Related Experiment Videos
Contraction of triton-treated culture cells. A calcium-sensitive contractile model
Abstract:
Using five species of mammalian and avian cells, the authors succeeded in preparing Triton-treated culture cells that contract upon addition of MgATP. The contraction of these Triton cell models was inhibited by N-ethylmaleimide-modified myosin subfragment-1, a specific inhibitor of actin-myosin interaction. Triton cell models adhered more strongly to the substratum than glycerinated ones. Triton cell models of mouse 3T3 and human MRC-5 cells showed Ca2+-sensitive contraction. They required Ca2+ of 1 microM or more for the contraction. Other Triton cell models and all glycerinated cell models did not require Ca2+ for the contraction. The Ca2+-dependent contraction of 3T3 and MRC-5 cell models was inhibited by chlorpromazine, an inhibitor of calmodulin. The Ca2+-sensitivity of the contraction was lost by pretreatment of these cell models with adenosine 5'-O-(3-thiotriphosphate) in the presence of Ca2+. These results agree with a hypothesis that Ca2+-calmodulin-dependent phosphorylation of myosin light chain regulates actin-myosin interactions in non-muscle cells.
Insights
Researchers developed Triton-treated cell models that contract with MgATP, revealing insights into non-muscle cell contraction mechanisms. These models show calcium-dependent contraction regulated by calmodulin and myosin phosphorylation.
Area of Science:
- Cell Biology
- Biochemistry
- Molecular Biology
Background:
- Non-muscle cells exhibit contractile properties crucial for various cellular functions.
- Understanding the molecular mechanisms regulating cell contraction is essential for cell biology.
- Previous models, like glycerinated cells, have limitations in mimicking native cellular responses.
Purpose of the Study:
- To develop and characterize novel Triton-treated cell models for studying cell contraction.
- To investigate the role of actin-myosin interactions in non-muscle cell contractility.
- To explore the involvement of calcium and calmodulin in regulating cell contraction.
Main Methods:
- Preparation of Triton-treated cell models from five mammalian and avian cell species.
- Assessing cell contraction upon addition of MgATP.
- Utilizing specific inhibitors like N-ethylmaleimide-modified myosin subfragment-1 and chlorpromazine.
- Investigating calcium sensitivity and the effect of adenosine 5'-O-(3-thiotriphosphate) pretreatment.
Main Results:
- Triton-treated cells contracted upon MgATP addition, with inhibition by myosin subfragment-1, confirming actin-myosin involvement.
- Triton models exhibited stronger adhesion compared to glycerinated models.
- Mouse 3T3 and human MRC-5 cell models displayed calcium-sensitive contraction (≥ 1 microM Ca2+).
- Calcium-dependent contraction was inhibited by chlorpromazine, indicating calmodulin's role.
- Loss of calcium sensitivity after adenosine 5'-O-(3-thiotriphosphate) pretreatment supported the regulatory hypothesis.
Conclusions:
- Triton-treated cell models provide a viable system for studying non-muscle cell contraction.
- Actin-myosin interactions are key mediators of contraction in these models.
- Calcium-calmodulin-dependent myosin light chain phosphorylation likely regulates actin-myosin interactions in non-muscle cells.