). ΔΨm then recovers as respiration is increased to maintain ΔΨm. This occurs four times after each CaCl2 addition. Following the fifth addition, the mitochondrial Ca2+ ion concentration reaches a critical level, at which point mitochondrial permeability transition occurs and ΔΨm dissipates rapidly. A small ΔΨm is evident following permeability transition, as the addition of 10 µM FCCP still induces a collapse of ΔΨm (Figure 1).
The mitochondrial calcium concentration (Fluo-4 signal) begins to increase following the second CaCl2 addition when the free [Ca2+] in the media reaches 0.32 μM (Figure 2). Each subsequent CaCl2 addition caused a progressive increase in mitochondrial calcium.
Images of the simultaneous measurements of ΔΨm (TMRM, red signal) and mitochondrial calcium (Fluo-4, green signal) are shown (Figure 3).
Figure 1: Measurement of mitochondrial ΔΨm in digitonin permeabilized 143B cells using TMRM. 143B cells were preloaded with Fluo-4, AM and TMRM before permeabilization with digitonin. A 1:100 dilution of 40 mM exogenous CaCl2 was added in sequential aliquots. The final free [Ca2+] in the media is indicated for each addition (*). FCCP was added to a final concentration of 10 µM after approximately 40 min. ΔΨm is shown in red, represented by the relative intensity (R.I.) of the TMRM signal. Data is mean ± s.d. n = 3. Figure is adapted with permission from reference12. Please click here to view a larger version of this figure.
Figure 2: Measurement of mitochondrial calcium in digitonin permeabilized 143B cells using Fluo-4, AM. 143B cells were preloaded with Fluo-4, AM and TMRM before permeabilization with digitonin. A 1:100 dilution of 40 mM exogenous CaCl2 was added in sequential aliquots. The final free [Ca2+] in the media is indicated for each addition (*). FCCP was added to a final concentration of 10 µM after approximately 40 min. Mitochondrial calcium is shown in green, represented by the relative intensity (R.I.) of the Fluo-4 signal. Data is mean ± s.d. n = 3. Figure is adapted with permission from reference12. Please click here to view a larger version of this figure.
Figure 3: Simultaneous measurement of mitochondrial calcium and ΔΨm in digitonin permeabilized 143B cells using Fluo-4, AM and TMRM. 143B cells were preloaded with Fluo-4, AM and TMRM before permeabilization with digitonin. A 1:100 dilution of 40 mM exogenous CaCl2 was added in sequential aliquots. The final free [Ca2+] in the media is indicated for each addition. Representative confocal images showing the simultaneous staining of ΔΨm (TMRM, red) and mitochondrial calcium (Fluo-4, green). Note that the Fluo-4 signal is hard to detect at the resting calcium concentration of 0.062 μM. White scale bars = 10 μm. Figure is adapted with permission from reference12. Please click here to view a larger version of this figure.
Calcium plays a critical role in many cell processes, including muscle contraction, neuronal signaling and cell proliferation13. Increases in cell calcium concentrations are often associated with energy demand, with calcium able to directly stimulate mitochondrial oxidative phosphorylation to raise ATP generation3. It is therefore essential that we have the ability to effectively monitor mitochondrial calcium accumulation and to be able to compare how this function is affected by both genetic factors and pharmacological agents.
Critical steps within the protocol
This protocol describes the ability to monitor mitochondria calcium accumulation and ΔΨm simultaneously with Fluo-4, AM and TMRM. During the staining process it is critical to incubate the cells at room temperature to optimize the effectiveness of Fluo-4, AM in detecting mitochondrial calcium. Incubation at room temperature reduces the activity of cytosolic esterases, allowing the uncharged Fluo-4, AM to cross the mitochondrial membranes and enter the mitochondrial matrix. Conversely, incubation at 37 °C enhances esterase activity in the cytosol, cleaving off the AM ester to leave a charged Fluo-4 dye that would not be able to cross the mitochondrial membranes effectively.
Once cell staining has been performed, the cells are immersed in IM imaging solution. This solution has a higher TMRM concentration compared to the initial RS staining solution. Now that the cells are permeabilized, the TMRM no longer equilibrates across the plasma membrane. Therefore, the TMRM concentration is raised in the IM imaging solution to achieve the correct equilibration across the mitochondrial inner membrane. It is also critical at this stage to include thapsigargin in the IM imaging solution. Thapsigargin blocks the endoplasmic reticulum (ER) Ca2+ATPase, ensuring that the Fluo-4 signal is not detecting ER calcium.
Modifications and troubleshooting
Some cell types can express the multidrug resistance transporter (also known as MDR1 or P-glycoprotein) on their plasma membranes. This protein is known to export fluorescent indicators, including AM ester derivatives, from the cytosol out of the cell14. If the transporter is expressed, both Fluo-4, AM and TMRM may be exported from the cell cytosol, resulting in sub-optimal staining. To block this effect, Verapamil can be added to the RS staining solution to block the multidrug resistance transporter, inhibiting Fluo-4, AM and TMRM export during the staining process.
By permeabilizing the cells to be analyzed, Fluo-4, AM can be used to detect mitochondrial Ca2+, with no competing signals from other organelles or the cytosol. We routinely use the non-ionic detergent digitonin for permeabilization at a concentration of 25 µg/ml. This concentration may need to be adjusted to ensure that solubilization of the mitochondrial membranes does not occur. This can be determined empirically by assessing the intensity of the TMRM signal at different concentrations of digitonin. If the concentration of digitonin is too high, the mitochondrial membrane will be partially solubilized, reducing the TMRM signal.
Limitations of the technique
One of the main benefits of this protocol is that by using Fluo-4, AM to detect mitochondrial calcium, TMRM can be used to simultaneously measure ΔΨm, with negligible spectral overlap between the two dyes. To ensure the specificity of Fluo-4 for mitochondrial calcium, the cells being examined need to be permeabilized to eliminate the cytosolic Fluo-4 signal. This permeabilization is one limitation of this technique. While the IM imaging solution closely matches the ionic strength of the cell cytosol, it can't completely replicate all of its constituents. This may affect the results if specific cytosolic components are required in the experimental system being examined. Furthermore, as the cells are permeabilized, the protocol may not be suitable for longer experiments greater than 1 hr.
Significance of the technique with respect to existing/alternative methods
Various fluorescent dyes have been developed for assessing mitochondrial calcium8. These dyes are simple to use and can provide useful data in a short space of time. While these dyes accumulate predominantly in the mitochondria, some can also accumulate in other organelles, including liposomes, or remain in the cell cytosol. Therefore, care must be taken to ensure that these other signals do not influence the mitochondrial calcium signal.
In this protocol, mitochondrial Ca2+ is measured in permeabilized cells in the presence of thapsigargin. The advantage of this methodology is that the cytosolic and ER calcium signals are eliminated. Furthermore, permeabilization allows the use of Fluo-4, AM to detect mitochondrial Ca2+. Fluo-4, AM has very little spectral overlap with TMRM, meaning that mitochondrial Ca2+ and ΔΨm can be measure simultaneously using these two dyes.
Mitochondrial Ca2+ can also be measured using genetically encoded fluorescent reporters8. These reporters can be targeted to the mitochondria, resulting in specific mitochondrial Ca2+ signals. Genetically encoded probes need to be introduced into the cells being studied, which in some cases can take considerable time and effort. Conversely, our protocol uses the dyes Fluo-4, AM and TMRM, allowing for fast and simple cell staining to measure mitochondrial Ca2+ and ΔΨm simultaneously.
Future applications or directions after mastering this technique
Mitochondria act as local calcium buffers to regulate intracellular calcium concentrations and the energy requirements of the cell. When this process is disrupted, excessive mitochondrial calcium uptake can induce mitochondrial permeability transition, resulting in the collapse of ΔΨm and the release of pro-apoptotic molecules that trigger cell death induction4.
We present a fast and straight-forward protocol for the examination of mitochondrial calcium and its relationship to ΔΨm and the induction of mitochondrial permeability transition. This can be used to investigate how these mitochondrial parameters influence pathogenesis in a wide range of human diseases, including diabetes15 and age-related neurodegeneration conditions such as Alzheimer's and Parkinson's Disease16. Furthermore, this protocol can be used to examine how mitochondrial calcium and ΔΨm are affected by genetic defects or environmental toxins, and also how these affects can be modulated by therapies or drugs which target the mitochondria. These types of future experiments can provide important new insights into the role that mitochondrial calcium plays in both human health and disease.
The authors have nothing to disclose.
We thank Dr Kirstin Elgass and Dr Sarah Creed from Monash Micro Imaging for technical assistance, and the Wellcome Trust and Medical Research Council UK for financial support. MMcK is supported the Australian Research Council Future Fellowship Scheme (FT120100459), the William Buckland Foundation, The Australian Mitochondrial Disease Foundation (AMDF), The Hudson Institute of Medical Research and Monash University. This work was supported by the Victorian Government Operational Infrastructure Support Scheme.
Dulbecco's Modified Eagle Medium (DMEM) | ThermoFisher | 10566016 |
fetal bovine serum (FBS) | ThermoFisher | 16000044 |
1x phosphate buffered saline (PBS) | ThermoFisher | 10010023 |
100x penicillin/streptomycin (p/s) | ThermoFisher | 15140122 |
0.25% Trypsin / 0.25% EDTA | ThermoFisher | 25200056 |
8-well chambered coverslip | ibidi | 80826 |
NaCl | Sigma-Aldrich | 793566 |
KCl | Sigma-Aldrich | P9541 |
MgSO4 | Sigma-Aldrich | 746452 |
KH2PO4 | Sigma-Aldrich | 795488 |
D-glucose | Sigma-Aldrich | G8270 |
CaCl2 | Sigma-Aldrich | 746495 |
HEPES | Sigma-Aldrich | H3375 |
MgCl2 | Sigma-Aldrich | M2670 |
EGTA | Sigma-Aldrich | E4378 |
HEDTA | Sigma-Aldrich | H8126 |
malate | Sigma-Aldrich | M1000 |
glutamate | Sigma-Aldrich | G1626 |
ADP | Sigma-Aldrich | A5285 |
Ca2+ free Hank’s buffered salt solution (HBSS) | ThermoFisher | 14175-095 |
tetramethylrhodamine, methyl ester, perchlorate (TMRM) | ThermoFisher | T668 |
Verapamil | Sigma-Aldrich | V4629 |
Fluo-4 acetoxymethyl ester (Fluo-4, AM) | ThermoFisher | F14201 |
dimethyl sulfoxide (DMSO) | ThermoFisher | D12345 |
carbonyl cyanide p-trifluoromethoxyphenylhydrazone (FCCP) | Sigma-Aldrich | C2920 |
digitonin | Sigma-Aldrich | D141 |
thapsigargin | Sigma-Aldrich | T9033 |
Pluronic F-127 | ThermoFisher | P3000MP |
hemacytometer | VWR | 631-0925 |
10 cm cell culture dishes | Corning | COR430167 |
75 cm2 cell culture flasks | Corning | COR430641 |