Assembly Mechanism of Dictyostelium Myosin II:  Regulation by K+, Mg2+, and Actin Filaments

• Published in: Biochemistry (Easton) Vol. 35; no. 48; pp. 15504 - 15514
• Main Authors: Mahajan, Rohit K, Pardee, Joel D
• Format: Journal Article
• Language: English
• Published: United States American Chemical Society 03.12.1996
• Subjects: Actins - metabolism; Animals; Buffers; Dictyostelium; Kinetics; Magnesium - metabolism; Models, Chemical; Myosins - chemistry; Myosins - metabolism; Potassium - metabolism; Potassium Chloride - pharmacology
• ISSN: 0006-2960; 1520-4995
• DOI: 10.1021/bi9618981

Regulated assembly of myosin II in Dictyostelium discoideum amoebae partially controls the orderly formation of contractile structures during cytokinesis and cell migration. Kinetic and structural analyses show that Dictyostelium myosin II assembles by a sequential process of slow nucleation and controlled growth that differs in rate and mechanism from other conventional myosins. Nuclei form by an ordered progression from myosin monomers to parallel dimers to 0.43 μm long antiparallel tetramers. Lateral addition of dimers to bipolar tetramers completes the assembly of short (0.45 μm) blunt-ended thick filaments. Myosin heads are not staggered along the length of tapered thick filaments as in skeletal muscle, nor are bipolar minifilaments formed as in Acanthamoeba. The overall assembly reaction incorporating both nucleation and growth could be kinetically characterized by a second-order rate constant (k obs ,N+G) of 1.85 × 104 M-1 s-1. Individual rate constants obtained for nucleation, k obs ,N = 4.5 × 103 M-1 s-1, and growth, k obs ,G = 2.5 × 104 M-1 s-1, showed Dictyostelium myosin II to be the slowest assembling myosin analyzed to date. Nucleation and growth stages were independently regulated by Mg2+, K+, and actin filaments. Increasing concentrations of K+ from 50 to 150 mM specifically inhibited lateral growth of dimers off nuclei. Intracellular concentrations of Mg2+ (1 mM) accelerated nucleation but maintained distinct nucleation and growth phase kinetics. Networks of actin filaments also accelerated the nucleation stage of assembly, mechanistically accounting for spontaneous formation of actomyosin contractile fibers via myosin assembly (Mahajan et al., 1989). The distinct assembly mechanism and regulation utilized by Dictyostelium myosin II demonstrates that myosins from smooth muscle, striated muscle, and two types of amoebae form unique thick filaments by different pathways.