Biochemistry zyxwvu 1993, 32, zyxwvu 1127-1 140 1127 Role of Calcium in the Adhesion and Fusion of Bilayers? D. E. Leckband,* C. A. Helm,$ and J. Israelachvili'g* Department of Chemical and Nuclear Engineering, University of California, Santa Barbara, California 931 06, and Institut fur Physikalische Chemie, Johannes Gutenberg-Universitiit, Weldenveg 11, 0-6500 Mainz. Germany Received March 27, 1992; Revised Manuscript Received September 30, 1992 ABSTRACT: The interaction forces and fusion mechanisms of mixed zwitterionic-anionic phospholipid bilayers were measured with the surface forces apparatus. The bilayers were 3:l mixtures of either dimyris- toylphosphatidylcholine and zyxwvut dimyristoylphosphatidylglycerol (DMPC/DMPG) or dilauroylphosphatidyl- choline and dilauroylphosphatidylglycerol (DLPC/DLPG), and experiments were carried out in NaCl solutions with and without CaC12. In NaCl solutions, the forces between either mixed bilayer system were consistent with the DLVO (Derjaguin-Landau-Verwey-Overbeek) theory of repulsive electrostatic and attractive van der Waals forces, and fusion did not occur. At high pH (>6) and in high (20 mM) NaCl concentrations, a short-range hydration force extending about 13 A was evident, indicative of Na+ binding to the surfaces. In the presence of this large hydration repulsion, the interbilayer adhesion was abolished. When CaCl2 was added to the bathing solutions in the presence or absence of NaC1, the bilayers phase separate into small domains, coinciding with the occurrence of a large, long-range attractive force. Fusion occurred readily between the more fluid domains. The phase separations and fusion events could be directly visualized by observing the shapes of the optical fringes used to measure the surface separation and the change in surface profiles with time. The ease of fusion between mixed bilayers in the presence of calcium correlated closely with the strength of the long-range attractive force. This force is attributed to the additional hydrophobic force between domains or domain boundaries due to the exposure of excess hydrophobic groups resulting from the Ca2+-induced condensation of the PG- headgroups. Fusion is, therefore, attributed to the enhanced hydrophobicity of phase-separated domains and only indirectly to ion binding, to an enhanced adhesion, or to dehydration effects. These results provide the first direct evidence of a molecular relationship between calcium-induced phase separation and bilayer fusion. The role of cations in the adhesion and fusion of bilayers containing anionic lipids has been a topic of intensive study for many years (Papahadjopoulos et al., 1974, 1990; Papa- hadjopoulos, 1978; Poste zyxwvutsrq & Nicolson, 1978; Diizgunes et al., 1984a,b;Sowers, 1987). Fusion is thought to occur as a result of a number of factors, most of which involve the close apposition of membranes due to a reduced interbilayer repulsion resulting from surface dehydration and/or inter- bilayer bridging by divalent ions (McIver, 1970; Portis et al., 1979, Parsegian & Rand, 1983; Rand & Parsegian, 1986; Feigenson, 1986;Leikin et al., 1987). Other factors that may reduce interbilayer repulsion are depletion attraction or dehydration induced by poly(ethy1ene glycol) (Boni & Hui, 1987), a reduction in repulsive interbilayer undulations by membrane tension, and osmotic stress effects (Evans & Metcalfe, 1984; Servuss & Helfrich, 1989). Bilayer fusion may also occur as a consequence of enhanced hydrophobic interactionsbetween bilayers arising from lipid packing defects due to lateral phase separations,the formation of intrabilayer phase intermediates, membrane-bound proteins, or bilayer depletion (Papahadjopoulos et al., 1974; Papahadjopoulos, 1978; Siege1 et al., 1989; Hui et al., 1981). It has been proposed, and recently demonstrated, that the increased hydrophobic attraction between stressed bilayers through exposure of the bilayer interior is a major driving force in the fusion mechanism (Ohki & Diizgunes, 1979;Ohki, 1982;Ohki & Ohshima, 1984; Helm et al., 1989). ~~~~~~~~~~~~~~~~ t D.E.L. was supported by NIH Grant PHS GM 47334, and C.A.H. * To whom correspondence should be addressed. 8 Johannes Gutenberg-Universitit. was supported by NSF Grant CTS90-15537. University of California. In regard to the mechanism of fusion, vesicle fusion has been modeled as a two-step process (Bentz et al., 1983). The vesicles first aggregate, and in the second step, bilayer destabilization and fusion occur. Divalent ions have been proposed to induce fusion primarily through membrane destabilization by inducing lateral phase separation or phase transitions, both as a result of specific ion-lipid interactions (Niretal., 1980;Diizgunesetal., l981,1984a,b). Incontrast, monovalent ions play no role in bilayer fusion, but they affect the overall fusion kinetics by altering the electrostatic forces between bilayers (Bentz et al., 1983). Calcium-induced lateral phase separation hasbeen proposed as an intermediate in bilayer fusion for several years (Pap- ahadjopouloset al., 1974,1977; Papahadjopoulos, 1978). Phase separations would result in packing fluctuations at domain boundaries, facilitating fusion by interaction of the exposed hydrophobic regions (Hui et al., 1981). The Occurrence of lateral phase separations in mixed lipid bilayers has since been correlated with their fusion susceptibility (Leventis et al., 1986; Silvius & Gagne, 1984a; Hui et al., 1983). In particular, differential scanning calorimetry studies of mixed vesicles with homologous lipid chain lengths indicated that the appearance of macroscopic lateral phase separation correlates directly with increased fusion rates within certain compositionranges (Silvius & Gagne, 1984a,Huiet al., 1983). Other systems, however, undergo fusion at compositionswhere calcium does not induce macroscopiclateral phase separation (Silvius & Gagne, 1984b;Diizgunes et al., 1984a,b). In such cases, fusion has been attributed to the formation of micro- domains, undectable by calorimetric measurements but observed by X-ray microprobeanalysis and by 31P-NMR (Hui et al., 1983). Additionally, it was also found that close 0006-2960/93/0432-1127%04.00/0 0 1993 American Chemical Society