The mechanism of lamellar-to-inverted hexagonal phase transitions: a study using temperature-jump cryo-electron microscopy.
The lamellar/inverted hexagonal (L alpha/HII) phase transition can be very fast, despite the drastic change in the topology of the lipid/water interfaces. The first structures to form in this transition may be similar to those that mediate membrane fusion in many lipid systems. To study the transition mechanism and other dynamic phenomena in membrane dispersions, we constructed an apparatus to rapidly trigger the transition and then vitrify the specimens to preserve the structure of transient intermediates. The apparatus applies millisecond-long temperature jumps of variable size to aqueous dispersions of lipids on electron microscope grids at times 9-16 ms before specimen vitrification. The vitrified specimens are then examined by cryo-transmission electron microscopy. Dispersions of egg phosphatidylethanolamine completed the transition within 9 ms when superheated by 20 K. Similar transition times have been observed in dioleoylphosphatidylethanolamine via time-resolved x-ray diffraction. N-monomethylated dioleoylphosphatidylethanolamine dispersions superheated to lesser extent exhibited slower transitions and more complex morphology. The structure of the first intermediates to form in the transition process could not be determined, probably because the intermediates are labile on the time scale of sample cooling and vitrification (< 1 ms) and because of the poor contrast developed by some of these small structures. However, the results are more compatible with a transition mechanism based on "stalk" intermediates than a mechanism involving inverted micellar intermediates. Temperature-jump cryo-transmission electron microscopy should be useful in studying dynamic phenomena in biomembranes, large protein complexes, and other colloidal dispersions. It should be especially helpful in studying the mechanism of protein-induced membrane fusion.
Siegel
D P
DP
Procter & Gamble Co., Miami Valley Laboratories, Cincinnati, Ohio 45239-8707.
Green
W J
WJ
Talmon
Y
Y
D055592:Biophysical Phenomena; D001703:Biophysics; D005615:Freezing; D007091:Image Processing, Computer-Assisted; D007700:Kinetics; D008081:Liposomes / Q000737:chemistry; D008563:Membrane Lipids / Q000737:chemistry*; D008854:Microscopy, Electron / Q000295:instrumentation; D008968:Molecular Conformation; D010713:Phosphatidylcholines / Q000737:chemistry; D010714:Phosphatidylethanolamines / Q000737:chemistry
D008081:Liposomes; D008563:Membrane Lipids; D010713:Phosphatidylcholines; D010714:Phosphatidylethanolamines; C020888:1,2-dielaidoylphosphatidylethanolamine; C017251:1,2-oleoylphosphatidylcholine