Laboratory of Cell Biology, National Heart, Lung, and Blood Institute, National Institutes of Health, Bethesda, MD 20892.
Laboratory of Physiology, National Heart, Lung, and Blood Institute, National Institutes of Health, Bethesda, MD 20892.
Proc Natl Acad Sci U S A. 2017 Aug 8;114(32):E6516-E6525. doi: 10.1073/pnas.1702375114. Epub 2017 Jul 24.
Addition of 1 mM ATP substantially reduces the light scattering of solutions of polymerized unphosphorylated nonmuscle myosin IIs (NM2s), and this is reversed by phosphorylation of the regulatory light chain (RLC). It has been proposed that these changes result from substantial depolymerization of unphosphorylated NM2 filaments to monomers upon addition of ATP, and filament repolymerization upon RLC-phosphorylation. We now show that the differences in myosin monomer concentration of RLC-unphosphorylated and -phosphorylated recombinant mammalian NM2A, NM2B, and NM2C polymerized in the presence of ATP are much too small to explain their substantial differences in light scattering. Rather, we find that the decrease in light scattering upon addition of ATP to polymerized unphosphorylated NM2s correlates with the formation of dimers, tetramers, and hexamers, in addition to monomers, an increase in length, and decrease in width of the bare zones of RLC-unphosphorylated filaments. Both effects of ATP addition are reversed by phosphorylation of the RLC. Our data also suggest that, contrary to previous models, assembly of RLC-phosphorylated NM2s at physiological ionic strength proceeds from folded monomers to folded antiparallel dimers, tetramers, and hexamers that unfold and polymerize into antiparallel filaments. This model could explain the dynamic relocalization of NM2 filaments in vivo by dephosphorylation of RLC-phosphorylated filaments, disassembly of the dephosphorylated filaments to folded monomers, dimers, and small oligomers, followed by diffusion of these species, and reassembly of filaments at the new location following rephosphorylation of the RLC.
添加 1mM 的 ATP 可显著减少聚合的非肌肉肌球蛋白 II（NM2）的无磷酸化非磷酸化肌球蛋白 II（NM2）溶液的光散射，并且这可通过调节轻链（RLC）的磷酸化来逆转。有人提出，这些变化是由于添加 ATP 后未磷酸化的 NM2 纤维的大量解聚为单体，以及 RLC-磷酸化后纤维的重新聚合。我们现在表明，在 ATP 存在下聚合的 RLC-非磷酸化和磷酸化的重组哺乳动物 NM2A、NM2B 和 NM2C 的肌球蛋白单体浓度的差异小到不足以解释其在光散射方面的显著差异。相反，我们发现，在向聚合的未磷酸化 NM2s 中添加 ATP 后，光散射的减少与单体的形成以及 RLC-非磷酸化纤维的裸露区的长度增加和宽度减小相关，此外还形成二聚体、四聚体和六聚体。RLC 磷酸化的 NM2s 的 ATP 添加的这两种效应均被 RLC 的磷酸化逆转。我们的数据还表明，与先前的模型相反，在生理离子强度下，RLC-磷酸化的 NM2s 的组装是从折叠的单体开始，到折叠的平行二聚体、四聚体和六聚体，这些二聚体、四聚体和六聚体展开并聚合成平行的纤维。该模型可以解释 NM2 纤维在体内的动态重定位，即 RLC-磷酸化的纤维去磷酸化，去磷酸化的纤维解聚为折叠的单体、二聚体和小寡聚体，然后这些物质扩散，以及 RLC 重新磷酸化后在新位置重新组装纤维。
