ASSEMBLY MECHANISM OF DICTYOSTELIUM MYOSIN-II - REGULATION BY K-FILAMENTS(, MG2+, AND ACTIN)

Regulated assembly of myosin II in Dictyostelium discoideum amoebae pa rtially controls the orderly formation of contractile structures durin g cytokinesis and cell migration. Kinetic and structural analyses show that Dictyostelium myosin II assembles by a sequential process of slo w 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 mu m long antiparalle l tetramers. Lateral addition of dimers to bipolar tetramers completes the assembly of short (0.45 mu m) blunt-ended thick filaments. Myosin heads are not staggered along the length of tapered thick filaments a s in skeletal muscle, nor are bipolar minifilaments formed as in Acant hamoeba. The overall assembly reaction incorporating both nucleation a nd growth could be kinetically characterized by a second-order rate co nstant (k(obs,N+G)) Of 1.85 x 10(4) M(-1) s(-1) Individual rate consta nts obtained for nucleation, k(obs,N) = 4.5 x 10(3) M(-1) s(-1), and g rowth, k(obs,G) = 2.5 x 10(4) M(-1) s(-1), showed Dictyostelium myosin II to be the slowest assembling myosin analyzed to date. Nucleation a nd growth stages were independently regulated by Mg2+, K+, and actin f ilaments. Increasing concentrations of K+ from 50 to 150 mM specifical ly inhibited lateral growth of dimers off nuclei. Intracellular concen trations of Mg2+ (1 mM) accelerated nucleation but maintained distinct nucleation and growth phase kinetics. Networks of actin filaments als o accelerated the nucleation stage of assembly, mechanistically accoun ting for spontaneous formation of actomyosin contractile fibers via my osin assembly (Mahajan et al., 1989). The distinct assembly mechanism and regulation utilized by Dictyostelium myosin II demonstrates that m yosins from smooth muscle, striated muscle, and two types of amoebae f orm unique thick filaments by different pathways.