Studies of Membrane Fusion and Vesicle Fission of Biomembranes using Giant Unilamellar Vesicles and Cubic Phase and Their Applications

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(1) Using the single giant uniLαmelLαr vesicle (GUV) method, we found that low concentrations (less than critical micelle concentration (CMC), but above their threshold concentrations) of amphiphiles with a single long hydrocarbon chain (i.e., single long chain amphiphiles) such as lysophosphatidylcholine (lyso-PC), lyosophosphatidic acid, octylglucoside, induced the vesicle fission of GUVs of lipid membranes in the liquid-ordered (lo) phase such as dipalmitoylphosphatidylcholine(DPPC)/cholesterol(6/4) membranes. To elucidate the effect of the inclusion of DOPC (i.e., an Lα phase forming lipid) in the lo phase-membrane of GUVs on the lyso-PC-induced vesicle fission of the DPPC/chol (6/4)-GUV, we investigated effect of low concentrations of lyso-PC on GUVs of DPPC/DOPC/chol membranes. With an increase in DOPC concentration in the membrane, the threshold concentration of lyso-PC to induce the vesicle fission increased. At and above 30 mol% DOPC, no vesicle fission occurred. On the basis … More of these results, we have proposed a hypothesis of the mechanism of the single long chain amphiphile-induced vesicle fission of the GUV of the lo phase-membrane.(2) We investigated the effect of H+ (i.e. pH effect) on the phase and structure of membranes of a negatively charged lipid, dioleoylphosphatidylserine (DOPS) and an electrically neutral lipid, monoolein (MO) mixtures under a physiological ion concentration condition, using small-angle X-ray scattering (SAXS). At neutral pH, DOPS/MO membranes containing high concentrations of DOPS were in the Lα phase. First, the pH effect on the phase and structure of the multiLαmelLαr vesicles (MLVs) of the DOPS/MO membranes preformed at neutral pH was investigated by adding various low pH buffers into the MLV suspension. For 20%-DOPS/80%-MO-MLVs, at and below pH 2.9, a transition from the Lα to cubic (Q224) phase occurred within 1 h. This phase transition was reversible ; a subsequent increase in pH to a neutral one in the membrane suspension transformed the cubic phase into the original Lα phase. Second, we found that a decrease in pH transformed Lαrge uniLαmelLαr vesicles of DOPS/MO membranes into the cubic phase after the membrane fusion between the LUVs under simiLαr conditions. We have proposed the mechanism of the low-pH induced phase transition, and also made a quantitative analysis on the critical pH of the phase transition. This finding is the first demonstration that a change in pH can induce a reversible phase transition between the Lα and cubic phases of lipid membranes within 1 h. We reasonably consider that pH-induced interconversion between the cubic and the Lα phases would also occur in cells.(3)We have proposed a novel method, the single GUV method to probe functions and dynamics of biomembranes and interactions of substances with biomembranes. We have succeeded in revealing the pore formation induced by antimicrobial peptide, maginin2, and the interactions of antibacterial substance, (-)-epigallocatechin gallate(ECGg) with lipid membranes. Less