Abstract
Membrane-bound protein complexes involving PFTs, released by virulent bacteria are known to form pores leading to host cell lysis. However, membrane remodelling and related repair processes during attack by PFTs remain largely unexplored. in our recent experiments [1] with GUVs exposed to prototypical cholesterol dependent cytolysin, Listeriolysin O (LLO), we were able to correlate changes in lipid dynamics to membrane bound conformational states of LLO oligomers. We now report non-monotonic variations in lipid diffusion due to interplay of lipid ejection and crowding by membrane bound oligomers of LLO. Our experiments monitor lipid dynamics and membrane micro-rheology over a broad range of protein-to-lipid ratios encompassing the dilute, intermediate as well as the crowded regime. This systematic variation of protein concentration reveals for the first time a unique non-monotonic behaviour of lipid diffusivity as a function of PFT concentration which falls outside the purview of existing membrane dynamics models. by monitoring the fluorescent intensity of lipids using confocal microscopy and number of lipids in FCS we provide direct evidence of lipid loss during pore formation. Not only do we observe the anticipated crowding induced decreasing lipid diffusivities at high protein concentration, but our results reveal a counter-intuitive increase in lipid diffusivity at intermediate LLO concentrations. We quantitatively capture these trends using an established model based on the dependence of membrane lipidxdiffusion on the available free area. We correlate changes in observed lipid diffusivity to the free area contributions from different PFT oligomeric states, thus providing unique insights connecting lipid dynamics to bound protein distributions. 1. Ilanila IP, Ramesh Cheerla, K Ganapathy Ayappa and Jaydeep K Basu, “Correlated protein conformational states and membrane dynamics during attack by pore-forming toxins”, Proc Nat Acad Sci. 116, 12839 (2019).
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