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Fig. 2. Characterization of phospholipid movement at a qualitative level in (plasma) membranes is frequently based on phospholipid probes. These analogs have a reporter group attached to a short fatty acid chain and maintain most of the properties of endogenous phospholipids, except that they are more water-soluble, which facilitates incorporation from the medium into the outer monolayer of the membrane (A). Transport of these probes is usually monitored by extracting with bovine serum albumin (BSA) the residual fraction of analogs not transported across the membrane. Depending on the head group and cell type, the lipid analog inserted into the outer plasma membrane leaflet can be internalized by spontaneous flip-flop, by protein-mediated translocation or endocytosis. In human fibroblasts, for example, disappearance of NBD-PS from the cell surface is predominantly due to fast translocation across the plasma membrane (and endosomal membranes), resulting in a labeling of various intracellular membranes (B). In contrast, NBD-SM is internalized via endocytic vesicles resulting in the appearance of intracellular fluorescent spots (C). Bar, 20 µm.
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