Chemistry and Physics of Lipids 165 (2012) 630–637 Contents lists available at SciVerse ScienceDirect Chemistry and Physics of Lipids j our na l ho me p age: www.elsevier.com/locate/chemphyslip Effect of temperature on the formation of liquid phase-separating giant unilamellar vesicles (GUV) Viktoria Betaneli a,1 , Remigiusz Worch a,b,∗,1 , Petra Schwille a a BIOTEC, Biophysics Research Group, Technical University Dresden, Tatzberg 47-51, 01307 Dresden, Germany b Laboratory of Biological Physics, Institute of Physics, Polish Academy of Sciences, Al. Lotnikow 32/46, 02-668 Warsaw, Poland a r t i c l e i n f o Article history: Received 29 March 2012 Received in revised form 12 June 2012 Accepted 13 June 2012 Available online 29 June 2012 Keywords: Giant unilamellar vesicles Protein reconstitution Lipid rafts Confocal microscopy Fluorescence correlation spectroscopy Lipid fluorescent probe a b s t r a c t Giant unilamellar vesicles (GUVs) are widely used as model systems to study both, lipid and membrane protein behavior. During their preparation by the commonly applied electroformation method, a num- ber of issues must be considered to avoid the production of artifacts due to a poor lipid hydration and protein degradation. Here we focus on the effect of preparation temperature on GUVs composed of the most commonly used domain-forming mixture dioleoylelphospatidylcholine/shingomyelin/cholesterol (DOPC/SM/chol) (2/2/1). Lower production temperatures are generally preferable when aiming at a func- tional reconstitution of proteins into the membrane. On the other hand, lower growth temperature is suspected to alter the lipid composition and the yield of phase-separating vesicles. By confocal imag- ing, we find that vesicles prepared significantly above and below the melting temperature T m have the same overall morphology, similar size distributions of vesicles and a similar area coverage by liquid- ordered (L o ) domains. However, a large population analysis indeed reveals a different overall yield of phase-separating vesicles. Two-focus scanning fluorescence correlation spectroscopy measurements did not show any divergence of lipid analog mobility in (L o ) and (L d ) phases in vesicles prepared at different temperatures, indicating that the lowered growth temperature did not influence the lipid organiza- tion within the two phases. Moreover, the expected advantages of lower preparation temperature for proteo-GUVs could be exemplified by the reconstitution of voltage dependent anion channel (VDAC) into DOPC/SM/chol GUVs, which aggregates at high, but not at low preparation temperatures. © 2012 Elsevier Ireland Ltd. All rights reserved. 1. Introduction Biological membranes are assemblies of various types of lipids and integral membrane proteins, but their complexity is a limiting factor for quantitative experiments. Therefore a variety of simpler artificial membrane models with controllable size, geometry and compositions were developed (for a review see Chan and Boxer, 2007). Giant unilamellar vesicles (GUVs) are especially useful for optical microscopy, mainly because of their cell-like size and cur- vature (for a recent review see Garcia-Saez et al., 2009 and Kahya, 2010). Apart from the studies devoted exclusively to lipid mixtures, GUVs are also used as a platform for membrane protein reconstitu- tion from previously prepared proteoliposomes. This approach was Abbreviations: DOPC, dioleoylphosphatidylcholine; SM, sphingomyelin; chol, cholesterol; RT, room temperature; FV, field of view; Lo, liquid-ordered; L d , liquid-disordered; FCS, fluorescence correlation spectroscopy; 2fsFCS, two-focus scanning FCS; DiD, 1,1 ′ -dioctadecyl-3,3,3 ′ ,3 ′ tetramethylindodicarbocyanine 4- chlorobenzenesulfonate salt. ∗ Corresponding author. Tel.: +48 22 843 66 01x2204; fax: +48 22 843 09 26. E-mail address: remiwo@ifpan.edu.pl (R. Worch). 1 Both authors contributed equally to this work. first advertized by Girard et al., successfully incorporating the Ca 2+ - ATPase and bacteriorhodopsin. Following similar protocols, several other proteins were successfully incorporated in the membranes of artificial vesicles (summarized in Kahya, 2010). However, GUV electroformation from proteoliposomes poses several limitations. One of them is the use of buffers with physio- logical salt concentration required for protein stability, which was a motivation for development of novel electroformation protocols (Pott et al., 2008; Shaklee et al., 2010). Another one is related with the preparation temperature, which is advised to be high enough to ensure melting and mixing of all lipids, followed by a slow cool- ing to achieve near-equilibrium states for phase behavior studies (Morales-Penningston et al., 2010). Indeed, it was reported that for phospholipids with high gel-to-liquid phase transition tempera- tures, the electroformation did not result in GUV production when performed at room temperature (Shimanouchi et al., 2009). The effect of growth temperature was shown to influence the misci- bility transition temperature in DOPC/DPPC mixtures containing 35% of cholesterol (Veatch and Keller, 2005). However, even more drastic changes were observed when a too low amount of lipids in the film was used (Veatch and Keller, 2005). Therefore differ- ent experimental methods produce vesicles with slightly different 0009-3084/$ – see front matter © 2012 Elsevier Ireland Ltd. All rights reserved. http://dx.doi.org/10.1016/j.chemphyslip.2012.06.006