  The phase separation in lipid bilayers that include negatively charged lipids
is examined experimentally. We observed phase-separated structures and
determined the membrane miscibility temperatures in several binary and ternary
lipid mixtures of unsaturated neutral lipid, dioleoylphosphatidylcholine
(DOPC), saturated neutral lipid, dipalmitoylphosphatidylcholine (DPPC),
unsaturated charged lipid, dioleoylphosphatidylglycerol
(DOPG$^{\scriptsize{(-)}}$), saturated charged lipid,
dipalmitoylphosphatidylglycerol (DPPG$^{\scriptsize{(-)}}$), and cholesterol.
In binary mixtures of saturated and unsaturated charged lipids, the combination
of the charged head with the saturation of hydrocarbon tail is a dominant
factor for the stability of membrane phase separation.
DPPG$^{\scriptsize{(-)}}$ enhances phase separation, while
DOPG$^{\scriptsize{(-)}}$ suppresses it. Furthermore, the addition of
DPPG$^{\scriptsize{(-)}}$ to a binary mixture of DPPC/cholesterol induces phase
separation between DPPG$^{\scriptsize{(-)}}$-rich and cholesterol-rich phases.
This indicates that cholesterol localization depends strongly on the electric
charge on the hydrophilic head group rather than on the ordering of the
hydrocarbon tails. Finally, when DPPG$^{\scriptsize{(-)}}$ was added to a
neutral ternary system of DOPC/DPPC/Cholesterol (a conventional model of
membrane rafts), a three-phase coexistence was produced. We conclude by
discussing some qualitative features of the phase behaviour in charged
membranes using a free energy approach.
