To study when and where acetylcholine receptor (AChR) clusters appear on developing rat myotubes in primary culture, we have made time-lapse movies of total internal reflection fluorescence (TIRF) overlaid with schlieren transmitted light images. The receptors, including the ones newly incorporated into the membrane, were labeled with rhodamine a-bungarotoxin (R-BT) continuously present in the medium. Since TIRF illuminates only cell-substrate contact regions where almost all of the AChR clusters are located, background fluorescence from fluorophores either in the bulk solution or inside the cells can be suppressed. Also, because TIRF minimizes the exposure of the cell interior to light, the healthy survival of the culture during imaging procedures is much enhanced relative to standard epi-(or trans-) illumination. During the experiment, cells were kept alive on the microscope stage at 37°C in an atmosphere of 10% C02. Two digital images were recorded by a CCD camera every 20 min: the schlieren image of the cells and the TIRF image of the clusters. After background subtraction, the cluster image was displayed in pseudocolors, overlaid onto the cell images, and recorded as 3 frames on a videotape. The final movies are thus able to summarize a week-long experiment in less than a minute. These movies and images show that clusters form often shortly after the myoblast fusion but sometimes much later, and the formation takes place very rapidly (a few hours). The clusters have an average lifetime of around a day, much shorter than the lifetime of a typical myotube. The brightest and largest clusters tend to be the longest-lived. The cluster formation seems to be associated with the contacts of myotubes at the glass substrate, but not with cell-cell contacts or myoblast fusion into myotubes. New AChR continuously appear in preexisting clusters: after photobleaching, the fluorescence of some clusters recovers within an hour. o 1994 WiIey-Liss, Inc.
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.
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