Opening-Up of Liposomal Membranes by Talin
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Proc. Natl. Acad. Sci. USA Vol. 95, pp. 1026–1031, February 1998 Biophysics Opening-up of liposomal membranes by talin AKIHIKO SAITOH,KINGO TAKIGUCHI*, YOHKO TANAKA, AND HIROKAZU HOTANI Division of Molecular Biology, Graduate School of Science, Nagoya University, Nagoya 464-01, Japan Edited by Donald M. Engelman, Yale University, New Haven, CT, and approved November 17, 1997 (received for review July 9, 1997) ABSTRACT Morphological changes of liposomes caused by generated by dissolving phospholipids in a chloroformy interactions between liposomal membranes and talin, a cytoskel- methanol solution, 98:2 (volyvol). Ten microliters each of 10 etal submembranous protein, were studied by direct, real-time mM phosphatidylethanolamine (PE) or phosphatidylcholine observation by using high-intensity dark-field microscopy. Sur- (PC) and phosphatidylglycerol (PG) or phosphatidylserine prisingly, when talin was added to a liposome solution, liposomes (PS) were mixed. The organic solvent was evaporated under a opened stable holes and were transformed into cup-shaped flow of nitrogen gas, and the lipids were further dried in vacuo liposomes. The holes became larger with increasing talin con- for at least 90 min. Forty microliters of buffer A (5 mM centration, and finally the cup-shaped liposomes were trans- TriszHCl, pH 8.0y1mMATPy5 mM DTT) was then added to formed into lipid bilayer sheets. These morphological changes the dried lipid films at 4°C. Upon liquid addition, the lipid films were reversed by protein dilution, i.e., the sheets could be transformed back into closed spherical liposomes. We demon- immediately started swelling to form liposomes. Swelling was strated that talin was localized mainly along the membrane facilitated by occasionally agitating the test tubes by hand. verges, presumably avoiding exposure of its hydrophobic portion After 30 min, the liposome suspensions were diluted 10-fold at the edge of the lipid bilayer. This is the first demonstration that with buffer A containing talin at various concentrations. We a lipid bilayer can stably maintain a free verge in aqueous added ATP in solution to examine the effect of actin on the solution. This finding refutes the established dogma that all lipid talin activity, because ATP is required to maintain the native bilayer membranes inevitably form closed vesicles and suggests activity of actin. that talin is a useful tool for manipulating liposomes. Liposomes were observed at 25°C with a dark-field micro- scope (BHF, Olympus, Tokyo). Images were recorded by using Phospholipids spontaneously assemble into bilayer mem- an SIT video camera (C-2400-08, Hamamatsu Photonics, branes in aqueous solution and necessarily form liposomes, Hamamatsu City, Japan) and were further processed with a which are closed-membrane vesicles (1). Liposomes often have digital image analyzer (IBAS, Zeiss) to enhance contrast. been studied as simplified models of biological membranes Protein. Talin was isolated from chicken gizzard according (2–5) and are now used as such in a number of applications to the method of Muguruma et al. (19). Samples were dialyzed from pharmacology to bioengineering (6), for example, as against 20 mM TriszHCl, pH 8.0y0.5 mM DTTy0.5 mM carriers of DNA vectors or anticancer drugs for internal phenylmethylsulfonyl fluoride (PMSF) and were then used. deliveries. However, studies of interaction mechanisms be- To make a concentration gradient of talin for microscope tween liposome membranes and biological components, such specimens, we used a flow cell made of a glass slide and a as DNA or protein, are now still in progress (5, 7, 8), and the coverslip that were firmly fixed together with spacers. To apply dynamic behavior of such complexes in solution has remained talin to liposomes, a drop of talin in buffer A was placed on an unclear. Therefore, real-time approaches by using optical open side of the flow cell, which had been filled with the microscopy to study the dynamic behavior of liposomes re- sulting from interactions between liposomal membranes and liposome solution. A gentle flow was then induced in the cell, biological elements are very important. thereby moving liposomes at various speeds. Slowly moving Liposomes can be visualized with several types of optical liposomes were followed in the microscope, and transforma- microscopes. In this study, we used high-intensity dark-field tions of liposomes at the buffer front containing talin were microscopy (9–11), because dark-field microscopy is the best monitored. Conversely, to dilute talin, a drop of buffer A was way to visualize the intact three-dimensional morphology and placed on an open side of the flow cell that had been filled with the dynamic behavior of individual lamellar liposomes in transformed liposomes, and the reverse processes were mon- solution, and only this type of microscopy provides real-time, itored as described above. high-contrast images. In practice, other types of high-contrast Actin was purified from rabbit skeletal muscle as described microscopes, such as phase contrast or differential interfer- previously (20). Anti-talin monoclonal antibody (T 3287) was ence, still yield poor contrast for individual lamellar liposomes. purchased from Sigma (21) and was used after 100- to 1,000- In this study, we investigated morphological changes of lipo- fold dilution with buffer A. somes caused by talin. Talin is an actin-binding, peripheral- Assay of Talin Activities. The binding activity of talin to membrane protein that localizes at focal contacts in cells and that liposomes was determined by the cosedimentation method. links actin filaments with plasma membranes through integrin Talin was added to a liposome solution and incubated at 25°C (12–15). It has also been reported that talin can bind to mem- for 1 hr. Unbound and bound proteins were separated by branes directly and can promote actin polymerization (16–18). centrifugation at 300,000 3 g for 15 min. To evaluate the effects of talin, the frequency of three kinds of membrane MATERIALS AND METHODS structures—spherical liposomes, cup-shaped, and sheet- Preparation and Observation of Liposomes. Liposomes shaped membranes—was determined by observation under were prepared as described previously (9–11). Lipid films were dark-field microscopy. The publication costs of this article were defrayed in part by page charge This paper was submitted directly (Track II) to the Proceedings office. Abbreviations: PC, phosphatidylcholine; PG, phosphatidylglycerol; payment. This article must therefore be hereby marked ‘‘advertisement’’ in PE, phosphatidylethanolamine; PS, phosphatidylserine; PMSF, phe- accordance with 18 U.S.C. §1734 solely to indicate this fact. nylmethylsulfonyl fluoride. © 1998 by The National Academy of Sciences 0027-8424y98y951026-6$2.00y0 *To whom reprint requests should be addressed. e-mail: j46037a@ PNAS is available online at http:yywww.pnas.org. nucc.cc.nagoya-u.ac.jp. 1026 Downloaded by guest on September 27, 2021 Biophysics: Saitoh et al. Proc. Natl. Acad. Sci. USA 95 (1998) 1027 FIG. 1. Transformed liposomes observed by dark-field microscopy in the presence of talin. Liposomes were prepared from PE and PG (1:1, mol:mol). (A) Spherical liposomes obtained in the absence of talin. (B) Cup-shaped liposomes observed in the presence of 0.4 mM talin; at this talin concentration, the molar ratio of protein to total phospholipids was about 1y1,000. (C) A sheet-shaped membrane has stuck to the coverslip in 1.5 mM talin. (D) A sequence of photographs showing a small spherical liposome entering a cup-shaped membrane through its opening; the spherical liposome was trapped for several minutes and then escaped through the same opening (data not shown). (E) Three-dimensional model of a cup-shaped membrane with a small spherical liposome nearby. (F) Sequential images of a freely fluctuating lipid bilayer membrane, taken at 67-ms intervals. (G) Models of the membrane corresponding to each photograph of F.(H and J) Liposomes that have three or two opening holes, respectively, observed in the presence of 1.5 mM talin. (I and K) Models corresponding to liposomes shown in photographs H and J, respectively. (Bars 5 2 mm.) Visualization of Talin Localization. To determine the local- activity as unlabeled talin as judged by the capability to transform ization of talin in transformed membranes, talin was labeled with liposomes. The localization of the labeled talin on liposomes was rhodamine iodoacetamide (R-492, Molecular Probes). Three determined with fluorescence optics assembled into a dark-field micromolar talin was incubated with 15 mM rhodamine iodoac- microscope (BHF, Olympus) (20). etamide at 37°C for 15 min. The reaction was stopped by addition of DTT to a final concentration of 5 mM, dialyzed against 20 mM RESULTS TriszHCl, pH 8.0y0.5 mM DTTy0.5 mM PMSF to remove the remaining fluorescent dye, and then concentrated by using a Morphological Change of Liposomes by Talin. Liposomes small volume concentrator (Centricon, Amicon). The dye-to- made from neutral and acidic phospholipids usually take a protein molar ratio was 0.3. The labeled talin had the same spherical shape at neutral pH and low-salt conditions, and they Downloaded by guest on September 27, 2021 1028 Biophysics: Saitoh et al. Proc. Natl. Acad. Sci. USA 95 (1998) bright and thick to look as if they are multilamellar. However, analysis of dark-field images indicates that liposomes prepared by our method are unilamellar. The light intensity scattered from liposomes depends very strongly on the number of membrane layers. It has already been shown that when unila- mellar liposomes are transformed into double-lamellar lipo- somes, the line-like images became more than 10-fold brighter (9). The line-like images of liposomes used in this study have a uniform brightness (Fig. 1A), and this brightness is almost identical to that of single-layered membranes from erythrocyte ghosts whose associated proteins have been removed by pro- tease treatment (9). When talin was added to the liposome solution, cup- shaped liposomes, which resembled a ‘‘U’’ in the dark-field image (Fig.