Influence of the glycocalyx on the size and mechanical properties of plasma membrane-derived vesicles

Abstract

Recent studies have reported that the overexpression of MUC1 glycoproteins on cell surfaces changes the morphology of cell plasma membranes and increases the blebbing of vesicles from them, supporting the hypothesis that entropic forces exerted by MUC1 change the spontaneous curvature of cell membranes. However, how MUC1 is incorporated into and influences the size and biophysical properties of plasma-membrane-blebbed vesicles is not understood. Here we report single-vesicle-level characterization of giant plasma membrane vesicles (GPMVs) derived from cells overexpressing MUC1, revealing a 40× variation in MUC1 density between GPMVs from a single preparation and a strong correlation between GPMV size and MUC1 density. By dispersing GPMVs in aqueous liquid crystals (LCs), we show that the elasticity of the LC can be used to strain individual GPMVs into spindle-like shapes, consistent with the straining of fluid-like membranes. To quantify the influence of MUC1 on membrane mechanical properties, we analyze the shapes of strained GPMVs within a theoretical framework that integrates the effects of MUC1 density and GPMV size on strain. We measure the spontaneous curvature of GPMV membranes to be 2-10 μm^-1 and weakly influenced by the 40× variation in MUC1 density, a conclusion we validate by performing independent experiments in which MUC1 is enzymatically removed from GPMVs. Overall, our study advances the understanding of heterogeneity in size and MUC1 density in GPMVs, and establishes single-vesicle-level methods for characterization of mechanical properties within a heterogeneous population of GPMVs. Furthermore, our measurements highlight differences between membrane properties of GPMVs and their parent cells.

Fig. 1. (A) (Upper) Schematic illustration of the plasma membrane of a mammalian cell. (Middle) Electron micrographs showing three plasma membrane morphologies of genetically modified human breast epithelial cells that accompany an increase in mucin density in the glycocalyx. (Lower) Schematic illustration of bending of membranes driven by entropic forces generated by mucins. (B) Predicted values of spontaneous curvature as a function of mucin density on cells. Reprinted from ref. , Copyright (2019), with permission from Elsevier.

Fig. 2. (A) The chemical structure of disodium cromoglycate (DSCG). (B) Schematic illustration of aggregates of DSCG molecules in an aqueous solution. (C) Illustration of orientational ordering of DSCG aggregates in a nematic phase. (D) Schematic illustration of the membranes of giant unilamellar vesicles consisting of 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC). (E) Bright-field micrograph and schematic illustration of a DOPC GUV strained in 15 wt% DSCG solution (nematic). (F) Schematic illustration of the plasma membranes of red blood cells. (G) Bright-field micrographs and schematic illustration of an RBC strained in 17.3 wt% DSCG solution (nematic).

Fig. 3. (A) Schematic illustration of the formation of giant plasma membrane vesicles (GPMVs), and bright-field micrographs of GPMVs in an isotropic buffer and nematic phase of 13.8 wt% DSCG. (B) Histogram depicting the distribution of sizes of a population of 1E7 GPMVs. (C) Histogram depicting the distribution of GFP fluorescence intensity per area (μm²) of a population of 1E7 GPMVs. (D), (E) Bright-field and fluorescence micrographs of 1E7 GPMV in 13.8 wt% DSCG showing aspect ratio and GFP fluorescence intensity per area of (D) 2.05 and 635 a.u. μm⁻², and (E) 1.49 and 5358 a.u. μm⁻², respectively. (F) Plot of aspect ratios of 1E7 GPMVs strained in 13.8 wt% DSCG as a function of GFP fluorescence intensity per μm².

Fig. 4. (A) Plot of surface area of GPMVs of 3 different GFP fluorescence intensity per μm² ranges. (B) Color-map of aspect ratio of 1E7 GPMVs strained in DSCG (13.8 wt%) as a function of their surface area and GFP fluorescence intensity per μm². (C) Plot of calculated values of spontaneous curvatures (C₀) of GPMVs of 3 different GFP fluorescence intensity per μm² ranges (data represent mean ± standard error) (assuming bending modulus, κ = 10⁻¹⁹ J, and interfacial energy density, τ = 5 × 10⁻⁶ N m⁻¹).

Fig. 5. (A) Schematic illustration of cleavage of MUC1 biopolymers from GPMV membranes by StcE mucinase enzymes. (B) Histogram plot from flow cytometry of fluorescence intensity of GFP on 1E7 GPMVs and 1E7 GPMVs treated with StcE mucinase. (C) Histogram plot of the surface area of the two populations of GPMVs. (D) Plot of aspect ratios of the two populations of GPMVs. (E) Plot of calculated values of spontaneous curvature on the two GPMV populations. (F) Plot of predicted aspect ratios of GPMVs with volumes, V = 100 μm³, 150 μm³, 200 μm³, for a range of values of spontaneous curvatures (assuming bending modulus, κ = 10⁻¹⁹ J, and interfacial energy density, τ = 5 × 10⁻⁶ N m⁻¹).