Selected publications

1. Mikhalyov I., Samsonov A. (2011). Lipid raft detecting in membranes of live erythrocytes. Biochim. Biophys. Acta 1808 (7), 1930–9 [+]

   The fluorescent probe N-(BODIPY(®)-FL-propionyl)-neuraminosyl-GM(1) (BODIPY-GM(1)) was used to detect lipid rafts in living red blood cells (RBCs) membranes. The probe was detected with fluorescence video microscopy and was found to be uniformly distributed along plasma membrane at room temperature (23°C). At 4°C some probe clearly phase-separated to yield detectable bright spots that were smaller than spatial resolution. As measured by spectrofluorometry, in addition to a major fluorescence peak caused by emissions from monomers, the probe exhibited a red-shifted peak that is characteristic of a BODIPY fluorophore at high local concentrations, indicating that some probe had clustered. Red-shifted fluorescence was the greatest at 4°C, intermediate at 23°C, and the smallest at 37°C. Treating the RBCs with methyl-β-cyclodextrin to remove cholesterol eliminated the red-shifted peak. This strongly indicates that the presence of cholesterol was essential for phase separation of the probe. Fluorometry experiments indicate that rafts exist at 23°C and at 37°C, even though the membrane appears to be uniform at the resolution of microscope. The distinct GM(1) patches distributed over entire membrane of the erythrocytes were observed at both 23°C and at 37°C in RBCs stained with Alexa FL 647 cholera toxin subunit B conjugate (CTB-A647 ). Based on both fluorometry and fluorescence microscopy, some rafts clearly exist at 37°C.

2. Sachl R., Mikhalyov I., Gretskaya N., Olżyńska A., Hof M., Johansson L.B. (2011). Distribution of BODIPY-labelled phosphatidylethanolamines in lipid bilayers exhibiting different curvatures. Phys Chem Chem Phys 13 (24), 11694–701 [+]

   In this paper we have investigated the behaviour of newly synthesised mono-palmitoyl- and dipalmitoyl-phosphatidylethanolamine probes (abbreviated as mPE and dPE, respectively) labelled in the polar headgroup region by either the FL-BODIPY or the 564/570-BODIPY fluorophore and solubilised in lipid systems that exhibit different curvatures. Because of the bulky BODIPY-groups, the monoacyl-form derivatives have a conic-like shape, whereas that for the diacyl derivatives is rather cylindrical. A careful analysis of time-resolved resonance energy transfer experiments by means of analytical models as well as Monte Carlo simulations shows that the mPE derivatives have a comparable affinity to highly curved bilayer regions (torroidal pores formed by magainin-2 in lipid bilayers, or the rims of discoid bicelles) and to planar bilayer regions (i.e. the flat region of lipid bilayers and bicelles). Furthermore, the monoacyl-probes are as compared to the diacyl-probes effectively closer to each other in a lipid bilayer, while none of these probes seems to be randomly distributed. Self-aggregation is most efficiently induced by the larger aromatic 564/570-BODIPY chromophore, but it is suppressed when using the diacyl instead of the monoacyl-form, and/or by attaching BODIPY-groups to the acyl-chain.

3. Mikhalyov I., Gretskaya N., Johansson L.B. (2009). Fluorescent BODIPY-labelled GM1 gangliosides designed for exploring lipid membrane properties and specific membrane-target interactions. Chem. Phys. Lipids 159 (1), 38–44 [+]

   New fluorophore-labelled G(M1) gangliosides have been synthesised and spectroscopically characterised. Spectroscopically different BODIPY groups were covalently linked, specifically to either the polar or the hydrophobic part of the ganglioside molecule. The absorption and fluorescence spectroscopic properties are reported for 564/571-BODIPY- and 581/591-BODIPY-labelled G(M1). Each of the different BODIPY groups is highly fluorescent and depolarisation experiments provide molecular information about the spatial distribution in lipid bilayers, as well as order and dynamics. From experiments performed on two spectroscopically different BODIPY:s, specific interactions can be revealed by monitoring the rate/efficiency of donor-acceptor electronic energy transfer. Systems of particular interest for applying these probes are e.g. mixtures of lipids, and peptides/proteins interacting with lipid membranes.

4. Shnyrova A.V., Ayllon J., Mikhalyov I.I., Villar E., Zimmerberg J., Frolov V.A. (2007). Vesicle formation by self-assembly of membrane-bound matrix proteins into a fluidlike budding domain. J. Cell Biol. 179 (4), 627–33 [+]

   The shape of enveloped viruses depends critically on an internal protein matrix, yet it remains unclear how the matrix proteins control the geometry of the envelope membrane. We found that matrix proteins purified from Newcastle disease virus adsorb on a phospholipid bilayer and condense into fluidlike domains that cause membrane deformation and budding of spherical vesicles, as seen by fluorescent and electron microscopy. Measurements of the electrical admittance of the membrane resolved the gradual growth and rapid closure of a bud followed by its separation to form a free vesicle. The vesicle size distribution, confined by intrinsic curvature of budding domains, but broadened by their merger, matched the virus size distribution. Thus, matrix proteins implement domain-driven mechanism of budding, which suffices to control the shape of these proteolipid vesicles.

5. Samsonov A.V., Mikhalyov I., Cohen F.S. (2001). Characterization of cholesterol-sphingomyelin domains and their dynamics in bilayer membranes. Biophys. J. 81 (3), 1486–500 [+]

   Lipids segregate with each other into small domains in biological membranes, which can facilitate the associations of particular proteins. The segregation of cholesterol and sphingomyelin (SPM) into domains known as rafts is thought to be especially important. The formation of rafts was studied by using planar bilayer membranes that contained rhodamine-phosphatidylethanolamine (rho-DOPE) as a fluorescent probe, and wide-field fluorescence microscopy was used to detect phase separation of the probe. A fluorescently labeled GM(1), known to preferentially partition into rafts, verified that rho-DOPE faithfully reported the rafts. SPM-cholesterol domains did not form at high temperatures but spontaneously formed when temperature was lowered to below the melting temperature of the SPM. Saturated acyl chains on SPMs therefore promote the formation of rafts. The domains were circular (resolution > or = 0.5 microm), quickly reassumed their circular shape after they were deformed, and merged with each other to create larger domains, all phenomena consistent with liquid-ordered (l(o)) rather than solid-ordered (s(o)) domains. A saturated phosphatidylcholine (PC), disteoryl-PC, could substitute for SPM to complex with cholesterol into a l(o)-domain. But in the presence of cholesterol, a saturated phosphatidylethanolamine or phosphatidylserine yielded s(o)-domains of irregular shape. Lipids with saturated acyl chains can therefore pack well among each other and with cholesterol to form l(o)-domains, but domain formation is dependent on the polar headgroup of the lipid. An individual raft always extended through both monolayers. Degrading cholesterol in one monolayer with cholesterol oxidase first caused the boundary of the raft to become irregular; then the raft gradually disappeared. The fluid nature of rafts, demonstrated in this study, may be important for permitting dynamic interactions between proteins localized within rafts.