Mahajan R K, Vaughan K T, Johns J A, Pardee J D
Department of Cell Biology and Anatomy, Cornell University Medical College, New York, NY 10021.
Proc Natl Acad Sci U S A. 1989 Aug;86(16):6161-5. doi: 10.1073/pnas.86.16.6161.
Because myosin thick filaments form in the actin-rich cortex of nonmuscle cells, we have examined the role of Dictyostelium actin filaments in the assembly of Dictyostelium myosin (type II). Fluorescence energy transfer and light-scattering assembly assays indicate that self-association of Dictyostelium myosin into bipolar thick filaments is kinetically regulated by actin filament networks. Regulation is nucleotide dependent but does not require ATP hydrolysis. Myosin assembly is accelerated approximately 5-fold by actin filaments when either 1 mM ATP or 1 mM adenosine 5'-[beta,gamma-imido]triphosphate (AMP-P[NH]P) is present. However, actin filaments together with 1 mM ADP abolish myosin assembly. Accelerated assembly appears to require transient binding of myosin molecules to actin filaments before incorporation into thick filaments. Fluorescence energy-transfer assays demonstrate that myosin associates with actin filaments at a rate that is equivalent to the accelerated myosin assembly rate, evidence that myosin to actin binding is a rate-limiting step in accelerated thick filament formation. Actin filament networks are also implicated in regulation of thick filament formation, since fragmentation of F-actin networks by severin causes immediate cessation of accelerated myosin assembly. Electron microscopic studies support a model of actin filament-mediated myosin assembly. In ADP, myosin monomers rapidly decorate F-actin, preventing extensive formation of thick filaments. In AMP-P[NH]P, myosin assembles along actin filaments, forming structures that resemble primitive stress fibers. Taken together, these data suggest a model in which site-directed assembly of thick filaments in Dictyostelium is mediated by the interaction of myosin monomers with cortical actin filament networks.
由于肌球蛋白粗丝在非肌肉细胞富含肌动蛋白的皮质中形成，我们研究了盘基网柄菌肌动蛋白丝在盘基网柄菌肌球蛋白（II型）组装中的作用。荧光能量转移和光散射组装分析表明，盘基网柄菌肌球蛋白自组装成双极粗丝在动力学上受肌动蛋白丝网络调控。这种调控依赖核苷酸，但不需要ATP水解。当存在1 mM ATP或1 mM腺苷5'-[β,γ-亚氨基]三磷酸（AMP-P[NH]P）时，肌动蛋白丝可使肌球蛋白组装加速约5倍。然而，肌动蛋白丝与1 mM ADP一起会抑制肌球蛋白组装。加速组装似乎需要肌球蛋白分子在掺入粗丝之前先与肌动蛋白丝短暂结合。荧光能量转移分析表明，肌球蛋白与肌动蛋白丝结合的速率等同于肌球蛋白加速组装的速率，这证明肌球蛋白与肌动蛋白的结合是加速形成粗丝的限速步骤。肌动蛋白丝网络也参与粗丝形成的调控，因为肌动蛋白切割蛋白使F-肌动蛋白网络断裂会导致加速的肌球蛋白组装立即停止。电子显微镜研究支持肌动蛋白丝介导的肌球蛋白组装模型。在ADP存在的情况下，肌球蛋白单体迅速结合到F-肌动蛋白上，阻止粗丝的大量形成。在AMP-P[NH]P存在的情况下，肌球蛋白沿着肌动蛋白丝组装，形成类似于原始应力纤维的结构。综上所述，这些数据提示了一个模型，即盘基网柄菌中粗丝的定点组装是由肌球蛋白单体与皮质肌动蛋白丝网络的相互作用介导的。
