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Reactivation of cell-free models of Physarum plasmodia after myosin reconstitution
1Institute of Cytology, University of Bonn/Federal Republic of Germany.
Abstract:
Thin-spread glycerol-extracted Physarum plasmodia were treated with N-ethylmaleimide (NEM) to block myosin-ATPase and contractility. After supplementing the models with purified plasmodial myosin, they could be reactivated and contracted upon addition of ATP. Fluorescently labeled actomyosin fibers ruptured during contraction, resulting in beaded or rod-like contraction centers. Glycerol-extracted plasmodia lose their negative Ca++-dependence during extraction. Reconstitution of NEM-treated models with plasmodial myosin partly restored this Ca++-sensitivity. Thus, either myosin or a factor associated with it seems to be involved in the Ca++-dependent regulation of cytoplasmic actomyosin contraction in Physarum. NEM-blocked models reconstituted with skeletal muscle myosin were not reactivated by ATP. The same plasmodia subsequently incubated with plasmodial myosin were able to contract.
Insights
Physarum plasmodia treated with N-ethylmaleimide (NEM) regained contractility with added myosin. This suggests myosin or associated factors regulate calcium-dependent cytoplasmic actomyosin contraction in Physarum.
Area of Science:
- Cell Biology
- Biochemistry
- Cytoskeletal Dynamics
Background:
- Physarum plasmodia exhibit cytoplasmic streaming driven by actomyosin contractions.
- Calcium ions (Ca++) play a crucial role in regulating these contractions.
- Glycerol extraction disrupts the Ca++-dependence of these contractile systems.
Purpose of the Study:
- To investigate the role of myosin in the Ca++-dependent regulation of Physarum actomyosin contraction.
- To determine if plasmodial myosin can restore contractility and Ca++-sensitivity to extracted and inhibited models.
Main Methods:
- Glycerol-extracted Physarum plasmodia were treated with N-ethylmaleimide (NEM) to inhibit myosin-ATPase and contractility.
- Reconstitution experiments involved adding purified plasmodial myosin or skeletal muscle myosin.
- Contractility was assessed by the addition of Adenosine Triphosphate (ATP) and Ca++ sensitivity was evaluated.
Main Results:
- NEM-treated plasmodia regained ATP-dependent contractility upon reconstitution with purified plasmodial myosin.
- Actomyosin fibers showed rupture and formation of beaded/rod-like structures during contraction.
- Reconstitution with plasmodial myosin partially restored Ca++-sensitivity, which was lost during glycerol extraction.
- Skeletal muscle myosin failed to reactivate NEM-blocked plasmodia.
Conclusions:
- Plasmodial myosin, or a closely associated factor, is implicated in the Ca++-dependent regulation of cytoplasmic actomyosin contraction in Physarum.
- The specific interaction of plasmodial myosin is essential for restoring both contractility and Ca++-sensitivity.
