This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. The subproject and investigator (PI) may have received primary funding from another NIH source, and thus could be represented in other CRISP entries. The institution listed is for the Center, which is not necessarily the institution for the investigator. A number of studies indicate that the eukaryotic cell membranes contain coexisting liquid-ordered and liquid-crystalline lipid domains. However, the current evidence for such phase separation is indirect and so far there has been no direct demonstration of differences in the order parameters for the lipids in these two types of regions or their relative population in the plasma membranes of live cells. In order to investigate this further, we carried out electron spin resonance studies employing several chain-labeled phosphatidylcholine spin labels, which have been incorporated into the plasma membranes of live RBL-2H3 mast cells. The results obtained by us provide direct evidence for the presence of two different types of lipid populations in the plasma membranes of whole, live RBL-2H3 cells. Nonlinear least squares simulation of the ESR spectra recorded at various temperatures between 5C and 37C indicated that the spin-labeled lipid probes experience two different types of environments and yielded the order parameters, rotational diffusion times and mole fraction of the spin label in the two environments. Similar studies on CHO cells and mouse 3T3 cells have shown that the plasma membranes of these cells also contain two types of domains differing significantly in their dynamic phase behavior. In addition, the order parameters and rotational diffusion rates observed for the three cell lines are different, suggesting that the lipid dynamics in different cells are different. These observations suggest that coexistence of lipid domains that differ significantly in their dynamic order in the plasma membrane is a general phenomenon, which is likely to be observed with different types of cells.
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