Mahajan R K, Pardee J D
Department of Cell Biology and Anatomy, Cornell University Medical College, New York, New York 10021, USA.
Biochemistry. 1996 Dec 3;35(48):15504-14. 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 microns long antiparallel tetramers. Lateral addition of dimers to bipolar tetramers completes the assembly of short (0.45 microns) 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 (kobs,N+G) of 1.85 x 10(4) M-1 s-1. Individual rate constants obtained for nucleation, kobs,N = 4.5 x 10(3) M-1 s-1, and growth, kobs,G = 2.5 x 10(4) 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.
盘基网柄菌（Dictyostelium discoideum）变形虫中肌球蛋白II的有序组装部分控制了胞质分裂和细胞迁移过程中收缩结构的有序形成。动力学和结构分析表明，盘基网柄菌肌球蛋白II通过缓慢成核和可控生长的连续过程进行组装，其速率和机制与其他传统肌球蛋白不同。核的形成是一个有序的过程，从肌球蛋白单体到平行二聚体，再到0.43微米长的反平行四聚体。二聚体横向添加到双极四聚体上完成了短（0.45微米）钝端粗丝的组装。肌球蛋白头部不像在骨骼肌中那样沿锥形粗丝的长度交错排列，也不像在棘阿米巴中那样形成双极微丝。包含成核和生长的整体组装反应在动力学上可以用二阶速率常数（kobs,N+G）1.85×10(4) M-1 s-1来表征。成核的个体速率常数kobs,N = 4.5×10(3) M-1 s-1，生长的个体速率常数kobs,G = 2.5×10(4) M-1 s-1，表明盘基网柄菌肌球蛋白II是迄今为止分析的组装最慢的肌球蛋白。成核和生长阶段分别受Mg2+、K+和肌动蛋白丝的调节。K+浓度从50 mM增加到150 mM会特异性抑制二聚体从核上的横向生长。细胞内Mg2+浓度（1 mM）加速了成核，但保持了不同的成核和生长阶段动力学。肌动蛋白丝网络也加速了组装的成核阶段，从机制上解释了通过肌球蛋白组装自发形成肌动球蛋白收缩纤维的现象（Mahajan等人，1989年）。盘基网柄菌肌球蛋白II所利用的独特组装机制和调节表明，来自平滑肌、横纹肌和两种变形虫的肌球蛋白通过不同途径形成独特的粗丝。
