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Experimental evidence for the contractile activities of Acanthamoeba myosins IA and IB
Abstract:
The low-shear viscosity of 5-30 microM F-actin was greatly increased by the addition of 0.1-0.5 microM unphosphorylated Acanthamoeba myosins IA and IB. The increase in viscosity was about the same in 2 mM ADP as in the absence of free nucleotide but was much less in 2 mM ATP. The single-headed monomolecular Acanthamoeba myosins were as effective as an equal molar concentration of two-headed muscle heavy meromyosin and much more effective than single-headed muscle myosin subfragment-1. These results suggest that Acanthamoeba myosins IA and IB can cross-link actin filaments as proposed in the accompanying paper (Albanesi, J. P., Fujisaki, H., and Korn, E. D. (1985) J. Biol. Chem. 260, 11174-11179) to explain the actin-dependent cooperative increase in actin-activated Mg2+-ATPase activity as a function of the concentration of myosin I. Superprecipitation occurred when phosphorylated myosin IA or IB was mixed with F-actin. In addition to myosin I heavy chain phosphorylation, superprecipitation required Mg2+ and ATP. ATP hydrolysis was linear during the time course of the superprecipitation, and inhibitors of ATP hydrolysis inhibited superprecipitation. A small, dense contracted gel was formed when the reaction was carried out in a cuvette, and a birefringent actomyosin thread resulted from superprecipitation in a microcapillary. The rate and extent of superprecipitation depended on the actin and myosin I concentrations with maximum superprecipitation occurring at an actin:myosin ratio of 7:1. These results provide strong evidence for the ability of Acanthamoeba myosins IA and IB to perform contractile and motile functions.
Insights
Acanthamoeba myosins IA and IB significantly increase actin filament viscosity and cause superprecipitation, indicating contractile functions. These single-headed myosins are highly effective cross-linkers.
Area of Science:
- Biochemistry
- Cell Biology
- Molecular Motors
Background:
- Actin-binding proteins are crucial for cellular structure and motility.
- Myosins are motor proteins that interact with actin filaments.
- Acanthamoeba myosins I (unphosphorylated and phosphorylated) are investigated for their functional properties.
Purpose of the Study:
- To investigate the effect of Acanthamoeba myosins IA and IB on F-actin viscosity.
- To determine the role of phosphorylation and ATP in myosin I-mediated actin interactions.
- To provide evidence for the contractile and motile functions of Acanthamoeba myosins I.
Main Methods:
- Viscometry was used to measure the effect of myosins on F-actin.
- Superprecipitation assays were performed with phosphorylated and unphosphorylated myosins.
- ATP hydrolysis rates and effects of inhibitors were monitored during superprecipitation.
Main Results:
- Unphosphorylated Acanthamoeba myosins IA and IB significantly increased F-actin low-shear viscosity.
- Myosin I's cross-linking ability was comparable to heavy meromyosin and superior to myosin subfragment-1.
- Phosphorylated myosins induced superprecipitation with F-actin, requiring Mg2+ and ATP hydrolysis.
Conclusions:
- Acanthamoeba myosins IA and IB function as effective cross-linkers of actin filaments.
- Myosin I heavy chain phosphorylation is essential for superprecipitation.
- These findings strongly support the contractile and motile capabilities of Acanthamoeba myosins IA and IB.