Novel approaches to study membrane organization: investigation of dependence between signal transductions and cell membrane organization in eukaryotic cells

The plasma membrane constitutes the boundary between the environment inside the cell and the surrounding milieu. It is a self-organised structure composed of lipids and proteins. Over time the scientists’ view of the plasma membrane organisation has evolved presenting different organisation concepts. One of them is the lipid raft hypothesis which defines lipid rafts as cholesterol- and sphingolipid- rich nanodomains within the plasma membrane, which localise and concentrate raft-associated proteins, in particular signalling proteins, to specific sites. Due to their distinctive lipid composition, these domains are more ordered than their fluid surroundings. Hence, these membrane domains constitute biophysically and biochemically discrete platforms. However, lipid nanodomains remain controversial, mainly because they are difficult to visualise directly in intact, live cells due to their small size and transient nature. To map membrane order in lipid vesicles and live cell membranes, fluorescent microscopy techniques combined with membrane order sensitive dyes were applied. Moreover, a novel quantitative analysis of fluorescence lifetime and lipid mobility was established. Firstly, the new family of environmentally sensitive fluorophores was tested to measure membrane order by generalised polarisation imaging and fluorescence lifetime imaging microscopy (FLIM). It was shown that higher GP values and longer lifetimes were registered in plasma membranes compared to intercellular membranes, indicating a high degree of membrane order in the plasma membrane compared to intercellular membranes. Further, lipid mobility was investigated by fluorescence spectral correlation spectroscopy (FSCS) successfully combined with the membrane order sensitive dye NR12S. The diffusion of the lipid marker was simultaneously detected in both lipid phases and the single-molecule cross-correlation signal was obtained indicating the dye molecules diffusing between lipid phases. Furthermore, the solvent relaxation of the membrane marker was investigated by spectrally resolved FLIM. The results suggested that membrane curvature and lipid compositions influence the solvent relaxation process. Finally, the protein organisation in the membrane was investigated, showing preferences in distribution due to a membrane order. In conclusion, the data presented in this PhD thesis suggest the coexistence of both lipid phases in the plasma membrane. Moreover, the nanodomains in the plasma membrane are temporally stable within a millisecond time scale.