SUBNANOSECOND POLARIZED FLUORESCENCE PHOTOBLEACHING - ROTATIONAL DIFFUSION OF ACETYLCHOLINE-RECEPTORS ON DEVELOPING MUSCLE-CELLS

Polarized fluorescence recovery after photobleaching (PFRAP) is a tech nique for measuring the rate of rotational motion of biomolecules on l iving, nondeoxygenated cells with characteristic times previously rang ing from milliseconds to many seconds. Although very broad, that time range excludes the possibility of quantitatively observing freely rota ting membrane protein monomers that typically should have a characteri stic decay time of only several microseconds. This report describes an extension of the PFRAP technique to a much shorter time scale. With t his new system, PFRAP experiments can be conducted with sample time as short as 0.4 mu s and detection of possible characteristic times of l ess than 2 mu s. The system is tested on rhodamine-alpha-bungarotoxin- labeled acetylcholine receptors (AChRs) on myotubes grown in primary c ultures of embryonic rat muscle, in both endogenously clustered and no nclustered regions of AChR distribution. It is found that similar to 4 0% of the AChRs in nonclustered regions undergoes rotational diffusion fast enough to possibly arise from unrestricted monomer Brownian moti on. The AChRs in clusters, on the other hand, are almost immobile. The effects of rat embryonic brain extract (which contains AChR aggregati ng factors) on the myotube AChR were also examined by the fast PFRAP s ystem. Brain extract is known to abolish the presence of endogenous cl usters and to induce the formation of new clusters. It is found here t hat rotational diffusion of AChR in the extract-induced clusters is as slow as that in endogenous clusters on untreated cells but that rotat ional diffusion in the nonclustered regions of extract-treated myotube s remains rapid.