Direct Visualization of Vesicle−Bilayer Complexes by Atomic Force Microscopy

Interactions between phospholipid vesicles and bilayers play a central role in cell physiology, enabling secretion, signaling, and intracellular transport. In many instances these processes require fusion between membranes. Considerable attention has been paid to acquiring the structural details of proteins that mediate fusion;1 however, less is known about the structure and organization of lipids during this process. In addition, the interaction between lipids and proteins is known to be important for fusion. Evidence for this comes from the fact that when fusogenic proteins are reconstituted into lipid bilayers, their activities are often a sensitive function of lipid type (reviewed in ref 2). Substantial evidence also implicates membrane cholesterol content in the ability of some enveloped viruses to fuse with and enter liposomes.3-5 Because of this, the physical mechanisms of membrane fusion in reconstituted lipid systems have received intense study with a variety of physical methodologies that includes fluorescence spectroscopy and microscopy,6 light scattering,7 and the surface forces apparatus.8 These studies have revealed that membrane fusionpropensitydependsonseveralparameters including fluidity, lysis tension, and bending modulus and have produced a wealth of quantitative data on the mechanical properties of bilayers formed from different lipid compositions. The atomic force microscope (AFM) is particularly wellsuited to the characterization of reconstituted complexes of biological macromolecules because of its ability to operate under buffer, in real time, and at the nanometer length scale. Thus, much effort has been made to use AFM to study lipid vesicles, planar bilayers, and other selfassembled lipid microstructures. Similarly, AFM would be an attractive tool with which to study structural intermediates in membrane fusion. Several investigators have presented AFM images of both purified synaptic vesicles and reconstituted phospholipid vesicles on solid substrates. The former studies9-11 have lent valuable insight into the mechanical properties of synaptic vesicles as well as how those properties change in the presence of biochemical effectors. The latter studies either have used receptor-ligand interactions to tether the vesicles to substrates such as gold12 and mica13 or have focused on the kinetics and mechanisms of formation of supported bilayers from phospholipid vesicles.14,15 These studies have helpedelucidate thevariousstagesofmembraneassembly. Nonetheless, definitive visualization of individual vesicles has been limited. Presumably, this is due in part to vesicle deformation during scanning and the presence of artifactual contributions from the AFM tip. Even less is known from AFM about the association of vesicles with a planar bilayer, including structural details of the processes of adsorption, wetting, and fusion. The same is true of vesicle-vesicle interactions; while aggregates of small unilamellar vesicles (SUVs) are well within the limits of AFM’s spatial resolution, freeze-fracture electron microscopy remains the most widely used method for directly visualizing interactions between SUVs. Obtaining highresolution images of vesicles in contact with each other and with planar membranes would serve as an important first step toward introducing AFM to the study of membrane fusion intermediates. Recent light microscopic studies with giant vesicles6 suggest that membrane fusion in vitro can proceed through an orderly sequence of adsorption, adhesion, wetting, and merger, any or all of which might be directly captured by the AFM. In this report, we present AFM images of vesicle-bilayer complexes formed by adsorbing SUVs onto mica to form a continuous membrane and allowing excess vesicles to settle onto the membrane. We use depth measurements to show that we can successfully form a continuous supported membrane, and we present mechanical and structural evidence that the adsorbed structures truly are individual lipid vesicles. We also show that the morphology of the adsorbed structures is a function of lipid composition.