Cellular membranes are often composed of phospholipid mixtures in which one or more components have a tendency to adopt a type II non-bilayer lipid structure such as the inverted hexagonal (H||) phase. The formation of a type II non-bilayer intermediate, the inverted lipid micel is proposed as the initial step in membrane fusion (Verkleij 1984, Siegel, 1986). In the various forms of cellular transport mediated by carrier vesicles (e.g. exocytosis, endocytosis) the regulation of membrane fusion, and hence of inverted lipid micel formation, is of vital importance.We studied the phase behaviour of simple and complex lipid mixtures by cryo-electron microscopy to gain more insight in the ultrastructure of different lipid phases (e.g. Pβ’, Lα, H||) and in the complex membrane structures arising after Lα < - > H|| phase changes (e.g. isotropic, cubic). To prepare hydrated thin films a 700 mesh hexagonal grid (without supporting film) was dipped into and withdrawn from a liposome suspension. The excess fluid was blotted against filter paper and the thin films that form between the bars of the specimen grid were immediately (within 1 second) vitrified by plunging of the carrier grids into ethane cooled to its melting point by liquid nitrogen (Dubochet et al., 1982). Surface active molecules such as phospholipids play an important role in the formation and thinning of these aqueous thin films (Frederik et al., 1989). The formation of two interfacial layers at the air-water interfaces requires transport of surface molecules from the suspension as well as the orientation of these molecules at the interfaces. During the spontaneous thinning of the film the interfaces approach each other, initially driven by capillary forces later by Van der Waals attraction. The process of thinning results in the sorting by size of the suspended material and is also accompanied by a loss of water from the thinner parts of the film. This loss of water may result in the concentration and eventually in partial dehydration of suspended material even if thin films are vitrified within 1 sec after their formation. Film formation and vitrification were initiated at temperatures between 20-60°C by placing die equipment in an incubator provided widi port holes for the necessary manipulations. Unilamellar vesicles were made from dipalmitoyl phosphatidyl choline (DPPC) by an extrusion method and showed a smooth (Lα) or a rippled (PB’.) structure depending on the temperature of the suspensions and the temperature of film formation (50°C resp. 39°C) prior to vitrification. The thermotropic phases of hydrated phospholipids are thus faithfully preserved in vitrified thin films (fig. a,b). Complex structures arose when mixtures of dioleoylphosphatidylethanol-amine (DOPE), dioleoylphosphatidylcholine (DOPC) and cholesterol (molar ratio 3/1/2) are heated and used for thin film formation. The tendency of DOPE to adopt the H|| phase is responsible for the formation of complex structures in this lipid mixture. Isotropic and cubic areas (fig. c,d) having a bilayer structure are found in coexistence with H|| cylinders (fig. e). The formation of interlamellar attachments (ILA’s) as observed in isotropic and cubic structures is also thought to be of importance in biological fusion events. Therefore the study of the fusion activity of influenza B virus with liposomes (DOPE/DOPC/cholesterol/ganglioside in a molar ratio 1/1/2/0.2) was initiated. At neutral pH only adsorption of virus to liposomes was observed whereas 2 minutes after a drop in pH (7.4 - > 5.4) fusion between virus and liposome membranes was demonstrated (fig. f). The micrographs illustrate the exciting potential of cryo-electron microscopy to study lipid-lipid and lipid-protein interactions in hydrated specimens.
Blotting-free and lossless cryo-electron microscopy grid preparation from nanoliter-sized protein samples and single-cell extracts
