Mechanistic insights allows high yield assembly of giant unilamellar vesicles in solutions of physiological ionic strengths and studies of the effects of intercellular variation on circadian timekeeping.

Abstract

Giant unilamellar vesicles or GUVs are single-walled closed phospholipid bilayer membranes that resemble minimal biological cells. A general understanding of the mechanism of solvent-free GUV assembly from lamellar lipid films on surfaces is lacking. Our group has developed a quantitative framework using sedimentation and confocal microscopy to reproducibly measure the “molar yield” and distribution of sizes of GUVs. The molar yield provides a quantitative metric to understand the effects of physical variables on the process of assembly. Using this technique, we show that the assembly of GUVs composed of zwitterionic lipids can be explained by the so called budding and merging (BNM) model. The model explains the evolution of yields as a function of i) increasing surface concentration of lipid, ii) increasing ionic strength of the solution, and iii) increasing charge of the membrane. The mechanistic insight that budding is temporally distinct from merging allows, via modulation of ionic strength, the ability to obtain high yields of GUVs in buffers of arbitrary ionic strengths and valencies, including of physiological ionic strengths. The GUV buds remain attached to the substrate until harvested, which allows for diffusive loading of protein cargo into the vesicles. We find that the distribution of protein concentrations in the vesicles mimics the intercellular distribution of protein in cells. The GUVs thus offer a tractable in vitro platform for studying the effects of variability on biophysical phenomena such as circadian timekeeping.