The mechanism of assembly of Acanthamoeba myosin-II minifilaments: minifilaments assemble by three successive dimerization steps.
We used 90 degrees light scattering, analytical ultracentrifugation, and electron microscopy to deduce that Acanthamoeba myosin-II minifilaments, composed of eight molecules each, assemble by a novel mechanism consisting of three successive dimerization steps rather than by the addition of monomers or parallel dimers to a nucleus. Above 200 mM KCl, Acanthamoeba myosin-II is monomeric. At low ionic strength (less than 100 mM KCl), myosin-II polymerizes into bipolar minifilaments. Between 100 and 200 mM KCl, plots of light scattering vs. myosin concentration all extrapolate to the origin but have slopes which decrease with increasing KCl. This indicates that structures intermediate in size between monomers and full length minifilaments are formed, and that the critical concentrations for assembly of these structures is very low. Analytical ultracentrifugation has confirmed that intermediate structures exist at these salt concentrations, and that they are in rapid equilibrium with each other. We believe these structures represent assembly intermediates and have used equilibrium analytical ultracentrifugation and electron microscopy to identify them. Polymerization begins with the formation of antiparallel dimers, with the two tails overlapping by approximately 15 nm. Two antiparallel dimers then associated with a 15-nm stagger to form an antiparallel tetramer. Finally, two tetramers associate with a 30-nm stagger to form the completed minifilament. At very low ionic strengths, the last step in the assembly mechanism is largely reversed and antiparallel tetramers are the predominant species. Alkaline pH, which can also induce minifilament disassembly, produces the same assembly intermediates as are found for salt induced disassembly.
Sinard
J H
JH
Department of Cell Biology and Anatomy, Johns Hopkins University School of Medicine, Baltimore, Maryland 21205.
Stafford
W F
WF
Pollard
T D
TD
D000048:Acanthamoeba / Q000378:metabolism*; D000818:Animals; D003198:Computer Simulation; D007700:Kinetics; D008027:Light; D046911:Macromolecular Substances; D008854:Microscopy, Electron; D008961:Models, Structural; D009218:Myosins / Q000378:metabolism*; D009994:Osmolar Concentration; D011189:Potassium Chloride / Q000494:pharmacology; D012542:Scattering, Radiation; D014461:Ultracentrifugation / Q000379:methods
D046911:Macromolecular Substances; D011189:Potassium Chloride; D009218:Myosins