Mitochondrial membrane potential (MMP) is the electrical potential difference across the inner mitochondrial membrane. It's created by the unequal distribution of ions across the membrane and active pumping of protons linked to respiration. MMP forms part of the driving force for the synthesis of ATP through chemiosmosis and is therefore essential to the metabolic state of the cell.
Our work focuses on the real-time analysis of MMP in mammalians cells by using fluorescent dyes. We particularly focused on evaluating the ability of thioflavin T (ThT) to report on MMP levels and considered the effect of imaging conditions on this. We found that ThT can act as an MMP indicator dye, when used at low micromolar concentrations with low blue-light exposure. With higher concentration and under certain imaging conditions, however, we showed that blue light used for the imaging of ThT can cause photoactivation of ThT and the loss of MMP. This effect is absent when ThT-containing cells are not imaged with blue light.
In the cover image of the December 13 issue of Biophysical Reports, we visually demonstrate these findings by imaging cells co-incubated with ThT and tetramethylrhodamine methyl ester (TMRM), a well-established MMP-indicating dye. We repeatedly exposed half of the cells in the field of view to blue and red light (used in the imaging of ThT and TMRM, respectively) and half of the cells to red light only. The repeated exposure with blue light led to the photoactivation of ThT and subsequent loss of MMP, which is seen by a loss in TMRM signal on the left side of the image and accumulation of ThT in the nucleoli. On the right side, instead, we can see that cells exposed to only red light maintained their MMP (indicated by the TMRM signal). There is clearly a dramatic difference in TMRM distribution between the two halves of the image. This difference effectively demonstrates the combined effects of specific dyes and light conditions on cellular function.
The presented research shows that ThT can act as an MMP indicator in mammalian cells when used at low concentrations and with low blue-light exposure. However, this dye can also disrupt MMP, depending additively on its concentration and blue-light exposure. These findings call for a re-evaluation of ThT use at micromolar range in live-cell analyses, but at the same time, present ThT as a potential tool to study MMP dynamics under conditions in which TMRM might have limitations (e.g., in studies using microfluidic devices in which TMRM can bind to the device structure) or in which controlled MMP disruption is sought.
Further information on our research can be found at https://warwick.ac.uk/fac/sci/lifesci/research/osslab.
— Emily Skates, Hadrien Delattre, Zoe Schofield, Munehiro Asally, and Orkun S. Soyer