Dispersal and reformation of acetylcholine receptor clusters of cultured rat myotubes treated with inhibitors of energy metabolism.

Bloch, Robert J.

doi:10.1083/jcb.82.3.626

Cited by 105 publications

(81 citation statements)

References 43 publications

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“…Aside from highlighting the technical features of TIRF, these time-lapse video sequences overlaying TIRF and schlieren illumination, along with the associated analyses of still frames, tend to confirm, extend, and quantify observations on AChR clusters on rat myotubes made by previous studies, notably those of Bloch and collaborators (Bloch, 1979(Bloch, ,1986Bloch and Geiger, 1980;Bloch and Pumplin, 1988;Bloch et al, 1989). These biological conclusions are as follows:…”

Section: Discussionsupporting

confidence: 78%

“…The average lifetime of a n individual cluster is only around 1 day, although [as previously noted by Bloch (1979)], clusters are generally present in cultures for about 3 days. The largest and brightest clusters tend to be longer-lived.…”

Section: Discussionmentioning

confidence: 79%

“…A detailed description of the culture preparation can be found in Axelrod (1980) and Bloch (1979). In brief, rat muscle cell cultures were prepared from the hind leg skeletal muscle tissues of neonatal rats.…”

Section: Materials and Methods Rat Muscle Cell Culture Preparationmentioning

confidence: 99%

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

Wang

Axelrod

1994

Developmental Dynamics

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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.

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Section: Discussionsupporting

confidence: 78%

Section: Discussionmentioning

confidence: 79%

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

Wang

Axelrod

1994

Developmental Dynamics

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“…The myotubes in these cultures developed as described in earlier studies (Heinemann et al, 1977;Koenig and Vigny, 1978;Bloch, 1979;Rieger et al, 1980). Myoblasts started fusing to form myotubes in 3 days, the myotubes began to fibrillate in 5 days, and, in 6 days, virtually all myotubes were fibrillating vigorously.…”

Section: Resultsmentioning

confidence: 76%

Cellular localization of the molecular forms of acetylcholinesterase in cultured embryonic rat myotubes

1982

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Three different methods were used to quantitate the external and intracellular acetylcholinesterase (AChE) in cultured embryonic rat myotubes. The results from these methods were in excellent agreement and showed that one-fourth of the AChE is external and three-fourths is intracellular in these cells. The molecular forms of AChE in the intracellular and external compartments then were analyzed. To isolate intracellular AChE, external AChE was inactivated irreversibly by treating myotubes at 2°C with 10 PM methanesulfonyl fluoride and 100 PM decamethonium for 1 hr. To isolate external AChE, myotubes at 2°C were first exposed to 1.0 PM echothiophate for 15 min to protect (diethylphosphorylate) external AChE and then treated with 1.0 mM methanesulfonyl fluoride for 30 min at 37°C to inactivate intracellular AChE irreversibly. Control myotubes and myotubes in which the external or intracellular enzyme had been isolated were extracted sequentially to separate globular, asymmetric, and nonextractable AChE. Individual globular and asymmetric forms then were analyzed by velocity sedimentation on sucrose gradients. The fractions in which external enzyme had been isolated as diethylphosphorylated AChE were reactivated with l-methyl-2-hydroxyiminomethylpyridinium prior to analysis. Our data indicate that intracellular and external AChE have different compositions. Intracellular AChE is 79% globular forms (20% 10 S and 59% 4 S), 17% asymmetric forms (6% 16 S, 5% 12.5 S, and 7% other), and 4% nonextractable enzyme. External AChE is 55% globular forms (26% 10 S and 29% 4 S), 32% asymmetric forms (12% 16 S, 10% 12.5 S, and 10% other), and 13% nonextractable enzyme; therefore external enzyme is composed of relatively more 10 S, 16 S, 12.5 S, and nonextractable AChE and relatively less 4 S AChE. The most striking finding to emerge from this study is that the globular and asymmetric forms are all predominantly intracellular in cultured embryonic rat myotubes. Our results indicate that 85% of the 4 S, 69% of the 10 S, 59% of the 16 S, and 56% of the 12.5 S forms are intracellular. These results support the hypothesis that the 10 S and asymmetric forms of AChE are assembled intracellularly from 4 S precursors.The enzyme acetylcholinesterase (AChE) occurs in a isolated from the electric organ of the eel Electrophorus number of molecular forms. Three of these forms are electricus. The three asymmetric forms are designated globular proteins and three are asymmetric. The globular Ad, As, and Alz and consist of one, two, forms are monomers, dimers, and tetramers of the basic or three tetrameric assemblies of identical catalytic subcatalytic subunit designated G1, Gz, and Gd (Bon et al., units attached to an elongated collagen-like tail Vigny et al., 1979). The structure of the asymmetric al. Rosenberry and Richardson, 1977; Anglister forms has been analyzed most carefully using AChE and Silman, 1978).

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“…In some cases, cells were pretreated for 5-6 h with 5 mM sodium azide to disperse the existing AChR clusters (4). Some cells were labeled and fixed after sodium azide treatment.…”

Section: Microinjectionmentioning

confidence: 99%

Microinjection of a monoclonal antibody against a 37-kD protein (tropomyosin 2) prevents the formation of new acetylcholine receptor clusters.

Marazzi

Bard

Klymkowsky

et al. 1989

The Journal of Cell Biology

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Dispersal and reformation of acetylcholine receptor clusters of cultured rat myotubes treated with inhibitors of energy metabolism.

Cited by 105 publications

References 43 publications

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

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

Cellular localization of the molecular forms of acetylcholinesterase in cultured embryonic rat myotubes

Microinjection of a monoclonal antibody against a 37-kD protein (tropomyosin 2) prevents the formation of new acetylcholine receptor clusters.

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