Abstract
Phospholipid ternary systems are useful model systems for understanding lipid-lipid interactions and their influence on biological properties such as cell signaling and protein translocation. Despite extensive studies, there are still open questions relating to membrane phase behavior, particularly relating to a proposed state of three-phase coexistence, due to the difficulty in clearly distinguishing the three phases. We look in and around the region of the phase diagram where three phases are expected and use a combination of different atomic force microscopy (AFM) modes to present the first images of three coexisting lipid phases in biomimetic cell lipid membranes. Domains form through either nucleation or spinodal decomposition dependent upon composition, with some exhibiting both mechanisms in different domains simultaneously. Slow cooling rates are necessary to sufficiently separate mixtures with high proportions of lo and lβ phase. We probe domain heights and mechanical properties and demonstrate that the gel (lβ) domains have unusually low structural integrity in the three-phase region. This finding supports the hypothesis of a “disordered gel” state that has been proposed from NMR studies of similar systems, where the addition of small amounts of cholesterol was shown to disrupt the regular packing of the lβ state. We use NMR data from the literature on chain disorder in different mixtures and estimate an expected step height that is in excellent agreement with the AFM data. Alternatively, the disordered solid phase observed here and in the wider literature could be explained by the lβ phase being out of equilibrium, in a surface kinetically trapped state. This view is supported by the observation of unusual growth of nucleated domains, which we term “tree-ring growth,” reflecting compositional heterogeneity in large disordered lβ phase domains.
Highlights
A major advance in membrane science in recent decades has been the realization that multicomponent membranes are not randomly mixed, but are laterally heterogeneous owing to lipid-lipid interactions [1]
Studying lipid phases in supported lipid bilayers (SLBs) has many advantages, such as the SLB’s ideal geometry for applying a wide variety of scattering and local probe techniques, and the averaging and annealing of a large population of absorbed vesicles, negating the variation in composition seen in giant unilamellar vesicles (GUVs) [33] it has some disadvantages
The solution is to use a very slow cooling rate so that the system remains as close to equilibrium as possible during the development of the phase structure. With lb phases, this leads to the formation of sparse and very large domains, too large to be observed by atomic force microscopy (AFM), which is an inherently high-resolution technique
Summary
A major advance in membrane science in recent decades has been the realization that multicomponent membranes are not randomly mixed, but are laterally heterogeneous owing to lipid-lipid interactions [1]. An established way to understand the conditions under which domains form is to compile a phase diagram based on experimental observation, using a model lipid system comprising just three components [5]. Such model systems give insights into the more complex phenomenon of lipid organization in native membranes and have their own applications, which include biosensing [6], drug-delivery [7], and nanofabrication [8]. A number of studies have used sphingomyelin [13] as the saturated lipid component, which, somewhat more complex in composition, carries with it the advantage of being a more realistic representation of the outer leaflet of the plasma membrane.
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