Real Time Fluorescence Microscopy Observations of Dehydration-Induced Domain Formation in Raft Forming Multi Lamellar Lipid Stacks

Abstract

Domain formation in multi-component lipid mixtures is a well-known phenomenon understood in terms of equilibrium phase separation of lipid components into distinct co-existing phases. Such phase-separated equilibrium domain morphologies have been extensively studied in mono- and bimolecular lipid configurations (e.g., Langmuir monolayers, and giant unilamellar vesicles), and to a lesser extent in multilamellar lipid configurations consisting of discrete, low number of lamellae (less than ten). We have run experiments that reveal pattern formation in thick lipid stacks consisting of several thousand lamellae. We observe the appearance of domains when a lipid cake - pre-hydrated by a prolonged exposure to humid air - are left standing in laboratory air. The domain patterns develop in concert with the evaporation of water from a nominally wet pre-hydrated lipids consisting of putative raft forming mixtures of egg-sphingomylein, Cholesterol, and DOPC. Our results suggest that the dehydration of water-saturated lipid stacks provides the driving force to induce the lateral phase separation of the lipid mixture into the so-called liquid-ordered (cholesterol and sphingomyelin-enriched) and liquid-disordered phases. Our experiments enable studies of the dynamics of the domain pattern formation and the interactions between domains (e.g., long-term fusion) at low hydrations. We use a combination of microscopy tools, including Atomic Force Microscopy, fluorescence confocal microscopy, and bright-field microscopy, to determine the influence of interaction between the line tension and key elastic properties of the lipid membrane on the characteristics of the domain patterns. The observed dehydration-induced phase separation may have important consequences for freeze-drying or desiccation of lipid mixtures and living cells.