Bilayer mixing, fusion, and lysis following the interaction of populations of cationic and anionic phospholipid bilayer vesicles.

Cationic, O-alkylphosphatidylcholines, recently developed as DNA transfection agents, form bilayers indistinguishable from those of natural phospholipids and undergo fusion with anionic bilayers. Membrane merging (lipid mixing), contents release, and contents mixing between populations of positive vesicles containing O-ethylphosphatidylcholine (EDOPC) and negative vesicles containing dioleolylphosphatidylglycerol (DOPG) have been determined with standard fluorometric vesicle-population assays. Surface-charge densities were varied from zero to full charge. All interactions depended critically on surface-charge density, as expected from the adhesion-condensation mechanism. Membrane mixing ranged from zero to 100%, with significant mixing (>10 <70%) occurring between cationic vesicles that were fully charged and anionic vesicles that had fractional surface charges as low as 0.1. Such mixing with membranes as weakly charged as cell membranes should be relevant to transfection with cationic lipids. Unexpectedly, lipid mixing was higher at high than at low ionic strength when one lipid dispersion was prepared from EDOPC plus DOPG (in different proportions), especially when the other vesicles were of EDOPC; this may somehow be a consequence of the ability of the former mixture to assume non-lamellar phases. Leakage of aqueous contents was also a strong function of charge, with fully charged vesicles releasing essentially all of their contents less than 1 min after mixing. EDOPC was more active in this regard than was DOPG, which probably reflects stronger intermolecular interactions of DOPG. Fusion, as measured by contents mixing, exhibited maximal values of 10% at intermediate surface charge. Reduced fusion at higher charge is attributed to multiple vesicle interactions leading to rupture. The existence of previously published data on individual interactions of vesicles of the same composition made it possible for the first time to compare pairwise with population interactions, confirming the likelihood of population studies to overestimate rupture and hemifusion and underestimate true vesicle fusion.

Cholesterol stabilizes hemifused phospholipid bilayer vesicles.

Cholesterol was found to inhibit full fusion of oppositely charged phospholipid bilayer vesicles by stabilizing the contacting membranes at the stage of the hemifused intermediate. Vesicles of opposite charge containing different amounts of cholesterol were prepared using cationic (1,2-dioleoyl-sn-glycero-3-ethylphosphocholine) and anionic (dioleoylphosphatidylglycerol) phospholipids. Pairwise interactions between such vesicles were observed by fluorescence video microscopy in real time after electrophoretically maneuvering the vesicles into contact. Hemifusion accounted for more than 80% of the observed events when the vesicles contained 33-50 mole% cholesterol. In contrast, vesicles containing only a small proportion of cholesterol (

Physical and biological properties of cationic triesters of phosphatidylcholine.

The properties of a new class of phospholipids, alkyl phosphocholine triesters, are described. These compounds were prepared from phosphatidylcholines through substitution of the phosphate oxygen by reaction with alkyl trifluoromethylsulfonates. Their unusual behavior is ascribed to their net positive charge and absence of intermolecular hydrogen bonding. The O-ethyl, unsaturated derivatives hydrated to generate large, unilamellar liposomes. The phase transition temperature of the saturated derivatives is very similar to that of the precursor phosphatidylcholine and quite insensitive to ionic strength. The dissociation of single molecules from bilayers is unusually facile, as revealed by the surface activity of aqueous liposome dispersions. Vesicles of cationic phospholipids fused with vesicles of anionic lipids. Liquid crystalline cationic phospholipids such as 1, 2-dioleoyl-sn-glycero-3-ethylphosphocholine triflate formed normal lipid bilayers in aqueous phases that interacted with short, linear DNA and supercoiled plasmid DNA to form a sandwich-structured complex in which bilayers were separated by strands of DNA. DNA in a 1:1 (mol) complex with cationic lipid was shielded from the aqueous phase, but was released by neutralizing the cationic charge with anionic lipid. DNA-lipid complexes transfected DNA into cells very effectively. Transfection efficiency depended upon the form of the lipid dispersion used to generate DNA-lipid complexes; in the case of the O-ethyl derivative described here, large vesicle preparations in the liquid crystalline phase were most effective.

Directly observed membrane fusion between oppositely charged phospholipid bilayers.

A novel method was developed for the direct examination of pairwise encounters between positively and negatively charged phospholipid bilayer vesicles. Giant bilayer vesicles (unilamellar, 4-20 micron in diameter) prepared from 1,2-dioleoyl-sn-glycero-3-ethylphosphocholine, a new cationic phospholipid derivative, were electrophoretically maneuvered into contact with individual anionic phospholipid vesicles. Fluorescence video microscopy revealed that such vesicles commonly underwent fusion within milliseconds (1 video field) after contact, without leakage. Fusion occurred at constant volume and, since flaccid vesicles were rare, the excess membrane was not available after fusion. Hemifusion (the outer monolayers of each vesicle fused while the inner monolayers remained intact) was inferred from membrane-bound dye transfer and a change in the contact area. Hemifusion was observed as a final stable state and as an intermediate to fusion of vesicles composed of charged phospholipids plus zwitterionic phospholipids. Hemifusion occurred in one of three ways following adhesion: either delayed with an abrupt increase in area of contact, immediately with a gradual increase in area of contact, or with retraction during which adherent vesicles dissociated from a flat contact to a point contact. Phosphatidylethanolamine strongly promoted immediate hemifusion; the resultant hemifused state was stable and seldom underwent complete fusion. Although sometimes single contacts between vesicles led to rupture of both, in other cases, a single vesicle underwent multiple fusion events. Direct observation has unequivocally demonstrated the fusion of two, isolated bilayer-bounded bodies to yield a stable, non-leaky product, as occurs in cells, in the absence of proteins.

Site-directed mutagenesis of either the highly conserved Trp-22 or the moderately conserved Trp-95 to a large, hydrophobic residue reduces the thermodynamic stability of a spectrin repeating unit.

As reported previously (MacDonald, R. I., Musacchio, A., Holmgren, R. A., and Saraste, M. (1994) Proc. Natl. Acad. Sci. U. S. A. 91, 1299-1303), an unfolded peptide was obtained by site-directed mutagenesis of Trp-22 to Ala in the cloned, wild type 17th repeating unit (alpha17) of chicken brain alpha-spectrin. Trp occurs in position 22 of nearly all repeating units of spectrin. In the present study, Trp-22 was mutated to Phe or to Tyr to compare thermodynamic stabilities of urea-induced unfolding of alpha16 and mutants thereof. alpha16 was chosen for this study instead of alpha17, because alpha16 has two tryptophans, allowing urea-induced unfolding to be tracked by the fluorescence of the Trp remaining in each mutant peptide and by circular dichroism in the far UV. The free energies of unfolding of W22Y and W22F were 50% that of alpha16, showing that Trp-22 is crucial in stabilizing the triple helical bundle motif of the spectrin repeating unit. Mutation of the moderately conserved Trp-95 of alpha16 to Val, which occupies position 95 in alpha17, also yielded a peptide with 50% of the free energy of unfolding of alpha16. Thus, the thermodynamic stability of a given spectrin repeating unit may depend on both moderately and highly conserved tryptophans. Different structural roles of Trp-22 and Trp-95 in alpha16 are suggested by the slightly higher wavelength of maximum emission of Trp-22, the greater acrylamide quenching of Trp-95 than Trp-22, and the longer lifetime of Trp-95. For comparison with alpha16, urea-induced unfolding of spectrin dimer isolated from human red cells was monitored by far UV-CD and by tryptophan fluorescence. Thermodynamic parameters could not be rigorously derived for the stability of spectrin dimer because unfolding of spectrin dimer involved more than two states, unlike unfolding of cloned repeating units. However, the similar midpoints of CD-monitored denaturation curves of alpha16 and spectrin dimer, i. e. 2.7 and 3.2 M urea, respectively, indicate that investigation of cloned repeating units of spectrin can provide physiologically relevant information on these structures.

The carboxyl-terminal region of factor IX is essential for its secretion.

The carboxyl-terminal region of factor IX (residues 403-415) contains several natural mutations which result in mild to severe forms of hemophilia B. A battery of factor IX minigene expression vectors carrying various mutations in the C-terminal region were constructed and studied by transient expression assay using HepG2 cells. Mutations included in this study are Y404P, I408N, T412N, T412S, T415G, T415S, T415L, and T415R as well as five selected naturally occurring mutations in the region, R403Q, R403W, Y404H, W407R, and T412K. In comparison to the normal factor IX, these mutations neither significantly affected the factor IX mRNA level nor affected the stability of the secreted factor IX in the culture medium but did decrease to various extents the intracellular and secreted factor IX protein levels as quantified by enzyme-linked immunosorbent assay. T415L, T415S, and T415R showed only minor reductions in the intracellular and minor to moderate reductions in the secreted factor IX levels. T415G showed only minor reduction in the intracellular factor IX level but substantial reduction in the secreted levels. R403Q, R403W, and T412S showed moderate reductions in both intracellular and secreted factor IX levels. Y404H, Y404P, W407R, I408N, T412K, and T412N also showed minor to moderate reductions in the intracellular factor IX levels but very severe reductions in the secreted factor IX level. In one stage clotting assays, secreted factor IX mutants showed specific activities very similar to that of the normal factor IX, suggesting that the carboxyl-terminal region is neither directly involved in the tenase complex formation required for factor X activation nor involved in the activation of factor IX itself. In comparison to the normal factor IX, secreted levels of factor IX mutants with mutations R403Q, Y404H, W407R, and T412K were also very similar to the plasma levels reported for the hemophilia B patients carrying the same mutations. Treatment of cells with proteasome inhibitors (ALLM and ALLN) added to the culture medium at 50 microM resulted in drastic increases of the intracellular mutant factor IX to the levels equivalent to that of the normal factor IX, while the secreted factor IX levels were little or only marginally affected. With a higher concentration of the inhibitors (100 microM), however, both the intracellular and secreted mutant factor IX were fully elevated to the normal factor IX levels. Intracellular and secreted levels of the normal factor IX were little affected by the low inhibitor concentration and only marginally, if at all, at the higher concentration (< or = 10%). Serine protease inhibitors did not significantly affect intracellular and secreted factor IX levels. These results indicate that the carboxyl-terminal region plays a critical role in the cellular secretion of factor IX and that the mutant factor IX proteins carrying specific mutations in this region are subjected to efficient elimination by the proteasome protein degradation mechanism. Furthermore, these results strongly support that HepG2 cells can be utilized as a robust in vitro assay system for studying factor IX biosynthesis, well mimicking the in vivo phenomenon.