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

Abstract

For endocytosis and exocytosis, membranes transition among planar, budding, and vesicular topographies through nanoscale reorganization of lipids, proteins, and carbohydrates. However, prior attempts to understand the initial stages of nanoscale bending have been limited by experimental resolution. Through the implementation of polarized localization microscopy, this article reports the inherent membrane bending capability of cholera toxin subunit B (CTxB) in quasi-one-component-supported lipid bilayers. Membrane buds were first detected with <50 nm radius, grew to >200 nm radius, and extended into longer tubules with dependence on the membrane tension and CTxB concentration. Compared to the concentration of the planar-supported lipid bilayers, CTxB was (12 ± 4)× more concentrated on the positive curvature top and (26 ± 11)× more concentrated on the negative Gaussian curvature neck of the nanoscale membrane buds. CTxB is frequently used as a marker for liquid-ordered lipid phases; however, the coupling between CTxB and membrane bending provides an alternate understanding of CTxB-induced membrane reorganization. These findings allow for the reinterpretation of prior observations by correlating CTxB clustering and diffusion to CTxB-induced membrane bending. Single-particle tracking was performed on single lipids and CTxB to reveal the correlations among single-molecule diffusion, CTxB accumulation, and membrane topography. Slowed lipid and CTxB diffusion was observed at the nanoscale bud locations, suggesting a local increase in the effective membrane viscosity or molecular crowding upon membrane bending. These results suggest inherent CTxB-induced membrane bending as a mechanism for initiating CTxB internalization in cells that could be independent of clathrin, caveolin, actin, and lipid phase separation.

Structured clustering of the glycosphingolipid GM1 is required for membrane curvature induced by cholera toxin

“…These are consistent with simulations that show that only three of the five putative binding sites of CTxB are typically bound to GM1 (36). The diffusion rates observed here are consistent with the variety of previously published average diffusion rates (11,14,(37)(38)(39)(40)(41)(42)(43). Furthermore, we observe a non-linear effect on the diffusion coefficient on increasing the stoichiometry of binding of GM1 to CTxB.…”

“…These results also have implications for the use of cholera toxin as a marker for the liquid-ordered (raft) lipid phase. CTxB is not a non-perturbing marker for ordered domains; it can induce phase separation in membranes close to a demixing point (54,59), stabilize ordered phases (59,60), sort to already curved membranes (6,11,12), and induce membrane curvature (10,11). Our current findings reveal that any measurements of the impact of CTxB on a membrane require precise control of the CTxB and GM1 concentrations because the diffusive, sorting, and curvature-inductive properties of CTxB depend on its binding stoichiometry.…”

“…Here, we test the idea that CTxB requires defined stoichiometry of binding to create denovo highly-curved membranes (6)(7)(8)(9)(10)(11) and to sort the toxin into such highly-curved membrane structures (11)(12)(13). Because CTxB internalization in vivo does not exclusively rely on either clathrin-or caveolin-dependent processes (14)(15)(16)(17)(18)(19)(20)(21), it is feasible that an inherent membrane bending capability of CTxB may drive cellular uptake (10,22).…”

Resolving the Effects of Nanoscale Membrane Curvature on Lipid Mobility

“…With custom MATLAB (MathWorks, Inc.) programming, membrane buds were modeled with a radius of curvature equals to 50 nm and varying heights above a surrounding planar membrane (hbud). The bud membrane was smoothly connected to the surrounding planar membrane with a radius of curvature equals to 20 nm along the principal plane radial from the bud center ( Figure 4), as done previously [18,21]. hbud = 0 represents the case of a planar membrane with no bud protrusion.…”

“…With these buds, the interplay of curvature and lipid phase has been clearly demonstrated [16,17]. Recent studies have combined the ease of fabrication with polystyrene beads and nanoscale dimensions of electron beam lithography, by draping SLBs over <50 nm radius polystyrene nanoparticles to reveal single-lipid dynamics [18][19][20] and curvature-based protein sorting [21]. Within the membrane bud, there is both positive and negative curvature that complicates understanding the correlation between shape and lipid dynamics ( Figure 2).…”