Cell shape is determined by the interplay between the lipid bilayer and the underlying network of protein polymers. We explored unknown determinants involved in cell morphogenesis as factors that transform phospholipid-based liposomes (diameter 5-20 microm). Unlabeled giant liposomes, observed through dark-field optics, were metastable in an aqueous suspension. In contrast, liposomes robustly protruded uniform tubules immediately after the addition of a brain extract to the suspension. The tubulation reaction was greatly facilitated when the liposomes contained PIP or PIP2. Biochemical analysis of the brain extract revealed that heteromeric complexes of septins, a family of polymerizing GTP/GDP-binding proteins, are responsible for the membrane transformation. Ultrastructural analysis established that each membrane tubule (diameter 0.43 +/- 0.079 microm) is braced by a circumferential array of septin filaments. Although submembranous septin assemblies are associated with diverse cortical morphogenesis from yeast to mammals, the biophysical basis for the septin-membrane interplay remains largely unknown. Further, there is a biochemical discrepancy between the fast septin remodeling in cells and their slow self-assembly in vitro. This membrane-facilitated fast septin assembly demonstrated for the first time by our unique experimental system should provide important clues to characterize these processes.

Dynamic behaviors of liposomes caused by interactions between liposomal membranes and surfactant were studied by direct realtime observation by using high-intensity dark-field microscopy. Solubilization of liposomes by surfactants is thought to be a catastrophic event akin to the explosion of soap bubbles in the air; however, the actual process has not been clarified. We studied this process experimentally and found that liposomes exposed to various surfactants exhibited unusual behavior, namely continuous shrinkage accompanied by intermittent quakes, release of encapsulated liposomes, opening up, and inside-out topological inversion. L iposomes (which are closed membrane vesicles) have been well studied as simplified models of biological membranes (1-5) and are now used in a number of applications (5, 6), for example, as carriers of drug or DNA delivery or as artificial membranes for reconstructing membranous enzyme activities (7-9). Recently, many important phenomena affecting lipid bilayers, including their detergent solubilization, have been explored by using liposomes; such studies promote a better understanding of the biophysical properties of bilayer membranes and moreover will improve the handling of membrane proteins when they are isolated from or reconstructed into lipid bilayers (10-13). However, studies of intermediate stages in the detergent solubilization of liposomes are only now in progress (14-17), and the interaction mechanism between membranes and surfactants has remained unclear. Therefore, real-time approaches by using optical microscopy to study the dynamic behavior of liposomes are very important.High-intensity dark-field microscopy has enabled us to obtain real-time high-contrast images of giant unilamellar liposomes in aqueous solutions (18)(19)(20)(21)(22). In this study, we used such techniques to characterize the interactions between liposomal membranes and surfactants. Eight kinds of liposomes and various types of surfactants ( Fig. 1) were mixed in all possible combinations in a mixing chamber to generate a concentration gradient of each surfactant for microscope specimens, and morphological changes of liposomes exposed to those surfactants were monitored (18, 23). In the absence of surfactant, liposomal membranes were spherical, and thermal fluctuations of their spherical shape were largely suppressed by the surface tension of their membranes. Hereafter, this morphological state of liposomes will be called tense. In this study, we found several unusual behaviors of liposomes (which are published as supplemental data on the PNAS web site, www.pnas.org). Materials and MethodsPreparation and Observation of Liposomes. To prepare giant unilamellar liposome, liposome (total 1 mM lipid concentration) was made of phosphatidylcholine (PC) or of PC and one of seven other lipids (1:1, mol͞mol) in Hepes buffer (10 mM HepesNaOH, pH 7.0), as described previously (18,21,22). Lipids were dissolved in a chloroform͞methanol solution, 98:2 (vol͞vol), and mixed. The organic solvent was evaporated unde...

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...

Suzuki et al. [Biochemistry 28, 6513-6518 (1989)] have shown that, when F-actin is mixed with inert high polymer, a large number of actin filaments closely align in parallel with overlaps to form a long and thick bundle. The bundle may be designated non-polar, as the constituent filaments are random in polarity (Suzuki et al. 1989). I prepared non-polar bundles of F-actin using methylcellulose (MC) as the high polymer, exposed them to heavy meromyosin (HMM) in the presence of ATP under a light microscope, and followed their morphological changes in the continuous presence of MC. It was found that bundles several tens of micrometers long contracted to about one-third the initial length, while becoming thicker, in half a minute after exposure to HMM. Subsequently, each bundle was split longitudinally into several bundles in a stepwise manner, while the newly formed ones remained associated together at one of the two ends. The product, an aster-like assembly of actin bundles, was morphologically quiescent; that is, individual bundles never contracted upon second exposure to HMM and ATP, although they were still longer than the F-actin used. Bundles in this state consisted of filaments with parallel polarity as examined by electron microscopy. This implies that non-polar bundles were transformed into assemblies of polar bundles with ATP hydrolysis by HMM. Importantly, myosin subfragment-1 caused neither contraction nor transformation. These results are interpreted as follows. In the presence of ATP, the two-headed HMM molecule was able to cross-bridge antiparallel actin filaments, as well as parallel ones.(ABSTRACT TRUNCATED AT 250 WORDS)

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