Impact of Cholesterol-MβCD on Glycan-Supported Lipid Bilayer Patch Dynamics

Abstract

Alterations in the cholesterol content of biological membranes can induce morphological and functional changes in cells, disrupting signaling pathways and potentially reducing cell survivability. Among these changes, addition of cholesterol to membranes via the carrier molecule methyl-β-cyclodextrin (MβCD) promotes the formation of high-curvature protrusions and invaginations. When cholesterol is inserted into laterally constrained model membranes composed of the phospholipid DOPC and supported on a glass substrate, similar tubular protrusions extend from the bi-layer surface. Furthermore, when cholesterol is inserted into laterally unconstrained, glass-supported bilayer patches, these patches radially expand, increasing in surface area. This expansion is quickly followed by two stages of area contraction: during the first, the patch returns to its initial size and shape. During the second, holes form in the patch membrane until the patch disappears entirely. To determine if the same biomechanical phenomenon drives protrusion formation in cellular membranes, lipid patches deposited on the physiologically relevant substrate dextran are exposed to various concentrations of cholesterol-MβCD. The dynamical remodeling responses of glass- and dextran-supported patches are compared to ascertain the biophysical mechanisms that govern bilayer expansion and contraction. Furthermore, the fluorescent marker TopFluor cholesterol is used in an attempt to monitor the partitioning of cholesterol during each phase of patch remodeling. The results of these studies indicate that cholesterol-induced increases in lipid packing are responsible for the expansion of dextran-supported patches, and the condensing effect of cholesterol is likely the cause for the initial contraction of lipid patches. Later stage patch contraction is facilitated by the removal of DOPC from the bilayer by MβCD. Furthermore, limitations of the simplified glass and dextran membrane models are identified and characterized in order to distill experimental features from genuine behavior of biological membranes.