Abstract
Although cholesterol is an essential component of mammalian membranes, resolution of cholesterol organization in membranes and organelles (i.e. lysosomes) of living cells is hampered by the paucity of nondestructive, nonperturbing methods providing real time structural information. Advantage was taken of the fact that the emission maxima of a naturally occurring fluorescent sterol (dehydroergosterol) were resolvable into two structural forms, monomeric (356 and 375 nm) and crystalline (403 and 426 nm). Model membranes (sterol:phospholipid ratios in the physiological range, e.g. 0.5-1.0), subcellular membrane fractions (plasma membranes, lysosomal membranes, microsomes, and mitochondrial membranes), and lipid rafts/caveolae (plasma membrane cholesterol-rich microdomain purified by a nondetergent method) contained primarily monomeric sterol and only small quantities (i.e. 1-5%) of the crystalline form. In contrast, the majority of sterol in isolated lysosomes was crystalline. However, addition of sterol carrier protein-2 in vitro significantly reduced the proportion of crystalline dehydroergosterol in the isolated lysosomes. Multiphoton laser scanning microscopy (MPLSM) of living L-cell fibroblasts cultured with dehydroergosterol for the first time provided real time images showing the presence of monomeric sterol in plasma membranes, as well as other intracellular membrane structures of living cells. Furthermore, MPLSM confirmed that crystalline sterol colocalized in highest amounts with LysoTracker Green, a lysosomal marker dye. Although crystalline sterol was also detected in the cytoplasm, the extralysosomal crystalline sterol did not colocalize with BODIPY FL C(5)-ceramide, a Golgi marker, and crystals were not associated with the cell surface membrane. These noninvasive, nonperturbing methods demonstrated for the first time that multiple structural forms of sterol normally occurred within membranes, membrane microdomains (lipid rafts/caveolae), and intracellular organelles of living cells, both in vitro and visualized in real time by MPLSM.
Highlights
Cholesterol is an essential component of mammalian membranes, resolution of cholesterol organization in membranes and organelles of living cells is hampered by the paucity of nondestructive, nonperturbing methods providing real time structural information
Multiphoton laser scanning microscopy (MPLSM) of living L-cell fibroblasts cultured with dehydroergosterol for the first time provided real time images showing the presence of monomeric sterol in plasma membranes, as well as other intracellular membrane structures of living cells
Multiphoton Laser Scanning Microscopy of L-cell Fibroblasts Cultured with Large Unilamellar Membrane Vesicles Containing DHE—To determine whether L-cells cultured with noncrystalline DHE might exhibit the absence of crystalline DHE and/or a different intracellular DHE distribution, L-cell fibroblasts were cultured for 2 days with medium supplemented with DHE (20 g/ml) in the form of large unilamellar vesicles (LUV) composed of POPC: DHE (65:35) as described under “Experimental Procedures.”
Summary
After 90 min at 39,000 rpm on an SW40Ti swinging bucket rotor and Beckman XL-90 ultracentrifuge (Beckman Instruments, Fullerton, CA), pure sterol crystals appeared at d Ͻ1.054 [13], whereas plasma membrane-enriched fractions appeared at the 36 –24% (w/v) sucrose interface [43]. Fluorescence measurements of organelles and associated membranes were performed with samples in 2 ml of filtered 10 mM PIPES buffer, pH 7.4, in a quartz cuvette with the temperature regulated to 37 Ϯ 0.3 °C through use of a water heating bath (Fisher). Determination of DHE Steady-state Polarization during Exchange Assays—Steady-state fluorescence polarization measurements of DHE in plasma membranes, endoplasmic reticulum, mitochondria, lysosomes, and lysosomal membranes at 37 °C were performed as described earlier (38 – 40). Standard curves for DHE in lysosomal-lysosomal membrane exchanges were determined earlier [39, 46]
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