Laurdan in Cell Membranes and Tissues

From the pioneering work of Yu et al. (1996) and Parasassi et al. (1997) LAURDAN was proposed to be very promising in exploring cell membranes. In these studies domains of sizes below, in the same range as and above the microscope resolution limit (0.3 ^m) were observed in OK cells, red blood cells and brush-border native membranes, respectively (Parasassi et al. 1997). tte LAURDAN GP differences observed in compositionally complex mixtures and artificial lipid ternary mixtures containing phospholipids, sphingomyelin and cholesterol were recently

Artificial mixtures

Fluid ordered / fluid disordered Gel/fluid

Natural iipid mixtures

Fig.9.5. Two-photon-excitation LAURDAN fluorescence images (taken at the polar region of the vesicle) of DOPC/cholesterol/sphingomyelin 1:1:1 molar displaying fluid ordered/fluid disordered phase coexistence (top left) and DMPC/DSPC 1:1 molar displaying gel/fluid phase coexistence (top right). GUVs composed of brush border membrane lipid extract (bottomleft) displaying fluid ordered/fluid disordered like phase coexistence. The same membrane after cholesterol extraction (bottom right) shows gel/fluid phase coexistence. The bar corresponds to 20 |am min F.l. (a.u.)

Fig.9.5. Two-photon-excitation LAURDAN fluorescence images (taken at the polar region of the vesicle) of DOPC/cholesterol/sphingomyelin 1:1:1 molar displaying fluid ordered/fluid disordered phase coexistence (top left) and DMPC/DSPC 1:1 molar displaying gel/fluid phase coexistence (top right). GUVs composed of brush border membrane lipid extract (bottomleft) displaying fluid ordered/fluid disordered like phase coexistence. The same membrane after cholesterol extraction (bottom right) shows gel/fluid phase coexistence. The bar corresponds to 20 |am used to interpret GP images in cell membranes (Gaus et al. 2003). In this report the LAURDAN GP function was used to directly observed transient micron-sized high GP regions surrounded by low GP areas in living macrophages (Fig. 9.7). ttis paper demonstrated the presence oflateral phase separation in living cells, supporting strongly the cholesterol effect observed in the model systems (Dietrich et al. 2001). Interestingly enough, this last result is in line with the works reported by Gousset et al. (2002) and Bernardino de la Serna et al. (2004), where micron-sized domains were also observed in platelets upon activation and in native pulmonary surfactant membranes, respectively. Although micron-sized domains are observed in the membranes mentioned before (macrophages, platelets, pulmonary surfactant), generalization of this phenomenon must be done cautiously. Following the literature in this respect, the presence of micron-sized domains in biological membranes seems

Fig. 9.6. Bovine lipid extract surfactant membranes. The thermogram obtained from differential scanning calorimetry experiments indicates a very broad and complex phase transition temperature region. LAURDAN intensity and LAURDAN GP images reveal additional information showing three different phase transitions (fluid ^ fluid/gel, fluid/gel ^ solid/gel) in the same temperature range used in the differential scanning calorimetry experiments. The diameter ofthe GUVs is approximately 30 |im

Fig. 9.6. Bovine lipid extract surfactant membranes. The thermogram obtained from differential scanning calorimetry experiments indicates a very broad and complex phase transition temperature region. LAURDAN intensity and LAURDAN GP images reveal additional information showing three different phase transitions (fluid ^ fluid/gel, fluid/gel ^ solid/gel) in the same temperature range used in the differential scanning calorimetry experiments. The diameter ofthe GUVs is approximately 30 |im to be the exception instead of the general case. Ms last fact can be simply related to the membrane composition and the particular functions of the membranes under favorable environmental conditions (such as temperature).

Lastly LAURDAN GP was also applied to tissues. Sun et al. (2004) have demonstrated that both LAURDAN multiphoton polarization and GP can be combined under a two-photon-excitation fluorescence microscope to characterize the structural changes of intercellular lipids in skin tissue. Ms work demonstrated how the treatment of oleic acid results in a skin surface with a more random packing of lipid molecules, which facilitates water penetration.

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