Crystallization of Cholesterol in Phospholipid Membranes Follows Ostwald’s Rule of Stages

Crystallization
of membrane-embedded components within phospholipid
bilayers represents a distinct class of phase transformation that
occurs in structurally organized, molecularly crowded, and dimensionally
constrained amphiphilic fluids. Using unstable supported lipid bilayerstransiently
assembled via surface-mediated fusion and spreading of bicellar precursors
containing supersaturating concentrations of cholesterolwe
monitor here the morphological evolution and dynamics of cholesterol
crystallization within the membrane media. We find that the three-dimensional
(3D) crystallization of cholesterol from an unstable two-dimensional
(2D) in-membrane state proceeds via well-defined sequence of intermediates,
including filaments, rods, helices, and 2D rectangular plates, before
transforming into three-dimensional quadrilateral crystalscharacteristic
triclinic habit of cholesterol monohydrate. Our observations thus
demonstrate that these structurally distinct cholesterol polymorphs
are related to one another, contrasting with the notion that they
represent disparate crystal habits stabilized by differences in lipid
environments. Moreover, these observations indicate that cholesterol
crystallization within the membrane media follows nonclassical multistep
crystallization governed by the heuristic “Ostwald’s
rule of stages”, which predicts that the crystallization kinetics
proceed down the free energy landscape in a multistage process where
each successive phase transition incurs the smallest loss of free
energy relative to its predecessor. Furthermore, we find that the
well-known cholesterol extracting agent, β-cyclodextrin, acts
by catalytically tipping the equilibrium in favor of crystal growth
adding cholesterol from the membrane phase to the crystal in a layer-by-layer
manner. Taken together, our results provide a new description of in-membrane
cholesterol crystallization and may pave for a screening tool for
identifying molecular candidates that target cholesterol crystals.