Modulation of Membrane Fusion by Membrane Fluidity: Temperature Dependence of Divalent Cation Induced Fusion of Phosphatidylserine Vesicles
Nejat Düzgüneş: 0000-0001-6159-1391
We have investigated the temperature dependence of the fusion of phospholipid vesicles composed of pure bovine brain phosphatidylserine (PS) induced by Ca2+ or Mg2+. Aggregation of the vesicles was monitored by 90° light-scattering measurements, fusion by the terbium/dipicolinic acid assay for mixing of internal aqueous volumes, and release of vesicle contents by carboxyfluorescein fluorescence. Membrane fluidity was determined by diphenylhexatriene fluorescence polarization measurements. Small unilamellar vesicles (SUV, diameter 250 Å) or large unilamellar vesicles (LUV, diameter 1000 Å) were used, and the measurements were done in 0.1 M NaCl at pH 7.4. The following results were obtained: (1) At temperatures (0-5 °C) below the phase transition temperature (Tc) of the lipid, LUV (PS) show very little fusion in the presence of Ca2+, although vesicle aggregation is rapid and extensive. With increasing temperature, the initial rate of fusion increases dramatically. Leakage of contents at the higher temperatures remains limited initially, but subsequently complete release occurs as a result of collapse of the internal aqueous space of the fusion products. (2) SUV (PS) are still in the fluid state down to 0 °C, due to the effect of bilayer curvature, and fuse rapidly in the entire temperature range from 0 to 35 °C in the presence of Ca2+. The initial rate of leakage is low relative to the rate of fusion. At higher temperatures (15 °C and above), subsequent collapse of the vesicles' internal space causes complete release. At 0-5 °C, collapse does not occur, and the extent of fusion is limited; as the vesicles grow in size and start to resemble LUV, the fusion process slows down. (3) With Mg2+, LUV (PS) aggregate massively in the entire temperature range from 0 to 35 °C, but fusion and release of contents do not occur. (4) SUV (PS) do fuse in the presence of Mg2+. The initial rate of leakage relative to fusion is severalfold higher than during Ca2+-induced fusion of SUV. At low temperatures, this results in an early and complete release of vesicle contents. At 20 °C and above, complete release does not occur. Fusion stops spontaneously as the vesicles grow in size, and part of the aqueous contents is retained in the vesicles for a prolonged period of time. The results point out that a cation-induced isothermal phase transition of the bilayer lipids is not a prerequisite for the fusion process. Prerequisite for fusion appears to be that the vesicle is in an overall fluid state when the cation has bound to its outer surface. © 1985, American Chemical Society. All rights reserved.
Modulation of Membrane Fusion by Membrane Fluidity: Temperature Dependence of Divalent Cation Induced Fusion of Phosphatidylserine Vesicles.
Biochemistry, 24(1), 8–14.