Real-time recording of the cellular effects of the anion transporter prodigiosin

Cheung, Shane, Wu, Dan, Daly, Harrison C., Busschaert, Nathalie, Morgunova, Marina, Simpson, Jeremy C., Scholz, Dimitri, Gale, Philip A. and O'Shea, Donal F. (2018) Real-time recording of the cellular effects of the anion transporter prodigiosin. Chem, 4 (4), 879-895. (doi:10.1016/j.chempr.2018.02.009).

Abstract

The unraveling of the cellular effects of anion transporters is key to their potential development as apoptosis-inducing or autophagy-disrupting therapeutics. Here, we conducted a systematic study of the cellular responses to the anion transporter prodigiosin by using a pH on/off responsive near-infrared (NIR)-fluorescent probe in HeLa and LAMP1-GFP-transfected HeLa cell lines. The sequence of localized and global cellular acidity changes and the resulting outcomes induced by the anion transporter were visualized with high temporal and spatial resolution. The results show that prodigiosin causes the pH within the lysosomal lumen to rise, after which a non-organelle-specific increase in acidity of the cytosol takes place, which prompts cells to undergo apoptosis. This was confirmed by the quantification of NIR-emissive lysosomes, the intra-cellular fluorescence intensity, and fluorescence volume over time. This NIR probe overcame the limitations of acridine orange, which to date have severely restricted researchers in this field. Lysosomes act as the cells’ refuse bin, but because nature is an avid recycler, this waste is not left curbside. Upcycling of pre-used cell materials first requires the conversion of these materials back into their constituent components within the acidic lysosomes, akin to the digestion of food. Here, we used molecular light bulbs that were switched off when they entered the cell but switched themselves on after accumulating in lysosomes. While watching, we added a substance to purposely disrupt lysosomal acidity and record how both individual lysosomes and the whole cell responded. This is important because the recycling process can be interrupted if the lysosome acidity is neutralized with catastrophic cellular consequences. Our hope is that being able to observe different lysosome disruptors in action will help find the best agents that shut down the recycling centers in cancer cells and limit their ability to survive. A systematic study of the cellular responses to the anion transporter prodigiosin has been conducted with a lysosomal-responsive NIR-fluorescent probe in HeLa and LAMP1-GFP-transfected HeLa cell lines. Results show that prodigiosin rapidly causes the pH within the lysosomal lumen to rise, after which a non-organelle-specific increase in acidity of the cytosol takes place. This was confirmed by the quantification of changes in the number of NIR-emissive lysosomes and intra-cellular fluorescence intensity over time.

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Contributors

Author: Philip A. Gale

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