Pyrene cholesterol reports the transient appearance of nonlamellar intermediate structures during fusion of model membranes

We have hypothesized that modulating the free energy of hydrophobic mismatch (HM) might be a principal means to control the fusion process and that it may be a role of cholesterol to counteract HM and make membranes fusogenic. To test these hypotheses, we examined the ability of cholesterol 1-pyrenebutyrate (PY-Ch) and other pyrene-containing fluorescent probes to report interstices formed during the L(alpha)-H(II) transition of DiPoPE in terms of changes in excimer/monomer (E/M) fluorescence ratios. We found a significant (>150%) increase in the PY-Ch E/M in the hexagonal phase relative to the lamellar phase, presumably resulting from redistribution of PY-Ch from the curved lamellar leaflets to coexisting HMs that constitute 20 vol % of this phase. All other probes showed a much smaller or even an opposite (PY-hexadecanoic acid) effect. The time course of the PY-Ch E/M ratio during fusion of DOPC/PE/Ch small unilamellar vesicles showed a transient increase with a subsequent decrease, consistent with fusion proceeding through intermediates with significant HM. The amplitude and position of the maximum in E/M correlated with the rate of contents mixing. An increase in E/M was not seen when lipid mixing occurred in the absence of contents mixing. Our results suggest that PY-Ch provides a tool for monitoring fusion intermediates that occur after the initial fusion intermediate but prior to pore formation, possibly by accumulating in regions associated with HM.