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Copyright © 2007 The Biophysical Society. All rights reserved.
Biophysical Journal, Volume 92, Issue 6, L46-L48, 15 March 2007

doi:10.1529/biophysj.106.098673

Biophysical Letters

Microscopic Detection of Thermogenesis in a Single HeLa Cell

Madoka Suzuki^*, 1, Vadim Tseeb^†, 1, Kotaro Oyama^† and Shin’ichi Ishiwata^*, †, ,  

^* Consolidated Research Institute for Advanced Science and Medical Care, Waseda University, Tokyo, Japan
^† Department of Physics, Faculty of Science and Engineering, Waseda University, Tokyo, Japan

Address reprint requests and inquiries to Shin’ichi Ishiwata.

¹ Madoka Suzuki and Vadim Tseeb contributed equally to this work.

In a long history of the physiological studies on thermogenesis, the thermodynamic parameters have mainly been examined for different parts of the body, tissues or organs, as a whole 1,2,3,4. Although several groups have succeeded in imaging the heat production in single cells by the direct incorporation of fluorescent dyes into each cell to detect temperature changes, these studies had obvious weak points: the fluorescence intensity was also sensitive to either the fluidity of the plasma membrane 5 or the pH value of solution 6. Thus, to overcome the technical problems in the study of thermogenesis in single cells, there was a need to devise a new tool to measure the local temperature in an aqueous solution, having high spatial resolution without interfering with any environmental parameters. For this purpose, we recently devised a microthermometer 7.

The directional flow of various kinds of ions in living cells driven by electrochemical potentials and energy-consuming pumping processes is hypothesized to result in the heat production 8,9. Steep temperature gradients in cells have recently attracted strong interest from cell biologists, especially concerning the effects of local intracellular thermogenesis on the rates of chemical reactions, the rate of the diffusion process (e.g., the transmitter-receptor mismatch in the brain 10), the speed of exocytosis 11, and so on. One of the most interesting targets in this respect is the thermogenesis coupled with the changes in intracellular free Ca²⁺ concentration ([Ca²⁺]_in), because up to 3% of the total energy production by a cell is estimated to be used even in the resting conditions, simply to maintain large [Ca²⁺] gradients between the cytoplasm and the lumens of endoplasmic/sarcoplasmic reticulum, as well as against the extracellular space 12,13. Heat is produced by the initiation of calcium pumping because the sarco/endoplasmic reticulum Ca²⁺-ATPase (SERCA) utilizes only a part of the energy of ATP hydrolysis to pump Ca²⁺, whereas the rest is dissipated in the form of heat ranging from 10 to 30 kcal/mol hydrolyzed ATP 14.

Although the integrated thermogenesis in a large number of cells studied by microcalorimetry has been reported 15, the sensitivity of this method is not sufficient to measure heat production in single living cells. Therefore, to reveal how the cellular heat production correlates with [Ca²⁺]_in and other important cellular parameters, the measurements should be performed on the level of individual cells.

In this work we present a simple approach to measure the real-time thermogenesis in a single HeLa cell with simultaneously monitoring [Ca²⁺]_in regulated by ionomycin, which is a powerful ionophore making cellular and intracellular membranes highly permeable to Ca²⁺16. In all experiments, two microthermometers were used: one was gently pressing the cell to ensure good contact with the cell membrane, and the other, separated at least 20μm from the cell, served as a reference thermometer (Figure 1 and Figure 2).

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Figure 1

Schematic illustration of the setup (A) and the obtained temperature versus fluorescence intensity relationship (B). (A) See Supplementary Material for details. (B) Fluorescence intensity was measured in several regions of the microthermometer: the tip (circles), the central part (triangles), and the root (crosses).

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Figure 2

(A) Phase-contrast image of a cell and the two pipettes. (Upper) Measurement pipette. (Lower) Reference pipette. (B and C) Fluorescence images of Fluo-4 and Eu-TTA, respectively. Scale bar, 10μm.

Ionomycin (20μl of 0.2mM solution) was added from one side of the dish (ϕ35mm) containing 2ml of the medium, to the final concentration 2μM. The addition of this small volume did not result in the temperature change of the medium or the mechanical noise. The dish was kept still throughout the measurement. Although the HeLa cells survive in the presence of ionomycin better than other types of cells 16, we found that 10min is the longest time the HeLa cells could survive in our conditions.

Next, we recorded three fluorescence signals: from Fluo-4 loaded into the cell to monitor [Ca²⁺]_in, and from Europium (III) thenoyltrifluoroacetonate trihydrate (Eu-TTA) dissolved in DMSO, in two microthermometers, to monitor the temperature. These two fluorescent dyes are compatible because their excitation spectra are well separated and the emission spectra are in the region of the wavelengths longer than 515nm (cf. Fig. 1 and Supplementary Material ). The temperature change was estimated with the use of the slope of the calibration curve, −0.0274/°C (Figure 1B), which indicates strong dependence of the Eu-TTA fluorescence on temperature compared with other dyes (e.g., −0.018/°C for the rhodamine fluorescence; 17).

Fig. 3 shows the recordings of [Ca²⁺]_in made simultaneously with the thermogenesis detected in a single HeLa cell upon application of ionomycin, at two different extracellular [Ca²⁺], 5mM (Figure 3A) and 1mM (Figure 3B). As the filter wheel and the charge-coupled device camera were not synchronized, the signals from Fluo-4 and Eu-TTA were separated by subsequent analysis with the use of a self-written macro in Microsoft Excel. The recordings made over the long period of observation include photobleaching caused by excitation light. However, because the degree of photobleaching was small for 50mM Eu-TTA (Fig. 3, Aa and Ab), it was well approximated by a single exponential and could therefore be removed from the raw traces.

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Figure 3

Time courses of the fluorescence intensity at different extracellular [Ca²⁺]. Ionomycin was added to the petri dish at time 0 in the presence of 5mM Ca²⁺ (A), 1mM Ca²⁺ (B), and 2mM Ca²⁺ with 2μM thapsigargin after the pretreatment with 2mM Ca²⁺ and 2μM thapsigargin for 1h (C). A part of the experiment in panel A is presented as Supplementary Movie 1 . Each dot represents the average value in the region of interest at the tip of the pipette in a single video frame. (Aa, Ad, Bb, and Cb) The fluorescence intensity of Eu-TTA in the pipette contacting the cell. (Ab, Ae, Bc, and Cc) Fluorescence intensity of Eu-TTA in the reference pipette. (Ac, Ba, and Ca) The fluorescence intensity of Fluo-4. The arrows show the moment at which Fluo-4 signal increased, and the arrowheads indicate the moment at which the initiation of the positive thermogenesis was detected.

When ionomycin reached the cell surface, the increment of [Ca²⁺]_in was observed (Fig. 3, Ac and Ba). As a result, the fluorescence intensity of Eu-TTA in a pipette contacting the cell decreased with some time delay (Fig. 3, Ad and Bb). No detectable change in the fluorescence intensity, however, occurred in the reference pipette (Fig. 3, Ae and Bc). The value of temperature change varied between the measurements depending on the area of the contact between the pipette and the cell. The largest observed temperature increase was ∼1°C.

The rate of the [Ca²⁺]_in increase was faster at higher extracellular [Ca²⁺] (Fig. 3, Ac and Ba), due to a larger gradient of [Ca²⁺] between the outside and the inside of the cell. The positive thermogenesis always followed the increase in [Ca²⁺]_in and, in addition, occurred earlier in the presence of 5mM Ca²⁺ than with 1mM (Fig. 3, Ac, Ad, Ba, and Bb). The values of the time delay (s, mean±SE (N)) were 28±5 5 for 5mM Ca²⁺ and 126±12 3 for 1mM.

What is the heat source for the thermogenesis observed here? It is reasonable to consider that SERCA pumping up Ca²⁺ into endoplasmic reticulum (ER) is involved in the heat production 14. The dependence of the time delay on the extracellular [Ca²⁺] (Table 1) would then be understandable, because SERCA will have to start operating to keep constant [Ca²⁺]_in after [Ca²⁺]_in exceeds a threshold level due to the influx of extracellular Ca²⁺. To confirm this consideration, we next examined the effect of thapsigargin, an inhibitor of SERCA for ER 18. In these experiments, cells were first incubated with 2μM thapsigargin for 1h in the presence of 2mM Ca²⁺, and then 2μM ionomycin was applied together with 2μM thapsigargin and 2mM Ca²⁺. As expected, we observed the influx of Ca²⁺ upon the application of ionomycin, but no temperature change was detected (Figure 3C).

In summary, we demonstrated that the coupling between the real-time thermogenesis and the SERCA's activity could be detected in single HeLa cells. We can therefore conclude that SERCA plays a key role in the cascade of cellular heat production. The next targets will be the quantitative determination of the temperature distribution inside the cell, the identification of the source(s) of the thermogenesis, and the elucidation of their physiological roles. The thermodynamic parameters for the cell, e.g., the thermal conductivity of the cytoplasm containing high concentrations of proteins, and of the inner structures with phospholipid bilayers, which must be different from the homogeneous medium, should also be clarified in future studies.

Finally, we stress that using a microthermometer is potentially a highly powerful technique for studying local thermogenesis in tissue experiments because it allows one to easily penetrate the tissue, such as a brain slice, with the simultaneous use of electronic, chemical, and optical setups to monitor other physiological parameters.