Cell Membrane Integrity Examined by Nonlinear Light Scattering

Abstract

Mechanosensitive (MS) channels are responsible for preserving the integrity of the cytoplasmic membrane (CM) in the face of environmental shifts in osmolarity in a variety of cell types. Our understanding of the spatial and temporal activity of MS channels can be improved by applying new techniques capable of in vivo monitoring of their activity. Previously, we have shown that second-harmonic light scattering (SHS) provides real-time information regarding molecular uptake at biological membranes and characterizes permeability changes induced in cellular membranes1-2. Herein, we use SHS to examine changes in the physical integrity of the E. coli CM induced by hypo-osmotic shock. Given that the MS channels in E. coli are likely to have open pore diameters of >1.8 nm they can easily allow passive diffusion of low molecular weight species. We characterize the molecular uptake kinetics of malachite green cation, MG+ (ca. 1 nm diameter) under different osmotic gradients. These include; 1) cells treated with PBS, 2) cells treated in distilled water, and 3) cells re-suspended in PBS after treatment with distilled water. Comparing the adsorption and transport rates of MG+ at the CM in these conditions reveals that MS channels contribute considerably to CM permeability under hyp-osmotic shock. Our results are consistent with previous reports that MS channels in E. coli can function as a conduit for ion entry (Ca2+, H+) and not just osmolyte efflux3. Interestingly the majority of the cells exposed to high hypo-osmotic pressure (ca. 6 atm.) preserve their integrity even after ∼ 20 minutes. We suggest that SHS may represent a new experimental paradigm for not only investigating cell membrane integrity in real-time but also for examining MS channel activators.REFERENCES1. Wilhelm., Chem. Phys. Lett. (2014).2. Wilhelm, Biochem. (2015).3. Cox, Nat. Commun. (2013).