Fluorescent studies of acetylcholine receptor clustering.

Abstract

We have used various novel fluorescent microscopy techniques to study the clustering mechanism of the acetylcholine receptor (AChR) in primary cultures of rat myotubes. First, we further developed a technique, Polarized Fluorescence Photobleaching Recovery (PFPR), for measuring rotational diffusion in solutions and membranes. The technique is applicable to a wide range of time scales, from microseconds to seconds. Furthermore, it does not require deoxygenation, thereby making it suitable for the study of protein dynamics on living membranes. The technique was experimentally verified on small latex beads with a variety of diameters, common fluorophore labels, and solvent viscosities. We then measured the rotational motion of the AChR (around an axis perpendicular to the plane of the membrane) in various aggregational states on the surface of living rat myotubes. We found that nonclustered receptors are an heterogeneous mixture of several rotating components: a very fast component (D$\sb{\rm r} >$ 5000 s$\sp{-1}$, $\cong$31%), two intermediate components (D$\sb{\rm r} \cong$ 700 $\pm$ 80 s$\sp{-1}, \cong$15%, and D$\sb{\rm r} \cong$ 2.9 $\pm$.6 s$\sp{-1}, \cong$30%) and an immobile component (D$\sb{\rm r} <$ 0.1 s$\sp{-1}, \cong$24%). Clustered receptors, as well as clusters treated with carbachol, a receptor agonist that has a cluster disaggregating effect, show no rotation up to 300 ms (D$\sb{\rm r} <$ 0.1 s$\sp{-1}$). Another fluorescent technique used, Total Internal Reflection Fluorescence (TIRF) allowed us to look selectively at cytoskeletal proteins that codistribute with the cluster receptors. We found that: (a) the 43K protein codistributes with the clustered receptor molecules; (b) $\alpha$-actinin, filamin and vinculin are found associated preferentially with the receptor-poor regions of the cluster; (c) actin is found in both, receptor-rich and receptor poor regions of clusters. Scanning Fluorescence Correlation Spectroscopy (S-FCS) was used to measure the number of receptors in the microclusters constituting the large endogenous clusters present in the cultured myotubes. The values measured ranged from 200 to 13000 receptors per microcluster. This large range of values is due to biological variability between clusters. In summary: the technique of PFPR can be used to measure molecular rotational diffusion rates; different states of aggregation of the AChRs in the surface of cultured rat myotubes can be correlated with different rotational behavior of the receptors; nonclustered receptors are free to rotate whereas clustered ones are rotationally immobile; and clustered receptors present a possible close association with actin and 43K protein and no direct association with vinculin, $\alpha$-actinin and filamin.