Biological & Soft Matter Seminar: Using synthetic cell models to elucidate transport mechanisms in biological systems

Abstarct:

Life depends on constant energy transduction mechanisms. For instance, in cellular membranes, ion fluxes generate electrochemical potential gradients that energize vital processes such as ATP synthesis, nutrient uptake, and neuronal transduction. 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. Nonetheless, there is a lack of quantitative methods for correlating ionic fluxes to electrochemical gradient buildup in these biomimetic vesicles. We developed 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. 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. 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 ions.