Phase Topology and Growth of Single Domains in Lipid Bilayers Marie-Ce ´cile Giocondi, † Ve ´ronique Vie ´, † Eric Lesniewska, ‡ Pierre-Emmanuel Milhiet, † Martin Zinke-Allmang, § and Christian Le Grimellec* ,† Centre de Biochimie Structurale, CNRS UMR 5048-Universite ´ Montpellier I, INSERM U414, 29 rue de Navacelles, 34090 Montpellier Cedex, France, Laboratoire de Physique, CNRS-URA 5027, Universite ´ Bourgogne, 21011 Dijon Cedex, France, and Department of Physics and Astronomy, University of Western Ontario, London, Ontario, Canada N6A 3K7 Received August 21, 2000. In Final Form: December 4, 2000 The time-dependent topology of domains in supported phospholipid bilayers of a binary mixture of dioleoylphosphatidylcholine and dipalmitoylphosphatidylcholine under a buffer solution has been studied by atomic force microscopy. We observe a transient regime of the phase separation until 45 min after a temperature quench from a miscible state of the system into the gel-liquid crystal coexistence region with the earliest observation after 20 min showing large gel-phase domains (containing ∼10 4 -10 6 molecules) of irregular shapes. The transient regime is characterized by a power law for the growth rate of the domain size (A) with n ) 3.0 ( 0.4 in A ∝ t 2/n . After 45 min, an asymptotic power law with n ) 20 ( 10 is observed and is linked to an inhibited domain growth. The evolution of individual domains suggests that domain growth in the transient regime is governed by a ripening mechanism. The growth inhibition is linked to the observation that the gel domains in each leaflet of the bilayer must grow simultaneously at the same sites as they remain superimposed on each other throughout the phase separation process. The organization of biological membranes in in-plane microdomains 1-6 is now believed to play a key role in the development and regulation of membrane functions. 7,8 Recent progress in the characterization of these domains suggests that the lateral phase separation of lipid molecules plays a dominant role in their formation and organization. 9,10 By use of lipid bilayers as a model for biomembranes, the lateral organization in phase-sepa- rated two-component bilayer systems has been investi- gated for more than two decades. Earliest studies based on the use of freeze-fracture electron microscopy and electron diffraction described domains in the µm 2 range for liposomes made of various phase-separated binary mixtures of phospholipids 11-14 but gave limited informa- tion about the growth of these domains. Since that time, many studies have focused on the nonequilibrium dynamic ordering process and topology of coexisting liquid crystal- line/gel phases in phosphatidylcholine mixtures. These studies included fluorescence recovery after photobleach- ing (FRAP) experiments which showed that the phase separation is a slow process which can take several hours. Accompanying Monte Carlo simulations provided a sta- tistical description of the lateral organization of the bilayer. 15-20 Interestingly, the FRAP experiments and Monte Carlo simulations pointed toward much smaller domain sizes (in the range of a few tens of nanometers) for the studied systems, in sharp contrast to earlier experiments. The individual shape, growth law, and in- plane distribution of domains in such mixtures have yet to be established. Such knowledge of the topology of the membrane domains is an important issue as it may control the kinetics and yield of reactions between membrane constituents. 21 The atomic force microscope (AFM) is a useful tool to probe the lateral organization of lipid mixtures on a mesoscopic length scale because of its capability to image structures in aqueous media with a resolution that extends from the molecular to the microscopic level. 22-24 Such applications have been demonstrated for the topography * To whom correspondence should be addressed: C. Le Grimellec, C.B.S., INSERM U414, 29, rue de Navacelles, 34090 Montpellier Cedex, France. Tel: 33 467 41 79 07. Fax: 33 467 41 79 13. E-mail: clg@cbs.univ-montp1.fr. † Centre de Biochimie Structurale, CNRS UMR 5048-Universite ´ Montpellier I. ‡ Laboratoire de Physique, CNRS-URA 5027, Universite ´ Bour- gogne. § Department of Physics and Astronomy, University of Western Ontario. (1) Jain, M. K.; White, H. B. Adv. Lipid. Res. 1977, 15, 1. 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