Microclustering patterns of acetylcholine receptors on myotubes studied by spatial fluorescence autocorrelation

Abstract

We have characterized the highly detailed internal features of acetylcholine receptor (AChR) clusters on cultured rat myotubes by applying a novel image analysis technique—spatial fluorescence autocorrelation. AChR clusters were observed with total internal reflection fluorescence (TIRF) under high magnification after labelling the AChRs with tetramethylrhodamine α‐bungarotoxin, and recorded as CCD images. AChR clusters (on the tens of microns size scale) appear to be composed of microclusters (on the tenths of micron size scale), and the microclusters are sometimes organized into paracrystalline arrays. These structural features may reflect those of a protein meshwork that may underlie each cluster. To characterize these features, we have applied a spatial fluorescence autocorrelation technique which allows us to extract six parameters from each cluster image: average size of microclusters, contrast of microclusters, average spatial period of the paracrystalline arrays, contrast of periodicity, average fluorescence intensity, and average anisotropy of microclusters. These parameters were then followed as a function of myotube age after plating, and upon treatments with drugs which previously have been shown to gradually eliminate clusters. The drugs used were oligomycin (an energy metabolism inhibitor) and carbachol (a nicotinic AChR agonist). We show that myotube age only slightly affects the image parameters based on the spatial autocorrelation. Oligomycin induced significant decreases in the size of microclusters, contrast of microclusters, spatial period, contrast of periodicity, and anisotropy, whereas the carbachol study showed significant increases in the size of the microclusters and the anisotropy of the microclusters. These results suggest that these two drugs may act through different mechanisms in eliminating clusters.

Advances in Image Correlation Spectroscopy: Measuring Number Densities, Aggregation States, and Dynamics of Fluorescently labeled Macromolecules in Cells

“…Wang and Axelrod [116] used spatial autocorrelation functions to examine acetylcholine receptors (AChR) on rat myotubes. The size, periodicity, and spatial anisotropy of the AChR clusters were measured from the correlation function.…”

Actin Dynamics at the Living Cell Submembrane Imaged by Total Internal Reflection Fluorescence Photobleaching

“…The spatial autocorrelation function G for a "generic" parameter A was calculated (Wang and Axelrod, 1994)…”

Image Correlation Spectroscopy. II. Optimization for Ultrasensitive Detection of Preexisting Platelet-Derived Growth Factor-β Receptor Oligomers on Intact Cells

“…One of the key advantages of the FCS method is that the measured autocorrelation function reflects and is sensitive to changes in the aggregation state of the fluorophore (Palmer and Thompson, 1989;Thompson, 1991;Meyer and Schindler, 1988;Petersen, 1986;Petersen et al, 1993). Recently, the availability of FCS methods has increased with the introduction of implementation on confocal laser scanning microscopes (CLSMs) (Petersen et al, 1993;Koppel et al, 1994) and variations involving total internal reflection illumination and imaging with charge-coupled device (CCD) detectors (Wang and Axelrod, 1994;Huang and Thompson, 1996).…”