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Thioflavin T indicates membrane potential in mammalian cells and can affect it in a blue light dependent manner

Emily Skates, Hadrien Delattre, Zoe Schofield, Munehiro Asally, View ORCID ProfileOrkun S. Soyer
doi: https://doi.org/10.1101/2021.10.22.465407
Emily Skates
1Bio-Electrical Engineering Innovation Hub, University of Warwick, Coventry, CV4 7AL, UK
2School of Life Sciences, University of Warwick, Coventry, CV4 7AL, UK
3Warwick Integrative Synthetic Biology Centre (WISB); University of Warwick, Coventry, CV4 7AL, UK
4Midlands Integrative Doctoral Training Program; University of Warwick, Coventry, CV4 7AL, UK
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Hadrien Delattre
2School of Life Sciences, University of Warwick, Coventry, CV4 7AL, UK
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Zoe Schofield
1Bio-Electrical Engineering Innovation Hub, University of Warwick, Coventry, CV4 7AL, UK
2School of Life Sciences, University of Warwick, Coventry, CV4 7AL, UK
3Warwick Integrative Synthetic Biology Centre (WISB); University of Warwick, Coventry, CV4 7AL, UK
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Munehiro Asally
1Bio-Electrical Engineering Innovation Hub, University of Warwick, Coventry, CV4 7AL, UK
2School of Life Sciences, University of Warwick, Coventry, CV4 7AL, UK
3Warwick Integrative Synthetic Biology Centre (WISB); University of Warwick, Coventry, CV4 7AL, UK
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  • For correspondence: M.Asally@warwick.ac.uk O.Soyer@warwick.ac.uk
Orkun S. Soyer
1Bio-Electrical Engineering Innovation Hub, University of Warwick, Coventry, CV4 7AL, UK
2School of Life Sciences, University of Warwick, Coventry, CV4 7AL, UK
3Warwick Integrative Synthetic Biology Centre (WISB); University of Warwick, Coventry, CV4 7AL, UK
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  • ORCID record for Orkun S. Soyer
  • For correspondence: M.Asally@warwick.ac.uk O.Soyer@warwick.ac.uk
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ABSTRACT

The fluorescent benzothiazole Thioflavin T (ThT) has a high binding affinity to protein aggregates and is used as a marker for the study of this process, most commonly in the context of neurodegenerative disease research and diagnosis. Recently, this same dye was shown to indicate membrane potential in bacteria due to its cationic nature. This finding prompted a question whether ThT fluorescence is linked to the membrane potential in mammalian cells, which would be important for appropriate utilisation of ThT in research and diagnosis. Here, we show that ThT localises into the mitochondria of HeLa cells in a membrane-potential dependent manner. Specifically, ThT colocalised in cells with a well-established mitochondrial membrane-potential indicator Tetramethylrhodamine methyl ester (TMRM) and gave similar temporal responses as TMRM to treatment with a protonophore, carbonyl cyanide-4-(trifluoromethoxy) phenylhydrazone (FCCP). Additionally, we found that presence of ThT together with exposure to blue light (λ=405 nm) exposure, but neither factor alone, caused depolarisation of mitochondrial membrane potential. This depolarisation effect was recapitulated by a mathematical model implementing the potential-dependent distribution of ThT and its light-dependent binding in mitochondria. These results show that ThT can act as a membrane potential dye in mammalian cells, when used at low concentrations and with low blue-light exposure, while it causes dissipation of the mitochondrial membrane potential at higher concentrations and in the presence of blue light excitation. This conclusion motivates a re-evaluation of ThT’s use at micromolar range in live-cell analyses, while indicating that this dye can enable future studies on the potential connections between membrane potential dynamics and protein aggregation.

Competing Interest Statement

The authors have declared no competing interest.

Footnotes

  • Funding key: This project is funded by the Biotechnology and Biological Sciences Research Council (BBSRC) (grant IDs BB/T010150/1 and BB/M017982/1) and University of Warwick funded Midlands Integrative Biosciences Training Partnership (MIBTP) (grant ID BB/M01116X/1) and a Cancer Research UK (CRUK) Pioneer Award (to OSS).

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The copyright holder for this preprint is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under a CC-BY-NC-ND 4.0 International license.
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Posted October 22, 2021.
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Thioflavin T indicates membrane potential in mammalian cells and can affect it in a blue light dependent manner
Emily Skates, Hadrien Delattre, Zoe Schofield, Munehiro Asally, Orkun S. Soyer
bioRxiv 2021.10.22.465407; doi: https://doi.org/10.1101/2021.10.22.465407
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Thioflavin T indicates membrane potential in mammalian cells and can affect it in a blue light dependent manner
Emily Skates, Hadrien Delattre, Zoe Schofield, Munehiro Asally, Orkun S. Soyer
bioRxiv 2021.10.22.465407; doi: https://doi.org/10.1101/2021.10.22.465407

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