Time‐lapse total internal reflection fluorescence video of acetylcholine receptor cluster formation on myotubes

Abstract

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.

The structural basis of function in Cys-loop receptors

“…Some of Axelrod's pioneering experiments with total internal reflection microscopy (TIRF) were performed on muscle nAChRs labelled with antibodies (Wang & Axelrod, 1994). Since then, many investigators have appreciated that TIRF is an optimal technique for resolving membraneassociate proteins.…”

Fluorescently-Labeled Estradiol Internalization and Membrane Trafficking in Live N-38 Neuronal Cells Visualized with Total Internal Reflection Fluorescence Microscopy

“…In the present study we use live N-38 neurons and total internal reflection fluorescence microscopy (TIRFM) to image the trafficking of the membrane impermeant ER-ligand E6BSA-FITC and image GFP-ERα near or at the cell surface in real time. For example, similar studies examining nicotinic acetylcholine receptor trafficking have used the receptor-specific ligand α-bungarotoxin in real-time [18–20]. …”

Total Internal Reflection Fluorescence Microscopy

“…7. Long-term fluorescence movies of cells during development in culture (Wang and Axelrod, 1994): Because the cells are exposed to excitation light only at their cell-substrate contact regions and not through their bulk, they tend to survive longer under observation, thereby enabling time-lapse recording of a week in duration. During this time, newly appearing cell surface receptors can be immediately marked by fluorescent ligands that are continually present in the full cell culture medium while maintaining a low fluorescence background.…”