title: Super-resolution Stimulated Emission Depletion-Fluorescence Correlation Spectroscopy Reveals Nanoscale Membrane Reorganization Induced by Pore-Forming Proteins.
abstract: Membrane-protein interactions play a central role in membrane mediated cellular processes ranging from signaling, budding, and fusion, to transport across the cell membrane. Of particular significance is the process of efficient protein olgomerization and transmembrane pore formation on the membrane surface; the primary virulent pathway for the action of antimicrobial peptides and pore forming toxins (PFTs). The suggested nanoscopic length scales and dynamic nature of such membrane lipid-protein interactions makes their detection extremely challenging. Using a combination of super-resolution stimulated emission depletion nanoscopy with fluorescence correlation spectroscopy (STED-FCS) we unravel the emergence of nanoscale lateral heterogeneity in supported bilayer membranes made up of 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC) and cholesterol upon interaction with the PFT, listeriolysin O (LLO). A distinct length scale-dependent dynamical crossover (<200 nm) from a Brownian diffusive regime is observed at 33 and 50% cholesterol compositions, indicating the partitioning of lipids into domains with variable cholesterol content. At 25% cholesterol content, this dyamical crossover is observed only in bilayers incubated with LLO providing evidence for the existence of sub ∼100 nm dynamical lipid nanodomains bound to LLO pore assemblies. By introducing asymmetry in cholesterol composition across the bilayer leaflets we infer that this domain formation is driven largely due to active cholesterol sequestration and transient trapping of lipids to the membrane bound motifs present in the toxins, en route to LLO oligomerization and subsequent pore formation. Bilayers prepared with labeled lipids present in either the proximal or distal leaflet allow us to track the dynamical perturbation in a leaflet-dependent manner upon LLO incubation. From the differences in the extent and intensity of the dynamical crossover as observed with STED-FCS, these experiments reveal that the affinity for cholesterol in the membrane binding motifs of the LLO subdomains induce cholesterol and lipid reorganization to a greater extent in the distal (upper) leaflet when compared with the proximal (lower) leaflet. The observed length scale-dependent membrane reorganization that occurs due to invasion by LLO could be generalized to other cholesterol-dependent cytolysins and emphasizes the significant advantage of using super-resolution STED nanoscopy to unravel complex lipid-protein interactions in membrane and cellular biophysics.
authors: Department of Physics, ‡Center for Biosystems Science and Engineering, ¶Department of Chemical Engineering, and §Department of Molecular Reproduction, Development and Genetics, Indian Institute of Science Bangalore 560 012, India.
authors: Nirod Kumar
authors: NK
authors: Sarangi
authors: Department of Physics, ‡Center for Biosystems Science and Engineering, ¶Department of Chemical Engineering, and §Department of Molecular Reproduction, Development and Genetics, Indian Institute of Science Bangalore 560 012, India.
authors: Ilanila I
authors: II
authors: P
authors: Department of Physics, ‡Center for Biosystems Science and Engineering, ¶Department of Chemical Engineering, and §Department of Molecular Reproduction, Development and Genetics, Indian Institute of Science Bangalore 560 012, India.
authors: K G
authors: KG
authors: Ayappa
authors: Department of Physics, ‡Center for Biosystems Science and Engineering, ¶Department of Chemical Engineering, and §Department of Molecular Reproduction, Development and Genetics, Indian Institute of Science Bangalore 560 012, India.
authors: Sandhya S
authors: SS
authors: Visweswariah
authors: Department of Physics, ‡Center for Biosystems Science and Engineering, ¶Department of Chemical Engineering, and §Department of Molecular Reproduction, Development and Genetics, Indian Institute of Science Bangalore 560 012, India.
authors: Jaydeep Kumar
authors: JK
authors: Basu
mesh_terms: D008055:Lipids / Q000737:chemistry; D036103:Nanotechnology; D018272:Porins / Q000737:chemistry* / Q000378:metabolism; D013050:Spectrometry, Fluorescence / Q000379:methods*
chemical_list: D008055:Lipids; D018272:Porins