The Function of Nonmuscle Myosin Heavy Chains

与导致A-/A-小鼠胚胎第6.5天（E）致死的内脏内胚层形成失败相反，用NM II-B或嵌合NM II替代恢复了正常的内脏内胚层。这一发现与NM II在细胞粘附中的作用一致，也证实了NM II在内脏内胚层功能中的重要的、异构体独立的要求。基因敲入小鼠在E9.5和12.5之间死亡，因为胎盘形成缺陷与异常血管生成和细胞迁移相关，揭示了NM II-A在胎盘发育中的独特功能。体外结果进一步支持NM II-A在定向细胞迁移和粘着斑形成中的需求。这些研究结果表明，在这些过程中，NM II-A的异构体特异性作用，使另一种异构体或嵌合NM II异构体的替代不太成功。这些取代的失败不仅与NM II-A和II-B的不同动力学性质有关，而且还与它们由C-末端结构域决定的亚细胞定位有关。这些结果突出了NM II的N-末端马达和C-末端杆域的功能及其在细胞-细胞和细胞-基质粘附中的不同作用。 我们已经成功地表达全长野生型和突变NM II蛋白使用Sf 9-杆状病毒系统。我们还表达了两种嵌合NM II蛋白Wang A等人PNAS 2010，107（33）：14645-50和GFP-NM II融合蛋白。我们发现：I）尽管全长NM II-A、II-B和II-C表现出生物化学差异，但通过负染色电子显微镜（EM）确定的细丝形态在三种旁系同源物中基本上无法区分。在ATP的存在下，所有三种旁系同源物显示出类似的采用10 S紧凑构象的能力。II）嵌合分子的EM图像显示，旁系同源物的尾域在细丝形成和10 S紧密构象的形成方面是可互换的; III）与先前的报道相反，全长NM IIA蛋白中点突变的存在，（N93 K，D1424 N，E1841 K）对丝的形成几乎没有或没有明显的影响; IV）GFP融合到NM II上使我们能够通过TIRF显微镜直接分析体外运动性。

In contrast with the failure of visceral endoderm formation resulting in embryonic day (E)6.5 lethality of A-/A- mice, replacement with NM II-B or chimeric NM IIs restores a normal visceral endoderm. This finding is consistent with NM II's role in cell adhesion and also confirms an essential, isoform-independent requirement for NM II in visceral endoderm function. The knock-in mice die between E9.5 and 12.5 because of defects in placenta formation associated with abnormal angiogenesis and cell migration, revealing a unique function for NM II-A in placenta development. In vitro results further support a requirement for NM II-A in directed cell migration and focal adhesion formation. These findings demonstrate an isoform-specific role for NM II-A during these processes, making replacement by another isoform, or chimeric NM II isoforms, less successful. The failure of these substitutions is not only related to the different kinetic properties of NM II-A and II-B, but also to their subcellular localization determined by the C-terminal domain. These results highlight the functions of the N-terminal motor and C-terminal rod domains of NM II and their different roles in cell-cell and cell-matrix adhesion. We have successfully expressed full length wild type and mutated NM II proteins using the Sf9-baculovirus system. We also expressed two chimeric NM II proteins Wang A et al. PNAS 2010, 107(33):14645-50 and GFP-NM II fusion proteins. We find that: I) although full length NM II-A, II-B and II-C exhibit biochemical differences, the morphology of the filaments determined by negative-staining electron microscopy (EM) is essentially indistinguishable among the three paralogs. In the presence of ATP all three paralogs display a similar ability to adopt the 10S compact conformation. II) EM images of chimeric molecules show that the tail domains of the paralogs are interchangeable in terms of filament formation and formation of the 10S compact conformation; III) In contrast to a previous report, the presence of point mutations in full length NM IIA proteins (N93K, D1424N, E1841K) causing human diseases has little or no obvious effects on filament formation; IV) GFP fused to NM II allows us to directly analyze in vitro motility by TIRF microscopy.