Fluorescent Imaging of Cellular Fluxes

 

Seeing is Believing!

The rapid bursts of ions exiting cells are vital for many physiological processes. The challenge is visualizing these multiple events from cells where the coordinated release of ions is the basis of neuronal, cardiac, and muscle excitability and where perturbation results in human disease. Conventional electrical methods either report on total ion flow from a particular cell (electrophysiology) or an ion concentration at a specific location (ion-sensitive electrodes); however, neither approach provides the spatiotemporal resolution to globally image and measure efflux from the entire landscape of a cell, an intact circuit, tissue, or brain. Therefore, we have been developing a novel cell surface approach to fluorescently visualize ions and metabolites exiting cells.

Real-time Imaging of Cellular Proton Fluxes

We are using glycocalyx engineering (pioneered by Bertozzi and co-workers) to covalently label the cell surface with small molecule fluorescent pH sensors (cartoon). Using voltage-clamp fluorimetry to simultaneously control and visualize proton transport, we observe robust fluorescent signals that correspond to cellular proton fluxes in both directions—outward and inward. The real-time kinetics of proton accumulation and depletion at the cell surface directly serves as a readout of ion channel and membrane transporter activity. Using our approach, we have observed proton transport through voltage-gated proton channels, chloride/proton antiporters, and omega proton fluxes associated with human disease. 

Relevant Publications

Zhang L, Zhang M, Bellve K, Fogarty KE, Castro MA, Brauchi S, Kobertz WR. Wheat germ agglutinin-conjugated fluorescent pH sensors for visualizing proton fluxes. J Gen Physiol. 2020 Jun 1;152(6):e201912498. doi: 10.1085/jgp.201912498. PMID: 31978216; PMCID: PMC7266149.

Velentzas PD, Zhang L, Das G, Chang TK, Nelson C, Kobertz WR, Baehrecke EH. The Proton-Coupled Monocarboxylate Transporter Hermes Is Necessary for Autophagy during Cell Death. Dev Cell. 2018 Nov 5;47(3):281-293.e4. doi: 10.1016/j.devcel.2018.09.015. Epub 2018 Oct 11. PMID: 30318245; PMCID: PMC6219939.

Bandara HMD, Hua Z, Zhang M, Pauff SM, Miller SC, Davie EAC, Kobertz WR. Palladium-Mediated Synthesis of a Near-Infrared Fluorescent K⁺ Sensor. J Org Chem. 2017 Aug 4;82(15):8199-8205. doi: 10.1021/acs.joc.7b00845. Epub 2017 Jul 14. PMID: 28664732; PMCID: PMC5715468.

Zhang L, Bellve K, Fogarty K, Kobertz WR. Fluorescent Visualization of Cellular Proton Fluxes. Cell Chem Biol. 2016 Dec 22;23(12):1449-1457. doi: 10.1016/j.chembiol.2016.10.013. Epub 2016 Dec 1. PMID: 27916567; PMCID: PMC5182157.