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PubMed1986

Asymmetric lateral mobility of phospholipids in the human erythrocyte membrane.

The fluorescent phospholipid 1-acyl-2-[12-(7-nitrobenz-2-oxa-1,3-diazol-4- yl)aminododecanoyl]phosphatidylcholine (NBD-phosphatidylcholine) and the corresponding aminophospholipid derivatives (NBD-phosphatidylethanolamine and NBD-phosphatidylserine) were introduced in the human erythrocyte membrane by a nonspecific phospholipid exchange protein purified from corn. The lateral mobility of the fluorescent phospholipids was measured by using an extension of the classical photobleaching recovery technique that takes advantage of a modulated fringe pattern and provides a high sensitivity. In intact erythrocytes and in ghosts resealed in the presence of ATP, the fluorescence-contrast curves after photobleaching decayed biexponentially corresponding to two lateral diffusion constants. With NBD-phosphatidylcholine, the majority of the signal corresponded to a "slow" component (1.08 X 10(-9) cm2/sec at 20 degrees C), whereas with the amino derivatives the majority of the signal corresponded to a "fast" component (5.14 X 10(-9) cm2/sec at 20 degrees C). If the ghosts were resealed without ATP, the fast component of the aminophospholipids disappeared. We interpret these results as follows: (i) Provided the cells or the ghosts contain ATP, the three fluorescent phospholipids distribute spontaneously between inner and outer leaflets as endogenous phospholipids, namely NBD-phosphatidylcholine is located in the outer leaflet, while both aminophospholipids are preferentially located in the inner leaflet. (ii) The viscosity of the inner leaflet of human erythrocyte membranes is lower than that of the outer leaflet.

Adenosine Triphosphate↗

PubMedDate not supplied

Physical techniques for the study of exocytosis in isolated cells.

Membrane traffic is an important aspect of cell biology which implies shuttle vesicles and multiple binding/fusion events. In spite of rapid progress at the biochemical level, the mechanism of fusion is still not understood. A detailed physical description of the phenomenon is possible at the level of the plasma membrane where secretory vesicles fuse with the cell membrane, a process known as exocytosis. This process is specially active in neurons (release of neurotransmitter) and in endocrine cells (release of hormones), where exocytosis is tightly regulated. Among the biophysical techniques developed, cell membrane capacitance measurements by the technique of patch-clamp and amperometry of the oxidizable secretory products have resulted in interesting information. These techniques have described the initial fusion pore, its fluctuations, the efflux of material through the pore and its irreversible expansion. Optical techniques, using bioluminescent and fluorescent probes are also in progress. For instance, the dye FM 1-43 binds to but is not translocated through biological membranes and it has been used to measure membrane surface, as done by capacitance measurement. Evanescent wave fluorescence microscopy has been recently introduced to analyse the behaviour of secretory granules in the vicinity of the plasma membrane.

Animals↗