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Morphological changes in cultured myotubes treated with agents that interfere with lysosomal function
Abstract:
Treatment of cultured muscle cells with the inhibitors of lysosomal function, leupeptin, and chloroquine, decrease the degradation of acetylcholine receptors (AChR) and causes accumulation of undegraded receptors intracellularly. Under these conditions the number of cytoplasmic coated vesicles, i.e. structures that appear to transport this receptor within the cultured muscle cell, increases in parallel. This study investigates the effects of leupeptin and chloroquine on the morphology of cultured myotubes in order to learn more about the turnover of acetylcholine (Ach) receptors and the origin of the coated vesicles. Chloroquine causes involution of the plasma membrane, disorganization in the arrangement of sarcomeres, vacuolization, and enlargement of dense lysosome-like bodies in myotubes. The diameter of dense bodies in untreated myotubes is 0.36 +/- 0.01 micrometer (mean +/- SEM) compared with 2 +/- 0.12 micrometer after 48 h of incubation with chloroquine. Leupeptin does not disrupt the normal architecture of sarcomeres and does not cause vacuolization of the myotubes. However, leupeptin does enlarge the dense bodies, although to a lesser extent than chloroquine (average diameter after 48 h treatment, 1.0 +/- 0.06 micrometer, p less than 0.01). Untreated myotubes appear to contain equal numbers of large and small coated vesicles. After chloroquine treatment 95% of coated vesicles are large (80-120 nm in diameter), whereas after leupeptin treatment the majority of coated vesicles are small (40-70 nm in diameter). After incubation with horseradish peroxidase (HRP) 62% +/- 9 of coated vesicles in chloroquine-treated cells contain the tracer, whereas in control cells only 11% +/- 4 of coated vesicles contain HRP reaction product. These observations indicate that chloroquine causes accumulation of coated vesicles and interferes with degradation of AChR by preventing fusion of lysosomes with coated vesicles originating by endocytosis.
Insights
Lysosomal inhibitors like chloroquine and leupeptin prevent acetylcholine receptor (AChR) degradation in muscle cells. This leads to receptor buildup and increased coated vesicles, suggesting impaired lysosome-vesicle fusion.
Area of Science:
- Cell biology
- Muscle cell physiology
- Receptor trafficking
Background:
- Lysosomes degrade cellular components, including acetylcholine receptors (AChR).
- Inhibitors of lysosomal function impede this degradation process.
- Coated vesicles are implicated in intracellular transport.
Purpose of the Study:
- To investigate the effects of leupeptin and chloroquine on cultured myotube morphology.
- To understand acetylcholine receptor turnover and the origin of coated vesicles.
- To elucidate the mechanism by which lysosomal inhibitors affect receptor degradation.
Main Methods:
- Treatment of cultured myotubes with leupeptin and chloroquine.
- Morphological analysis using microscopy.
- Quantification of coated vesicle size and content (horseradish peroxidase uptake).
Main Results:
- Chloroquine induced plasma membrane involution, sarcomere disorganization, vacuolization, and enlarged lysosome-like bodies.
- Leupeptin enlarged dense bodies but did not cause vacuolization or sarcomere disruption.
- Chloroquine treatment led to accumulation of large coated vesicles, while leupeptin treatment resulted in smaller coated vesicles. Chloroquine-treated cells showed increased horseradish peroxidase uptake in coated vesicles.
Conclusions:
- Chloroquine interferes with AChR degradation by inhibiting lysosome-coated vesicle fusion.
- Leupeptin and chloroquine differentially affect myotube morphology and coated vesicle characteristics.
- These findings provide insights into receptor turnover and the role of coated vesicles in muscle cells.