Single-lipid tracking on nanoscale membrane buds: The effects of curvature on lipid diffusion and sorting

Abstract

Nanoscale membrane curvature in cells is critical for endocytosis/exocytosis and membrane trafficking. However, the biophysical ramifications of nanoscale membrane curvature on the behavior of lipids remain poorly understood. Here, we created an experimental model system of membrane curvature at a physiologically-relevant scale and obtained nanoscopic information on single-lipid distributions and dynamics. Supported lipid bilayers were created over 50 and 70 nm radius nanoparticles to create membrane buds. Single-molecule localization microscopy was performed with diverse mixtures of fluorescent and non-fluorescent lipids. Variations in lipid acyl tales length, saturation, head-group, and fluorescent labeling strategy were tested while maintaining a single fluid lipid phase throughout the membrane. Monte Carlo simulations were used to fit our experimental results and quantify the effects of curvature on the lipid diffusion and sorting. Whereas varying the composition of the non-fluorescent lipids yielded minimal changes to the curvature effects, the labeling strategy of the fluorescent lipids yielded highly varying effects of curvature. Most conditions yield single-population Brownian diffusion throughout the membrane; however, curvature-induced lipid sorting, slowing, and aggregation were observed in some conditions. Head-group labeled lipids such as DPPE-Texas Red and POPE-Rhodamine diffused >2.4× slower on the curved vs. the planar membranes; tail-labeled lipids such as NBD-PPC, TopFluor-PPC, and TopFluor-PIP2, as well as DiIC and DiIC displayed no significant changes in diffusion due to the membrane curvature. This article is part of a Special Issue entitled: Emergence of Complex Behavior in Biomembranes edited by Marjorie Longo.

Membrane Thinning Induces Sorting of Lipids and the Amphipathic Lipid Packing Sensor (ALPS) Protein Motif

“…Biological membranes are highly dynamic and heterogeneous lipid bilayer barriers that physically separate the functional compartments of cells (McMahon and Gallop, 2005). The role of membrane curvature in the spatial organization of membrane-associated proteins (Hatzakis et al, 2009;Bhatia et al, 2010;Antonny, 2011;Singh et al, 2012;Nguyen et al, 2017;Nepal et al, 2018;Bhaskara et al, 2019) and the membrane's local lipid composition (Derganc, 2007;Jiang and Powers, 2008;Sorre et al, 2009;Tian and Baumgart, 2009;Baumgart et al, 2011;Callan-Jones et al, 2011;Baoukina et al, 2018;Harayama and Riezman, 2018;Woodward et al, 2018) (and vice versa!) are wellestablished.…”

Single-lipid dynamics in phase-separated supported lipid bilayers

“…For example, FCS shows little effects from nanoscale membrane curvature relative to the curvaturedependent lipid mobility change in simulations (Kabbani et al, 2017). By mapping the singlelipid step lengths to locations on the membrane, the lipid mobility relative to membrane curvature has been analyzed (Kabbani and Kelly, 2017a;Woodward et al, 2018;Woodward and Kelly, 2020).…”

Nanoscale membrane curvature sorts lipid phases and alters lipid diffusion

“…The dish was chilled to 4 °C for 15 min before gentle rinsing with 5 mL of 4 °C, 200 mM sucrose. The creation of SLBs over nanoparticles for engineered curvature has been performed previously (26,27,(29)(30)(31).…”

“…The optical setup included an inverted IX83 microscope with a 100x, 1.49 NA objective (Olympus), a 2x emission path magnification (OptoSplit, Cairn Research), and an iXon 897-Ultra EMCCD camera (Andor Technology), as described previously (26,30). A Hg lamp with an excitation filter (BrightLine single-band filters, Semrock) provided illumination for diffractionlimited images.…”

“…Diffusion typically slows on cylindrical membranes when the radii are smaller than 50 nm (25). Some fluorescent lipids slow on spherical membranes with a 50-nm radius (26,27). The effects of curvature on lipid diffusion are correlated to the fluorophore location on the labeled lipid; head-group-labeled lipids diffuse 3x faster on flat versus curved membranes, whereas tail-labeled lipids diffuse at the same rate on flat and curved membranes (26).…”