Multiple Regulatory Steps Control Mammalian Nonmuscle Myosin II Assembly in Live Cells
Multiple Regulatory Steps Control Mammalian Nonmuscle Myosin II Assembly in Live Cells
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DOI：
10.1091/mbc.e08-04-0372
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发表时间：
2009-01-01
影响因子：
3.3
通讯作者：
Egelhoff, Thomas T.
中科院分区：
文献类型：
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作者：
Breckenridge, Mark T.;Dulyaninova, Natalya G.;Egelhoff, Thomas T.
为了更好地了解哺乳动物细胞中控制非肌肉肌球蛋白II(NM-II)组装的机制，我们创建了突变的NM-IIA结构，以允许在活细胞中测试两个广泛研究的细丝组装控制模型。缺少RLC结合结构域(Delta IQ2)的GFP-NM-IIA结构破坏了10S隔离单体状态的稳定，导致在扩散过程中单体回收严重缺陷，以及在极化迁移过程中从后面到前沿。缺乏非螺旋尾片(Delta尾片)的GFP-NM-IIA结构适合于前沿组装，但过度组装，表明在最初招募后从片层拆卸方面存在缺陷。Delta尾段表型被GFP-NM-IIA构建的GFP-NM-IIA重述，该结构在定位的尾段磷酸化位点(S1943A)上携带突变，验证了尾段和尾段磷酸化在正常的板层肌球蛋白II组装控制中的重要性。这些结果表明，6S/10S构象变化和尾片都有助于肌球蛋白II在哺乳动物细胞中的定位和组装。这项工作进一步提供了细胞洞察力，有助于解释与人类Myh9相关疾病患者的R1933-Stop等位基因相关的血小板和白细胞缺陷。
To better understand the mechanism controlling nonmuscle myosin II (NM-II) assembly in mammalian cells, mutant NM-IIA constructs were created to allow tests in live cells of two widely studied models for filament assembly control. A GFP-NM-IIA construct lacking the RLC binding domain (Delta IQ2) destabilizes the 10S sequestered monomer state and results in a severe defect in recycling monomers during spreading, and from the posterior to the leading edge during polarized migration. A GFP-NM-IIA construct lacking the nonhelical tailpiece (Delta tailpiece) is competent for leading edge assembly, but overassembles, suggesting defects in disassembly from lamellae subsequent to initial recruitment. The Delta tailpiece phenotype was recapitulated by a GFP-NM-IIA construct carrying a mutation in a mapped tailpiece phosphorylation site (S1943A), validating the importance of the tailpiece and tailpiece phosphorylation in normal lamellar myosin II assembly control. These results demonstrate that both the 6S/10S conformational change and the tailpiece contribute to the localization and assembly of myosin II in mammalian cells. This work furthermore offers cellular insights that help explain platelet and leukocyte defects associated with R1933-stop alleles of patients afflicted with human MYH9-related disorder.