Opening-up of liposomal membranes by talin

Abstract

Morphological changes of liposomes caused by interactions between liposomal membranes and talin, a cytoskeletal submembranous protein, were studied by direct, real-time observation by using high-intensity dark-field microscopy. Surprisingly, when talin was added to a liposome solution, liposomes opened stable holes and were transformed into cup-shaped liposomes. The holes became larger with increasing talin concentration, and finally the cup-shaped liposomes were transformed into lipid bilayer sheets. These morphological changes were reversed by protein dilution, i.e., the sheets could be transformed back into closed spherical liposomes. We demonstrated that talin was localized mainly along the membrane verges, presumably avoiding exposure of its hydrophobic portion at the edge of the lipid bilayer. This is the first demonstration that a lipid bilayer can stably maintain a free verge in aqueous solution. This finding refutes the established dogma that all lipid bilayer membranes inevitably form closed vesicles and suggests that talin is a useful tool for manipulating liposomes.Phospholipids spontaneously assemble into bilayer membranes in aqueous solution and necessarily form liposomes, which are closed-membrane vesicles (1). Liposomes often have been studied as simplified models of biological membranes (2-5) and are now used as such in a number of applications from pharmacology to bioengineering (6), for example, as carriers of DNA vectors or anticancer drugs for internal deliveries. However, studies of interaction mechanisms between liposome membranes and biological components, such as DNA or protein, are now still in progress (5,7,8), and the dynamic behavior of such complexes in solution has remained unclear. Therefore, real-time approaches by using optical microscopy to study the dynamic behavior of liposomes resulting from interactions between liposomal membranes and biological elements are very important.Liposomes can be visualized with several types of optical microscopes. In this study, we used high-intensity dark-field microscopy (9-11), because dark-field microscopy is the best way to visualize the intact three-dimensional morphology and the dynamic behavior of individual lamellar liposomes in solution, and only this type of microscopy provides real-time, high-contrast images. In practice, other types of high-contrast microscopes, such as phase contrast or differential interference, still yield poor contrast for individual lamellar liposomes.In this study, we investigated morphological changes of liposomes caused by talin. Talin is an actin-binding, peripheralmembrane protein that localizes at focal contacts in cells and that links actin filaments with plasma membranes through integrin (12-15). It has also been reported that talin can bind to membranes directly and can promote actin polymerization (16-18). MATERIALS AND METHODSPreparation and Observation of Liposomes. Liposomes were prepared as described previously (9-11). Lipid films were generated by dissolving phospholipids in a chloroform͞...

Equilibrium Theory and Geometrical Constraint Equation for Two-Component Lipid Bilayer Vesicles

“…Recently, the opening up process of lipid bilayer vesicles by talin was observed by Saitoh et al [20] and Nomura et al [21]. When the density and exterior environment were changed, the opening vesicle could not only become a membrane plane but also reform a spherical vesicle.…”

Microscopic observations reveal that fusogenic peptides induce liposome shrinkage prior to membrane fusion

“…The lipid films started swelling immediately to form liposomes, and the swelling was facilitated by agitating the test tubes occasionally by hand. Liposomes were observed at 25°C by using a dark-field BHF microscope (Olympus) as described (12)(13)(14)(15). Images were recorded by using an SIT video camera (C-2400-08; Hamamatsu Photonics, Hamamatsu City, Japan), further processed with IP-LAB software (Scanalytics, Fairfax, VA), and analyzed with IMAGE (National Institutes of Health, Bethesda) and PHOTOSHOP software (Adobe Systems, Mountain View, CA).…”

“…Giant liposomes were prepared as described (13)(14)(15). A total of 200 g of PC, or a 1:1 mixture of PC and PG (mol͞mol), was dissolved in 98:2 chloroform͞methanol (vol͞vol).…”

“…Giant liposomes can be visualized by several optical microscopic methods (9-11). However, we have used dark-field microscopy because it gives the best high-contrast images of giant liposomes compared with other methods (8,(12)(13)(14)(15).…”

“…Giant liposomes can be visualized by several optical microscopic methods (9-11). However, we have used dark-field microscopy because it gives the best high-contrast images of giant liposomes compared with other methods (8,(12)(13)(14)(15).To induce membrane fusion, we used three different peptides: HA, E5, and K5. HA is a 20-aa peptide derived from the N-terminal fusion peptide region of the influenza hemagglutinin HA2 subunit, whereas E5 and K5 are negatively and positively charged analogs of HA, respectively.…”