ABCA1 transporter reduces amphotericin B cytotoxicity in mammalian cells.

Amphotericin B (AmB) belongs to a group of polyene antibiotics commonly used in the treatment of systemic mycotic infections. A widely accepted mechanism of action of AmB is based on the formation of an oligomeric pore structure within the plasma membrane (PM) by interaction with membrane sterols. Although AmB binds preferentially to ergosterol, it can also bind to cholesterol in the mammalian PM and cause severe cellular toxicity. The lipid content and its lateral organization at the cell PM appear to be significant for AmB binding. Several ATP-binding cassette (ABC) transporters, including ABCA1, play a crucial role in lipid translocation, cholesterol redistribution and efflux. Here, we demonstrate that cells expressing ABCA1 are more resistant to AmB treatment, while cells lacking ABCA1 expression or expressing non-active ABCA1MM mutant display increased sensitivity. Further, a FLIM analysis of AmB-treated cells reveals a fraction of the antibiotic molecules, characterized by relatively high fluorescence lifetimes (> 6 ns), involved in formation of bulk cholesterol-AmB structures at the surface of ABCA1-expressing cells. Finally, lowering the cellular cholesterol content abolishes resistance of ABCA1-expressing cells to AmB. Therefore, we propose that ABCA1-mediated cholesterol efflux from cells induces formation of bulk cholesterol-AmB structures at the cell surface, preventing AmB cytotoxicity.

Nanoscale resolution in infrared imaging of protein-containing lipid membranes.

The precise imaging of biomolecular entities contributes to an understanding of the relationship between their structure and function. However, the resolution of conventional infrared microscopic imaging is diffraction limited and does not exceed a few micrometres. Atomic force microscopy, on the other hand, can detect infrared absorption down to the sub-micrometer level. In the present report, we demonstrate that for multi-bilayer lipid samples containing the plant photosynthetic pigment-protein complex LHCII, the resolution of this latter technique can be better than 20 nm. Such a high resolution is attributable to two factors: (i) the relatively high infrared absorption by the complex that is integrated perpendicular to the plane of the multilayer film, and (ii) the distinctly different mechanical properties and thermal conductivity of the lipid and protein components of the sample.

Rotation of the lever arm of Myosin in contracting skeletal muscle fiber measured by two-photon anisotropy.

The rotation of the lever arm of myosin cross-bridges is believed to be responsible for muscle contraction. To resolve details of this rotation, it is necessary to observe a single cross-bridge. It is still impossible to do so in muscle fiber, but it is possible to investigate a small population of cross-bridges by simultaneously activating myosin in a femtoliter volume by rapid release of caged ATP. In earlier work, in which the number of observed cross-bridges was limited to approximately 600 by confocal microscopy, we were able to measure the rates of cross-bridge detachment and rebinding. However, we were unable to resolve the power stroke. We speculated that the reason for this was that the number of observed cross-bridges was too large. In an attempt to decrease this number, we used two-photon microscopy which permitted observation of approximately 1/2 as many cross-bridges as before with the same signal/noise ratio. With the two-photon excitation, the number of cross-bridges was small enough to resolve the beginning of the power stroke. The results indicated that the power stroke begins approximately 170 ms after the rigor cross-bridge first binds ATP.

Effect of 13-cis violaxanthin on organization of light harvesting complex II in monomolecular layers.

Lutein, neoxanthin and violaxanthin are the main xanthophyll pigment constituents of the largest light-harvesting pigment-protein complex of photosystem II (LHCII). High performance liquid chromatography analysis revealed photoisomerization of LHCII-bound violaxanthin from the conformation all-trans to the conformation 13-cis and 9-cis. Maximally, the conversion of 15% of all-trans violaxanthin to a cis form could be achieved owing to the light-driven reactions. The reactions were dark-reversible. The all-trans to cis isomerization was found to be driven by blue light, absorbed by chlorophylls and carotenoids, as well as by red light, absorbed exclusively by chlorophyll pigments. This suggests that the photoisomerization is a carotenoid triplet-sensitized reaction. The monomolecular layer technique was applied to study the effect of the 13-cis conformer of violaxanthin and its de-epoxidized form, zeaxanthin, on the organization of LHCII as compared to the all-trans stereoisomers. The specific molecular areas of LHCII in the two-component system composed of protein and exogenous 13-cis violaxanthin or 13-cis zeaxanthin show overadditivity, which is an indication of the xanthophyll-induced disassembly of the aggregated forms of the protein. Such an effect was not observed in the monomolecular layers of LHCII containing all-trans conformers of violaxanthin and zeaxanthin. 77 K chlorophyll a fluorescence emission spectra recorded from the Langmuir-Blodgett (L-B) films deposited to quartz from monomolecular layers formed with LHCII and LHCII in the two-component systems with all-trans and 13-cis isomers of violaxanthin and zeaxanthin revealed opposite effects of both conformers on the aggregation of the protein. The cis isomers of both xanthophylls were found to decrease the aggregation level of LHCII and the all-trans isomers increased the aggregation level. The calculated efficiency of excitation energy transfer to chlorophyll a from violaxanthin assumed to remain in two steric conformations was analyzed on the basis of the chlorophyll a fluorescence excitation spectra and the mean orientation of violaxanthin molecules in LHCII (71 degrees with respect to the normal to the membrane), determined recently in the linear dichroism experiments [Gruszecki et al., Biochim. Biophys. Acta 1412 (1999) 173-183]. The calculated efficiency of excitation energy transfer from the violaxanthin pool assumed to remain in conformation all-trans was found to be almost independent on the orientation angle within a variability range. In contrast the calculated efficiency of energy transfer from the form cis was found to be strongly dependent on the orientation and varied between 1.0 (at 67.48 degrees ) and 0 (at 70.89 degrees ). This is consistent with two essentially different, possible functions of the cis forms of violaxanthin: as a highly efficient excitation donor (and possibly energy transmitter between other chromophores) or purely as a LHCII structure modifier.

Lutein and zeaxanthin as protectors of lipid membranes against oxidative damage: the structural aspects.

Two main xanthophyll pigments are present in the membranes of macula lutea of the vision apparatus of primates, including humans: lutein and zeaxanthin. Protection against oxidative damage of the lipid matrix and screening against excess radiation are the most likely physiological functions of these xanthophyll pigments in macular membranes. A protective effect of lutein and zeaxanthin against oxidative damage of egg yolk lecithin liposomal membranes induced by exposure to UV radiation and incubation with 2, 2'-azobis(2-methypropionamidine)dihydrochloride, a water-soluble peroxidation initiator, was studied. Both lutein and zeaxanthin were found to protect lipid membranes against free radical attack with almost the same efficacy. The UV-induced lipid oxidation was also slowed down by lutein and zeaxanthin to a very similar rate in the initial stage of the experiments (5-15 min illumination) but zeaxanthin appeared to be a better photoprotector during the prolonged UV exposure. The decrease in time of a protective efficacy of lutein was attributed to the photooxidation of the carotenoid itself. Both lutein and zeaxanthin were found to slightly modify mechanical properties of the liposomes in a very similar fashion as concluded on the basis of H(1) NMR and diffractometric measurements of pure egg yolk membranes and membranes pigmented with the xanthophylls. Linear dichroism analysis of the mean orientation of the dipole transition moment of the xanthophylls incorporated to the lipid multibilayers revealed essentially different orientation of zeaxanthin and lutein in the membranes. Zeaxanthin was found to adopt roughly vertical orientation with respect to the plane of the membrane. The relatively large orientation angle between the transition dipole and the axis normal to the plane of the membrane found in the case of lutein (67 degrees in the case of 2 mol% lutein in EYPC membranes) was interpreted as a representation of the existence of two orthogonally oriented pools of lutein, one following the orientation of zeaxanthin and the second parallel with respect to the plane of the membrane. The differences in the protective efficacy of lutein and zeaxanthin in lipid membranes were attributed to a different organization of zeaxanthin-lipid and lutein-lipid membranes.

Xanthophyll pigments in light-harvesting complex II in monomolecular layers: localisation, energy transfer and orientation

Monomolecular layers of the largest light-harvesting pigment-protein complex of Photosystem II (LHCII) were formed at the argon-water interface. The molecular area of the LHCII monomer in monomolecular layers determined from the isotherms of compression is found to be close to 14 nm2, which corresponds well to the molecular dimensions of the protein evaluated on the basis of crystallographic studies. Monolayers of LHCII were deposited on a glass support by means of the Langmuir-Blodgett technique and subjected to spectroscopic studies: electronic absorption spectrophotometry and spectrofluorometry. The fluorescence excitation spectra of chlorophyll a in monolayers of LHCII were analysed using gaussian deconvolution. Comparison of the absorption and fluorescence excitation spectra enabled calculation of the rate of excitation energy transfer in the system. Excitation energy was found to be transferred to chlorophyll a from chlorophyll b with 97% efficiency, from neoxanthin with 85%, from lutein with 62% and from violaxanthin with at least 54% efficiency. The analysis of the position of the 0-0 absorption band of the xanthophylls revealed that neoxanthin is located in the same protein environment as lutein but in a different environment than violaxanthin. The analysis of fluorescence excitation spectra of chlorophyll a in LHCII, recorded with the excitation light beam polarised in two orthogonal directions, enabled the determination of the mean orientation angle of the accessory xanthophyll pigments with respect to the plane of the sample. The mean orientation of lutein found in this study (approx. 51 degrees ) corresponds well to the crystallographic data. Neoxanthin was found to adopt a similar orientation to lutein. The transition dipole moment of violaxanthin was found to form a mean angle of 71 degrees with the axis spanning two polar regions of the protein, perpendicular to the plane of the monolayer, suggesting planar orientation of this pigment with respect to the plane of the thylakoid membrane. These experimentally determined xanthophyll orientations are discussed in terms of importance of peripheral xanthophyll pigments in supramolecular organisation of LHCII and the operation of the xanthophyll cycle within the thylakoid membrane.