Article pubs.acs.org/JPCB
Link between Fluorescent Probe Partitioning and Molecular Order of Liquid Ordered-Liquid Disordered Membranes Sherry S. W. Leung† and Jenifer Thewalt*,‡,†,§ †
Department of Physics, Simon Fraser University, Burnaby, BC V5A 1S6, Canada Department of Molecular Biology and Biochemistry, Simon Fraser University, Burnaby, BC V5A 1S6, Canada
‡
ABSTRACT: Fluorescence microscopy is an important technique for studying lipid membranes and is increasingly being used for examining liquid orderedliquid disordered phase coexistence. Liquid−liquid phase coexistence is a phenomenon of biological interest because it led to the lipid raft hypothesis, which postulates the existence of lateral heterogeneities in cell membranes. Observation of membrane heterogeneity relies on differential distribution of fluorescent membrane markers, but this can also modify the phase behavior, complicating the observation. We have used 2H NMR to measure the physical changes to 35:35:30 (mol/mol) DOPC/DPPC-D62/chol membranes introduced by fluorescent probes Laurdan and naphthopyrene. We measured miscibility transition temperature (Tmix) and DPPC-D62 chain order for a range of probe concentrations. We found that up to 0.5 mol% of the equipartitioning probe Laurdan does not influence DPPC-D62 acyl chain order or phase behavior. In contrast, 2.0 mol% Laurdan slightly increases the fraction of DPPC-D62 in the liquid disordered phase below the Tmix and increases Tmix by 1 °C. Conversely, the nominally liquid ordered phase preferring probe naphthopyrene slightly perturbs the membrane even at concentrations as low as 0.3 mol%. This suggests that the strength of fluorescent probe partitioning between liquid ordered and liquid disordered phases correlates with the degree of perturbation to membrane phase behavior.
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INTRODUCTION Fluorescence microscopy and spectroscopy can provide dynamic and structural information about membranes, and are widely used in studies of living cells and model membranes. For lipid membrane phase studies, a small amount of fluorescent probe usually has to be added. Probes which partition unequally between coexisting phases are used to provide contrast, and are typically added in small amounts (0.1−1 mol%). In thermodynamic terms, the probe may be seen as an ideally dilute solute, and is thus often presumed to be a silent observer.1 Although earlier monolayer2 and giant vesicle3 studies supported this assumption, there is now ample evidence to suggest that membrane and probe behavior are not independent of each other. Membrane composition can alter how a probe partitions, and probes can affect membrane behavior.4−6 Baumgart et al.6 showed that 26 different fluorescent probes reported different membrane behavior for giant unilamellar vesicles made with a typical model raft lipid mixture dioleoylphosphatidylcholine (DOPC), sphingomyelin (Sm), and cholesterol (chol). Although earlier reports found that 35:35:30 (mol/mol) DOPC/DPPC/chol membranes with 0.2 and 2 mol% TR-DPPE had the same Tmix,7 it was later shown with 2H NMR that an intermediate amount of TRDPPE substantially raised Tmix, compared with the probe-free membrane (private communications). Atomistic molecular dynamics also showed that TR-DPPE increases the miscibility transition temperature (Tmix) of an analogous generic ternary lipid membrane.8 In earlier work, we used 2H NMR to show © XXXX American Chemical Society
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