Several peripheral membrane proteins are known to form membrane pores through multimerization. In many cases, in biochemical reconstitution experiments, a complex distribution of oligomeric states has been observed that may, in part, be irrelevant to their physiological functions. This phenomenon makes it difficult to identify the functional oligomeric states of membrane lipid interacting proteins, for example, during the formation of transient membrane pores. Using fibroblast growth factor 2 (FGF2) as an example, we present a methodology applicable to giant lipid vesicles by which functional oligomers can be distinguished from nonspecifically aggregated proteins without functionality. Two distinct populations of fibroblast growth factor 2 were identified with (i) dimers to hexamers and (ii) a broad population of higher oligomeric states of membrane-associated FGF2 oligomers significantly distorting the original unfiltered histogram of all detectable oligomeric species of FGF2. The presented statistical approach is relevant for various techniques for characterizing membrane-dependent protein oligomerization.

• MeSH
• Cell Membrane metabolism   MeSH
• Fibroblast Growth Factor 2 * metabolism   MeSH
• Lipids MeSH
• Membrane Proteins * metabolism   MeSH
• Membranes MeSH
• Protein Multimerization MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH
• Names of Substances
• Fibroblast Growth Factor 2 * MeSH
• Lipids MeSH
• Membrane Proteins * MeSH

Many membrane proteins are thought to function as dimers or higher oligomers, but measuring membrane protein oligomerization in lipid membranes is particularly challenging. Förster resonance energy transfer (FRET) and fluorescence cross-correlation spectroscopy are noninvasive, optical methods of choice that have been applied to the analysis of dimerization of single-spanning membrane proteins. However, the effects inherent to such two-dimensional systems, such as the excluded volume of polytopic transmembrane proteins, proximity FRET, and rotational diffusion of fluorophore dipoles, complicate interpretation of FRET data and have not been typically accounted for. Here, using FRET and fluorescence cross-correlation spectroscopy, we introduce a method to measure surface protein density and to estimate the apparent Förster radius, and we use Monte Carlo simulations of the FRET data to account for the proximity FRET effect occurring in confined two-dimensional environments. We then use FRET to analyze the dimerization of human rhomboid protease RHBDL2 in giant plasma membrane vesicles. We find no evidence for stable oligomers of RHBDL2 in giant plasma membrane vesicles of human cells even at concentrations that highly exceed endogenous expression levels. This indicates that the rhomboid transmembrane core is intrinsically monomeric. Our findings will find use in the application of FRET and fluorescence correlation spectroscopy for the analysis of oligomerization of transmembrane proteins in cell-derived lipid membranes.

• MeSH
• Cell Membrane metabolism   MeSH
• Dimerization MeSH
• Humans MeSH
• Membrane Lipids metabolism   MeSH
• Membrane Proteins * metabolism   MeSH
• Protein Multimerization MeSH
• Fluorescence Resonance Energy Transfer * MeSH
• Check Tag
• Humans MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH
• Names of Substances
• Membrane Lipids MeSH
• Membrane Proteins * MeSH

Changes of membrane organization upon cross-linking of its components trigger cell signaling response to various exogenous factors. Cross-linking of raft gangliosides GM1 with cholera toxin (CTxB) was shown to cause microscopic phase separation in model membranes, and the CTxB-GM1 complexes forming a minimal lipid raft unit are the subject of ongoing cell membrane research. Yet, those subdiffraction sized rafts have never been described in terms of size and dynamics. By means of two-color z-scan fluorescence correlation spectroscopy, we show that the nanosized domains are formed in model membranes at lower sphingomyelin (Sph) content than needed for the large-scale phase separation and that the CTxB-GM1 complexes are confined in the domains poorly stabilized with Sph. Förster resonance energy transfer together with Monte Carlo modeling of the donor decay response reveal the domain radius of ~8 nm, which increases at higher Sph content. We observed two types of domains behaving differently, which suggests a dual role of the cross-linker: first, local transient condensation of the GM1 molecules compensating for a lack of Sph and second, coalescence of existing nanodomains ending in large-scale phase separation.

• MeSH
• Models, Chemical * MeSH
• Cholera Toxin chemistry   MeSH
• Membrane Fluidity * MeSH
• G(M1) Ganglioside chemistry   MeSH
• Lipid Bilayers chemistry   MeSH
• Membrane Microdomains chemistry   ultrastructure   MeSH
• Molecular Conformation MeSH
• Models, Molecular MeSH
• Cross-Linking Reagents chemistry   MeSH
• Phase Transition MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH
• Names of Substances
• Cholera Toxin MeSH
• G(M1) Ganglioside MeSH
• Lipid Bilayers MeSH
• Cross-Linking Reagents MeSH

Fluorescence correlation spectroscopy (FCS) is a single molecule technique used mainly for determination of mobility and local concentration of molecules. This review describes the specific problems of FCS in planar systems and reviews the state of the art experimental approaches such as 2-focus, Z-scan or scanning FCS, which overcome most of the artefacts and limitations of standard FCS. We focus on diffusion measurements of lipids and proteins in planar lipid membranes and review the contributions of FCS to elucidating membrane dynamics and the factors influencing it, such as membrane composition, ionic strength, presence of membrane proteins or frictional coupling with solid support.

• Keywords
• biomembranes, confocal microscopy, fluorescence fluctuation spectroscopy, giant unilamellar vesicles, lateral diffusion, supported lipid bilayers,
• MeSH
• Diffusion MeSH
• Spectrometry, Fluorescence * MeSH
• Microscopy, Confocal MeSH
• Lipid Bilayers chemistry   MeSH
• Membrane Lipids chemistry   MeSH
• Unilamellar Liposomes chemistry   MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH
• Review MeSH
• Names of Substances
• Lipid Bilayers MeSH
• Membrane Lipids MeSH
• Unilamellar Liposomes MeSH