Fluorescent probe partitioning in giant unilamellar vesicles of ‘lipid raft’ mixtures

Abstract

Direct visualization of raft-like l(o) (liquid-ordered) domains in model systems and cells using microscopic techniques requires fluorescence probes with known partitioning preference for one of the phases present. However, fluorescent probes may display dissimilar partitioning preferences in different lipid systems and can also affect the phase behaviour of the host lipid bilayer. Therefore a detailed understanding of the behaviour of fluorescent probes in defined lipid bilayer systems with known phase behaviour is essential before they can be used for identifying domain phase states. Using giant unilamellar vesicles composed of the ternary lipid mixture DOPC (1,2-dioleoyl-sn-glycero-3-phosphocholine)/DPPC (1,2-dipalmitoyl-sn-glycero-3-phosphocholine)/cholesterol, for which the phase behaviour is known, we examined nine commonly used fluorescent probes using confocal fluorescence microscopy. The partitioning preference of each probe was assigned either on the basis of quantification of the domain area fractions or by using a well-characterized l(d) (liquid-disordered)-phase marker. Fluorescent probes were examined both individually and using dual or triple labelling approaches. Most of the probes partitioned individually into the l(d) phase, whereas only NAP (naphtho[2,3-a]pyrene) and NBD-DPPE [1,2-dipalmitoyl-sn-glycero-3-phosphoethanolamine-N-(7-nitro-2-1,3-benzoxadiazol-4-yl] preferred the l(o) phase. We found that Rh-DPPE (Lissamine rhodamine B-1,2-dipalmitoyl-sn-glycero-3-phosphoethanolamine) increased the miscibility transition temperature, T(mix). Interestingly, the partitioning of DiIC18 (1,1'-dioctadecyl-3,3,3',3'-tetramethylindocarbocyanine perchlorate) was influenced by Bodipy-PC [2-(4,4-difluoro-5,7-dimethyl-4-bora-3a,4a-diaza-s-indacene-3-pentanoyl)-1-hexa-decanoyl-sn-glycero-3-phosphocholine]. The specific use of each of the fluorescent probes is determined by its photostability, partitioning preference, ability to detect lipid phase separations and induced change in T(mix). We demonstrate the importance of testing a specific fluorescent probe in a given model membrane system, rather than assuming that it labels a particular lipid phase.

In Situ Visualization of Lipid Raft Domains by Fluorescent Glycol Chitosan Derivatives

“…To see whether glycol chitosan-FITC can visualize the lipid raft domains in a more cell-like geometry, GUVs composed of DOPC/SM/Chol (1:1:1) containing 1 mol% of LR-DOPE were prepared and observed under a confocal fluorescence microscope after interacting with 20 µg/mL glycol chitosan-FITC. The appearance of the fluorescent lipid-labeled GUVs(Figure 6) was similar to that in the previous work 36. The confocal imaging results of GUVs also indicate that glycol chitosan-FITC could stain lipid raft domains preferentially(Figure 6), in line with that of SLBs (Figure 2).…”

Behaviour of NBD-head group labelled phosphatidylethanolamines in POPC bilayers: a molecular dynamics study

“…25 Alternatively, NBD probes can be used as acceptors to chromophores including diphenylhexatriene 26,27 or trans-parinaric acid, 28 or even as donors and acceptors simultaneously in homo-FRET studies. 20 Several fluorescence spectroscopic and microscopic studies have also indicated that, contrary to the vast majority of membrane fluorescent probes, doubly saturated long-chained NBD-diC n PE partitions favourably to liquid ordered (lo) phases in systems displaying lo/liquid disordered phase coexistence, 22,24,29,30 although the phase preference depends both on the probe acyl chain length and the composition of the underlying lipid mixture. 17,31 Despite their wide use in membrane biophysics, the behaviour of NBD-diC n PE probes has so far lacked a characterization using MD.…”

Fluorescence Techniques to Study Lipid Dynamics

“…Second, the quantum efficiency and brightness of most of the organic dyes are higher than for fluorescent proteins. Cholesterol (Boldyrev et al 2007;Holtta-Vuori et al 2008;Marks et al 2008;Oreopoulos and Yip 2009), Sphingomyelin (Marks et al 2008;Eggeling et al 2009;Tyteca et al 2010), GM1 (Coban et al 2007;Eggeling et al 2009;Mikhalyov et al 2009), PC, and PE (Baumgart et al 2007;Juhasz et al 2010) are some of the lipids that are often conjugated to organic dyes. Additionally, fluorescently labeled membrane-binders, like choleratoxin, are used to label, for example, the GMs on the cell surface (Middlebrook and Dorland 1984).…”