Preferential binding and re-organization of nanoscale domains on model lipid membranes by pore-forming toxins: insight from STED-FCS

Abstract

Potential nanodomains in cellular membranes are widely believed to be targeted by proteins and other biomolecules to enable execution of critical cellular functions. However, characterization of these nanodomains remains elusive, primarily due to the diffraction limit of a conventional optical microscope. Herein using super-resolution STED microscopy coupled with fluorescence correlation spectroscopy (STED-FCS), we provide experimental evidence of lipid nanodomains present in model membranes comprised of phosphocholine-cholesterol binary mixture, and its reorganization induced by pore-forming toxins (PFTs). In this study, we used two different types of PFTs, namely α-PFT (cytolysin A) and β-PFT (listeriolysin O), that preferentially associate as well as reorganize cholesterol-containing saturated and unsaturated phosphocholine membranes. The emergence of nanodomains due to the lipidlipid and lipid-PFT interactions was quantified at a length scales of ~50-150 nm using FCS diffusion law enabled by variation of spot sizes in the super-resolution STED microscopy. Our results shed light on the usefulness of super-resolution microscopy to quantify the underlying nanoscale domains in model membranes with implications for a wide variety of membranemediated cellular events observed in real cell membranes.

Bacterial pore-forming proteins induce non-monotonic dynamics due to lipid ejection and crowding

“…The non-monotonic nature is revealed in this system as well although there are quantitative differences in terms of actual values of D as well the dynamical cross-over concentration C p . This points to a common microscopic origin for the observed dynamical cross-over in these lipid membranes induced by LLO while the difference in actual cross-over concentrations C p is likely related to the intrinsic fluidity difference between the two lipid compositions as well as to the organisation and availability of cholesterol for LLO pore formation as has been observed earlier (9, 10, 41).…”

Pore Forming Protein Induced Biomembrane Reorganization and Dynamics: A Focused Review

“…when compared to similar domains in pristine bilayers (Sarangi and Basu, 2019). Given the generality of the FCS diffusion law plots and their utility in providing insights about the microscopic nature of membrane dynamics all subsequent plots of spot variation STED-FCS in this review will be discussed based on the τ D vs. d 2 plot although the representation in terms of D and/or D app can also be equivalently used.…”

“…Upon ClyA interaction, the domains with fast and slow dynamics reorganised to result in slowed down lipid diffusion. Adapted from Sarangi and Basu (2019).…”

“…In situations for α < 1 we refer to D as D app . For the special case, when t 0 < 0, the size of the nanodomains ω is extracted ( Favard et al, 2011 ; Sarangi et al, 2017 ; Sarangi and Basu, 2019 ) by setting τ D = 0 in Eq. 10 to obtain, …”

“…The influence of LLO binding and pore formation on lipid lateral diffusion was explored using confocal FCS on supported lipid bilayers (SLBs) ( Sarangi et al, 2016a ; Sarangi et al, 2016b ; Sarangi and Basu, 2018 ; Sarangi and Basu, 2019 ; Ilanila et al, 2021 ) and giant unilamellar vesicles (GUVs) ( Ponmalar et al, 2019 ). In some cases, FRET has also been used to correlate the protein bound states with the observed lipid dynamics.…”