Diffusely distributed acetylcholine receptors can participate in cluster formation on cultured rat myotubes

On aneurally cultured rat primary myotubes, 10% of the acetylcholine receptors (AcChoR) are found to be aggregated and immobilized in endogenous clusters while the remainder are diffusely distributed and partially mobile. This paper reports that AcChoR in clusters can be gathered from AcChoR in diffuse areas during the course of normal myotube development. AcChoR were fluorescently labeled with rhodamine-conjugated alpha-bungarotoxin, and all existing clusters in a circumscribed region of the culture dish were irreversibly photobleached by a slightly defocused laser beam, the movement of which was controlled by a lens mounted on a joystick translator. This procedure leaves intact only the fluorescent label on the diffusely distributed AcChoR. Observation of the myotubes after several hours of incubation revealed cluster fluorescence redevelopment. This cluster fluorescence must have consisted of AcChoR that previously were diffusely distributed. The majority (but not all) of cluster fluorescence redevelopment occurred in the location of a previously bleached cluster. About half of the redeveloped clusters have an annular shape. The major conclusions of this study are (i) diffusely distributed AcChoR can become clustered; (ii) endogenous clusters appear to form, at least in part, by "trapping" receptors as they diffuse in from surrounding regions; (iii) cluster formation is an ongoing process in cultured rat myotubes; and (iv) colchicine (a microtubule-disrupting agent) inhibits cluster formation.