Revealing the Effects of Nanoscale Membrane Curvature on Lipid Mobility

Abstract

Recent advances in nanoengineering and super-resolution microscopy have enabled new capabilities for creating and observing membrane curvature. However, the effects of curvature on single-lipid diffusion have yet to be revealed. The simulations presented here describe the capabilities of varying experimental methods for revealing the effects of nanoscale curvature on single-molecule mobility. Traditionally, lipid mobility is revealed through fluorescence recovery after photobleaching (FRAP), fluorescence correlation spectroscopy (FCS), and single particle tracking (SPT). However, these techniques vary greatly in their ability to detect the effects of nanoscale curvature on lipid behavior. Traditionally, FRAP and FCS depend on diffraction-limited illumination and detection. A simulation of FRAP shows minimal effects on lipids diffusion due to a 50 nm radius membrane bud. Throughout the stages of the budding process, FRAP detected minimal changes in lipid recovery time due to the curvature versus flat membrane. Simulated FCS demonstrated small effects due to a 50 nm radius membrane bud that was more apparent with curvature-dependent lipid mobility changes. However, SPT achieves a sub-diffraction-limited resolution of membrane budding and lipid mobility through the identification of the single-lipid positions with ≤15 nm spatial and ≤20 ms temporal resolution. By mapping the single-lipid step lengths to locations on the membrane, the effects of membrane topography and curvature could be correlated to the effective membrane viscosity. Single-fluorophore localization techniques, such SPT, can detect membrane curvature and its effects on lipid behavior. These simulations and discussion provide a guideline for optimizing the experimental procedures in revealing the effects of curvature on lipid mobility and effective local membrane viscosity.

Nanoscale Membrane Budding Induced by CTxB and Detected via Polarized Localization Microscopy

“…Finally, it is feasible that the membrane bending itself affects the local effective lipid viscosity and the free diffusion of lipids and proteins through the membrane buds. This effect was observed when membrane curvature was engineered by draping SLBs over nanoparticles and the lipid diffusion was measured with SPT (25,43) but not when measured with fluorescence recovery after photobleaching (FRAP) (44), as expected (34). In comparing the diffusion coefficient measured by SPT to that measured by FRAP or fluorescence correlation spectroscopy, it is critical to consider the differences in sensitivity to detecting mobile versus immobile diffusers, length-and timescale-dependent processes, and subpopulations of diffusers (33,34).…”

“…This effect was observed when membrane curvature was engineered by draping SLBs over nanoparticles and the lipid diffusion was measured with SPT (25,43) but not when measured with fluorescence recovery after photobleaching (FRAP) (44), as expected (34). In comparing the diffusion coefficient measured by SPT to that measured by FRAP or fluorescence correlation spectroscopy, it is critical to consider the differences in sensitivity to detecting mobile versus immobile diffusers, length-and timescale-dependent processes, and subpopulations of diffusers (33,34). The SPT results presented here are consistent with prior SPT results and, as expected, report a slower diffusion rate than FRAP or fluorescence correlation spectroscopy measurements (43,45).…”

“…The sequential localizations of single fluorophores were analyzed to reveal the diffusion rate of individual molecules versus membrane topography. A camera blur was caused by the single-frame exposure time (t exp ) being comparable to the time between frames (Dt) (33,34). The diffusion coefficient (D) was calculated from the D fit according to…”

“…Diffusion coefficients from SPT are typically extracted by fitting the mean squared displacement versus Dt. However, fitting a whole trajectory to a single diffusion coefficient blurs the effects of nanoscale curvature with the lipid trajectory sampling both curved and flat membranes (34). Further description of the SPT procedure and single-step fitting is provided in the Supporting Material.…”

Variations in Plasma Membrane Topography Can Explain Heterogenous Diffusion Coefficients Obtained by Fluorescence Correlation Spectroscopy

“…Theoretical studies have concluded that membrane curvature per se will slow down the diffusion of membrane proteins since it influences the packing in the two leaflets and hence the interaction between the protein and the lipids ( Gov, 2006 ; Yoshigaki, 2007 ; Kabbani et al, 2017 ). This should also be considered as an explanation for variations in τ D , since close to endless combinations of curvature in the focal volume are possible for the same membrane area.…”