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Using digitally analyzed fluorescence videomicroscopy, we have examined the behavior of acetylcholine receptors and concanavalin A binding sites in response to externally applied electric fields. The distributions of these molecules on cultured Xenopus myoballs were used to test a simple model which assumes that electrophoresis and diffusion are the only important processes involved. The model describes the distribution of concanavalin A sites quite well over a fourfold range of electric field strengths; the results suggest an average diffusion constant of ~ 6 × 10 -t° cm2/s. At higher electric field strengths, the asymmetry seen is substantially less than that predicted by the model. Acetylcholine receptors subjected to electric fields show distributions substantially different from those predicted on the basis of simple electrophoresis and diffusion, and evidence a marked tendency to aggregate. Our results suggest that this aggregation is due to lateral migration of surface acetylcholine receptors, and is dependent on surface interactions, rather than the rearrangement of microfilaments or microtubules. The data are consistent with a diffusion-trap mechanism of receptor aggregation, and suggest that the event triggering receptor localization is a local increase in the concentration of acetylcholine receptors, or the electrophoretic concentration of some other molecular species. These observations suggest that, whatever mechanism(s) trigger initial clustering events in vivo, the accumulation of acetylcholine receptors can be substantially enhanced by passive, diffusionmediated aggregation. T HE migration of cell-surface molecules is a problem of general interest in developing biological systems. The cell membrane is a fluid matrix consisting of a heterogeneous lipid bilayer, peripheral and integral membrane proteins, and specialized molecular assemblies linking intracellular and membrane-bound components (Singer and Nicholson, 1972; Marchesi, 1985). Man)' proteins and lipids diffuse freely in the plane of the membrane, and show a globally uniform distribution early in development (Frye and Edidin, 1970; Poo and Cone, 1974; Edidin, 1975). At later stages some of these molecules are concentrated in local regions of the plasma membrane by mechanisms not fully understood. An important example in neurobiology is the induction of acetylcholine receptor (AChR) ~ clusters at the developing neuromuscular junction (see Schuetze and Role, 1987, for review). In this case it is clear that at least some pre-existing, dispersed AChRs are recruited into the forming aggregate (Anderson et al., 1977; Kuromi and Kidokoro, 1984; Ziskind-Conhaim et al., 1984; Role et al., 1985). 1. Abbreviations used in this paper: ct-Bgt, alpha-Bungarotoxin; AChR, acetylcholine receptor; con A, concanavalin A; FLR-con A, fluorescein labeled concanavalin A; TMR-a-Bgt, tetra-methyl-rhodamine labeled alphaBungarotoxin. A useful technique for the study of macromolecular migration is the in situ electrophoresis of membrane components in response to externally applied electric fields (Jaffe, 1977; Poo and Robinson, 1977; Poo et al., 1978). This technique perturbs molecular distributions independently of cell contacts, and permits quantitative analyses of important migration parameters. In particular, myoball (spherical muscle cell) cultures have been used to advantage in testing theoretical predictions concerning electrophoretic mobility, diffusion, and aggregate stability of concanavalin A (con A) ligand binding sites and AChRs (Orida and Poo, 1978, 1980, 1981; Poo et al., 1979). Because myobaUs are nearly spherical, quantitation of component concentration about a two-dimensional perimeter is sufficient to characterize the distribution of the component in three dimensions. In the above-mentioned experiments, electrophysiological and microfluorimetric techniques were used to analyze the distribution of cell surface components after experimental manipulations. The increased availability of sophisticated computing and video-acquisition hardware has made possible a more detailed analysis of the distribution of cell surface molecules labeled with fluorescent probes. We report here the use of digitally analyzed videomicroscopy to extend the accuracy and resolution of data describing the distribution of con © The Rockefeller University Press, 0021-9525/88/05/1723/12 $2.00 The Journal of Cell Biology, Volume 106, May 1988 1723-1734 1723 A-binding sites and AChRs after exposure to electric fields. The use of these techniques permits rigorous comparison to theoretical predictions based on electrophoresis and diffusion. The approach quantitatively demonstrates significant aggregation of AChRs over and above that caused by electric fields; this phenomenon is of great interest in light of the dramatic accumulation of AChRs at the developing neuromuscular junction. Additional experiments examining the requirements for the aggregation phenomenon are also presented. Materials and Methods