Giant vesicles get energized: correlating membrane potentials to ion transport through fluorescence analysis

by Dr. Ran Tivony

Dept of Chemical Engineering

at Biological and soft-matter physics

Thu, 09 May 2024, 12:10
Sacta-Rashi Building for Physics (54), room 207

Abstract

Life depends on constant energy transduction mechanisms. In cellular membranes, ion fluxes generate electrochemical potential gradients that energize the synthesis of ATP. However, despite the importance of unravelling the link between energy transduction mechanisms and cellular activity, quantification of ion fluxes and electrochemical gradients in the complex environment of live cells is extremely challenging. Conversely, synthetic cell models like giant unilamellar vesicles (GUVs) are free of the structural complexity of cells and thus are ideal for studying ion transport under tightly controlled conditions. In this talk, I will describe a fluorescence-based approach for quantifying ion fluxes and the resultant variation of electrochemical potential gradients across the membrane of single GUVs. To gain maximal control over the size and membrane composition of these micron-sized liposomes (i.e., GUVs), we developed an integrated microfluidic platform that is capable of high-throughput production and purification of monodispersed GUVs [1]. By combining our microfluidic platform with quantitative fluorescence analysis, we determined the permeation rate of two biologically important electrolytes – protons (H+) and potassium ions (K+) – and were able to correlate their flux with electrochemical gradient accumulation across the lipid bilayer of single GUVs [2,3]. Through applying similar analysis principles, we also determined the permeation rate of K+ across two archetypal ion channels, gramicidin A and outer membrane porin F (OmpF). We then showed that the translocation rate of H+ across gramicidin A is four orders of magnitude higher than that of K+ unlike in the case of OmpF where similar transport rates were evaluated for both ions3.


References

[1] Tivony, R., Fletcher, M., Al Nahas, K., & Keyser, U. F. (2021). A microfluidic platform for sequential assembly and separation of synthetic cell models. ACS synthetic biology, 10(11), 3105-3116.
[2] Tivony, R., Fletcher, M., & Keyser, U. F. (2022). Quantifying proton-induced membrane polarization in single biomimetic giant vesicles. Biophysical Journal, 121(12), 2223-2232.
[3] Fletcher, M., Zhu, J., Rubio-Sánchez, R., Sandler, S. E., Nahas, K. A., Michele, L. D., ... & Tivony, R. (2022). DNA-based optical quantification of ion transport across giant vesicles. ACS nano, 16(10), 17128-17138.

Created on 04-05-2024 by Feingold, Mario (mario)
Updaded on 04-05-2024 by Feingold, Mario (mario)