Disorder within disordered regions of protein-free lipid bilayers.

Abstract

Optical microscopy-based reports on protein-free lipid bilayers with heterogeneous compositions generally consider observed phase separations as thermodynamic endpoints; while MD simulations indicate kinetic characteristics of domain formations. Here, we study GUVs of POPC:DOPE (9:1) and POPC:DPPC (9:1), labeled with disordered-phase probe Liss-RhodPE (1/1000 w/w), using confocal imaging. In addition to confirming previously well-reported disorder and/or phase-separations in vesicular membranes of above compositions, we report the discovery of non-uniform probe distributions within disordered phases, indicating varying degrees of disorder (VDD) for the same vesicular membrane. We further examine dynamics of probe distributions in VDD regions of vesicular membranes. Regression analysis of time-dependent changes in average pixel intensities of probe molecules (avPIs) in different regions of interest (ROIs) allowed quantitative characterization of local (within ROIs) and global (overall vesicular membrane) motion of probe molecules within disordered phases. Optics-related artifacts (e.g., photobleaching, excitation and/or emission fluctuations) were ruled out by noting negligible variations in whole-vesicle avPIs with time. However, avPIs within certain ROIs showed considerable changes with time. In fact, in the same vesicular membrane, avPIs in different ROIs within disordered phases show (a) linear dependence on time (indicating diffusive motion), (b) dependence on square of time (indicating convective motion), and (c) other dependencies on time (e.g., logarithmic, exponential, power, 1/time etc.) - all clubbed together as non-diffusive-non-convective motions. Thus, we rigorously establish existence of further (at least three) levels of heterogeneity, based on dynamics of probe molecules, within disordered phases of protein-free lipid bilayers. Such heterogeneity has previously been ascribed only to biologically active membranes due to presence of membrane proteins. Interestingly, while diffusive motion may be linked to overall membrane viscosity, convective motion may be linked to concepts such as line-tension resulting from heterogeneous compositions.