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.

Correlated protein conformational states and membrane dynamics during attack by pore-forming toxins

“…Understanding the mechanistic pathways for PFTs has relied largely on structural data gleaned from cryo-electron microscopy (cryo-EM) and X-ray crystallography, where detergents induce oligomerization to stabilize pore structures (5,6). Since CDCs and LLO in particular act by binding to membrane cholesterol, model lipid membranes have been used to ascertain the presence of prepore and pore structures with atomic force microscopy (AFM) (7)(8)(9)(10). The LLO pore states can exist either as partially oligomerized arcs or complete pores, with sizes ranging from 40 to 80 nm (9) and selective influx of dyes into giant unilamellar vesicles (GUVs) exposed to LLO confirm the presence of functional oligomeric intermediates (10).…”

“…Vesicle-leakage experiments can probe the kinetics of pore formation and cellular lysis without revealing direct information about oligomeric intermediates or lipid response (18)(19)(20)(21)(22). Recent superresolution-stimulated emission depletionfluorescence correlation spectroscopy (FCS) experiments (7,(23)(24)(25) on supported lipid bilayers (SLBs) have revealed the extent of induced dynamic heterogeneity and protein-mediated lipid reorganization due to pore formation. Deviations from Brownian diffusion are observed at length scales between 100 and 200 nm.…”

Background reduction in STED-FCS using coherent-hybrid STED

“…The most commonly used depletion pattern is a ring-shaped focus, which constrains the lateral resolution but leaves unchanged the axial resolution (2D STED). This depletion pattern has been extensively used to study two-dimensional systems like cellular membranes [4]- [8], but faces severe limitations when studying three-dimensional diffusion, due to varying axial cross-sections and high background originating from undepleted areas [5], [9] that leads to an increase in the apparent number of molecules in the observation volume [5], [10]. Alternatively, a bottle-shaped depletion beam can be used to essentially constrain the axial resolution (z-STED), but is more sensitive to optical aberrations [11], [12], which can be mitigated with adaptive optics [13].…”

z-STED imaging and spectroscopy to investigate nanoscale membrane structure and dynamics

“…Fluorescence correlation spectroscopy (FCS) is such a spectroscopic tool used to measure molecular mobility in membranes 15,16 , which is an important parameter to understand the molecular dynamics in cells [15][16][17][18][19][20] . Combination of STED with FCS (STED-FCS) has been used extensively to address the nanoscale membrane structure [21][22][23][24][25][26][27] . Recently STED has also been combined with spectral imaging and polarity-sensitive probes to quantitatively study the nanoscale physiochemical properties of the membrane 11 .…”