The Functions and Properties of Nonmuscle Myosin Heavy Chains

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。我们还表达了两个嵌合NM II蛋白和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。 我们已经确定了一个基因靶向位点（6 kb），在Myh 9基因的外显子2的区域，显示了一个非常高的和可重复的频率的HR在小鼠胚胎干细胞（ES）（95%在这种情况下对1-10%在大多数情况下）。据我们所知，这是迄今为止报告的最高比率。我们的初步研究表明，没有证据表明特定的DNA序列是负责这种高靶向效率，因为同源臂的逐渐缩短导致靶向频率相应降低。进一步研究表明，在小鼠ES细胞、诱导多能干细胞（iPS）和小鼠胚胎成纤维细胞（MEF）中，同一靶位点的GT效率表现为ES>iPS>MEF细胞，且随着靶位点3从外显子2向外显子2和3之间的内含子再向外显子3的转移，GT频率逐渐降低。这些结果暗示了染色体结构和可能的表观遗传修饰对GT效率的影响。此外，我们的研究结果具有重要的应用，因为Myh 9位点可以在不存在整合位点影响的情况下为转基因插入提供安全港，从而可以更准确地比较多个转基因品系。在我们目前的研究中，我们通过以下方式利用Myh 9基因座处的高频率HR：1）产生遗传替代小鼠模型以研究例如非肌肉肌球蛋白II的同种型和结构域特异性（Zhang Y等人，Blood，2012，119：238-50）。到目前为止，至少有5个小鼠模型已用于这些目的。2)创建成功模拟人类中发现的Myh 9-RD表型的Myh 9相关疾病（Myh 9-RD）小鼠模型。3)从小鼠胚胎干细胞获得高纯度心肌细胞。为此，将编码由心脏特异性启动子控制的嘌呤霉素抗性的盒整合到Myh 9基因座中。4)将shRNA表达盒整合到该位点用于基因特异性敲低。在每种情况下，高HR频率促进了所需ES细胞克隆的分离。

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 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. We have identified a gene targeting locus (6 kb), in the region of exon 2 of the Myh9 gene, that displays an extremely high and repeatable frequency of HR in mouse embryonic stem (ES) cells (95% in this case vs 1-10% in most cases). To our knowledge this is the highest rate that has been reported to date. Our initial investigations indicated no evidence for a specific DNA sequence that is responsible for this high targeting efficiency since a gradual shortening of the homologous arms results in a corresponding reduction of targeting frequency. Further studies using different cell lines including mouse ES cells, induced pluripotent stem (iPS) cells and mouse embryonic fibroblasts (MEFs) showed that the GT efficiency at the same targeted site exhibits an order of ES>iPS>MEF cells, and that GT frequency gradually decreases with the shift of the targeted sites 3 from exon 2 to the intron between exon 2 and 3 and then to exon 3 in mouse ES cells. These results imply the influence of chromosome structure and possible epigenetic modification on GT efficiency. Additionally, our findings have important applications as the Myh9 locus can provide a safe harbor for transgene insertions in the absence of the influence of the integration site, allowing multiple transgenic lines to be more accurately compared. In our current study we took advantage of the high frequency of HR at the Myh9 locus by: 1) generating genetic replacement mouse models to study the isoform and domain specificity of, for example, nonmuscle myosin IIs (Zhang Y et al., Blood, 2012, 119:238-50). So far, at least 5 mouse models have been produced for these purposes. 2) Creating Myh9 related disease (Myh9-RD) mouse models which successfully mimic the Myh9-RD phenotype found in humans. 3) Obtaining high purity cardiomyocytes derived from mouse embryonic stem cells. To this end, a cassette encoding Puromycin resistance controlled by a cardiac-specific promoter was integrated into the Myh9 locus. 4) Integrating a shRNA expression cassette into this site for gene-specific knockdown. In each case, the high HR frequency facilitated isolation of the desired ES cell clones.