Search

There has been a great effort to study lipid lateral organization in biomembranes in the past decades, in order to unravel the structural basis and functional significance of membrane lipid domains. However, in both respects fundamental doubts still persist, and recent results have widened this topic well beyond lipid rafts. In particular, the detection of sphingolipid-enriched gel domains in the yeast Saccharomyces cerevisiae plasma membrane, which are not the prototypical liquid ordered, sterol-enriched lipid rafts, will be described. The critical role of ordered lipid domains will be demonstrated with biophysical studies of membrane lipid organization in living cells and in model systems, concerning mammalian and fungal membranes. Membrane interactions with different types of bioactive molecules will be briefly presented, including endogenous molecules such as the hormone epinephrine or membrane proteins, as well as drugs, including anticancer and antitubercular compounds. Strategies to tackle the complexity of living cell membranes will be discussed, in an attempt to reach a compromise between lipid lamellar phases in artificial or reconstituted systems and observations in living cells. The development of new and improved biomimetic systems might provide answers to some of the open questions in the lipid domains field. Therefore, new lipid bilayer membrane models containing lipid domains stably formed on a conducting support (gold), where powerful surface and electrochemical techniques can be employed, will also be presented. The redox behavior of the catecholamine hormone epinephrine studied in such system showed that the lipid bilayer has a crucial role for the hormone chemical stability.

Lipid domains are part of the current description of cell membranes and their involvement in many fundamental cellular processes is currently acknowledged. However, their study in living cells is still a challenge. Fluorescence lifetimes have and will continue to play an important role in unraveling the properties and function of lipid domains, and their use in vivo is expected to increase in the near future, since their extreme sensitivity to the physical properties of the membrane and the possibility of optical imaging are particularly suited to deal with the hurdles that are met by researchers. In this review, a practical guide on the use of fluorescence lifetimes for the study of this subject is given. A section is devoted to studies in vitro, particularly membrane model systems, and how they are used to better design and correctly interpret results obtained in living cells. Criteria are presented for selecting suitable probes to solve each problem, drawing attention to factors sometimes overlooked and which may affect the fluorescence lifetime such as subcellular distribution and concentration of the probe. The principal groups of lifetime probes for lipid domains: (i) fluorescent lipid analogues, (ii) other lipophilic probes, and (iii) fluorescent proteins, and respective applications are briefly described and lab tips about the labeling of living cells are provided. The advantages and complementarities of spectroscopy (cuvette) work and fluorescence lifetime imaging microscopy are presented and illustrated with three selected case studies: (i) the finding of a new type of lipid rafts in yeast cells; (ii) the detection of liquid ordered type heterogeneity in animal cells below optical resolution; and (iii) establish a role for the transmembrane domain of influenza virus hemagglutinin with cholesterol-enriched domains in mammalian cells.