THE MECHANISM OF LAMELLAR-TO-INVERTED HEXAGONAL PHASE-TRANSITIONS - ASTUDY USING TEMPERATURE-JUMP CRYOELECTRON MICROSCOPY

The lamellar/inverted hexagonal (L(alpha)/H-II) phase transition can b e very fast, despite the drastic change in the topology of the lipid/w ater interlaces. The first structures to form in this transition may b e similar to those that mediate membrane fusion in many lipid systems. To study the transition mechanism and other dynamic phenomena in memb rane dispersions, we constructed an apparatus to rapidly trigger the t ransition and then vitrify the specimens to preserve the structure of transient intermediates. The apparatus applies millisecond-long temper ature jumps of variable size to aqueous dispersions of lipids on elect ron microscope grids at times 9-16 ms before specimen vitrification. T he 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 t imes have been observed in dioleoylphosphatidylethanolamine via time-r esolved x-ray diffraction. N-monomethylated dioleoylphosphatidylethano lamine dispersions superheated to lesser extent exhibited slower trans itions and more complex morphology. The structure of the first interme diates to form in the transition process could not be determined, prob ably because the intermediates are labile on the time scale of sample cooling and vitrification (<1 ms) and because of the poor contrast dev eloped by some of these small structures. However, the results are mor e compatible with a transition mechanism based on ''stalk'' intermedia tes than a mechanism involving inverted micellar intermediates. Temper ature-jump cryo-transmission electron microscopy should be useful in s tudying dynamic phenomena in biomembranes, large protein complexes, an d other colloidal dispersions. It should be especially helpful in stud ying the mechanism of protein-induced membrane fusion.