ABSTRACT
Although plasma membrane (PM) cholesterol-rich and -poor domains have been isolated by subcellular fractionation, the real-time arrangement of cholesterol in such domains in living cells is still unclear. Therefore, dehydroergosterol (DHE), a naturally occurring fluorescent sterol, was incorporated into cultured L-cell fibroblasts. Two PM markers, the enhanced cyan fluorescent protein (ECFP-Mem) and 3'-dioctadecyloxacarbocyanine perchlorate [DiOC(18)(3)], were used to distinguish DHE localized at the PM of living cells. Spatial enrichment of DHE in the PM of living cells was visualized in real time by multiphoton laser scanning microscopy (MPLSM). Quantitative models and image-processing techniques were developed for statistical analysis of the distribution of DHE within the PM. The PM was resolved from the cytoplasm in a two-step process, and a smooth trajectory reference of the PM was refined by statistical regression and moments-based techniques. Thus, DHE intensities over the PM were measured following the major DHE intensity distributions. Spatial distributions of DHE within the PM were examined by a statistical inference technique, complete spatial randomness (CSR). For PM regions densely populated with DHE, the distributions of DHE exhibited statistical arrangements that were not spatial random (i.e., homogeneous Poisson process) or regular but, instead, exhibited strong cluster patterns. In effect, real-time MPLSM imaging data for the first time demonstrated that sterol enrichment occurred in clustered regions in the PM, consistent with the existence of cholesterol-rich domains in the plasma membrane of living cells.
