A novel isoform of myosin 18A (Myo18A) is an essential sarcomeric protein in mouse heart
A novel isoform of myosin 18A (Myo18A) is an essential sarcomeric protein in mouse heart
复制标题
DOI：
10.1074/jbc.ra118.004560
复制
发表时间：
2019-05-03
影响因子：
4.8
通讯作者：
Hanley, Peter J.
中科院分区：
文献类型：
--
作者：
Horsthemke, Markus;Nutter, Lauryl M. J.;Hanley, Peter J.
虽然肌球蛋白 18B (Myo18B) 已知是一种关键的肌节蛋白，但肌球蛋白 18A (Myo18A) 的功能尚不清楚，尽管它与细胞运动和高尔基体形状有关。在这里，我们证明小鼠中 Myo18a 的纯合缺失（纯合 tm1a、tm1b 或 tm1d 等位基因）是胚胎致死的。与 Myo18b 类似，Myo18a 在胚胎心脏中高表达，而小鼠心脏限制性 Myo18a 缺失会导致胚胎死亡。令人惊讶的是，通过蛋白质印迹分析，我们无法使用定制的 C 端抗体检测小鼠心脏中已知的 Myo18A、Myo18A 和 Myo18A 亚型。然而，替代的抗Myo18A抗体检测到了比预期更大的蛋白质，并且RNA-Seq分析表明一种新的Myo18A转录物在小鼠心室肌细胞（和人类心脏）中表达。克隆和测序表明，这种心脏异构体（称为 Myo18A）缺乏 Myo18A 的含有 PDZ 的 N 末端，但包含一个替代的 N 末端延伸和一个长的富含丝氨酸的 C 末端。 EGFP 标记的 Myo18A 在定位于肌节 A 带水平的心室肌细胞中表达，Myo18a 敲除胚胎在第 10.5 天表现出杂乱的肌节，具有波状粗丝。我们还生成了骨髓限制性 Myo18a 敲除小鼠，以研究 Myo18A 在非肌肉细胞中的作用，例如巨噬细胞，其表达的 Myo18A 多于 Myo18A，但没有检测到细胞形状、运动性或高尔基体形状的缺陷。总之，我们鉴定了一种以前未被识别的肌节成分，即 Myo18A 的一种大型新型亚型（表示为 Myo18A）。因此，XVIII 类肌球蛋白的两个成员都是心脏肌节的重要组成部分。
Whereas myosin 18B (Myo18B) is known to be a critical sarcomeric protein, the function of myosin 18A (Myo18A) is unclear, although it has been implicated in cell motility and Golgi shape. Here, we show that homozygous deletion (homozygous tm1a, tm1b, or tm1d alleles) of Myo18a in mouse is embryonic lethal. Reminiscent of Myo18b, Myo18a was highly expressed in the embryo heart, and cardiac-restricted Myo18a deletion in mice was embryonic lethal. Surprisingly, using Western blot analysis, we were unable to detect the known isoforms of Myo18A, Myo18A and Myo18A, in mouse heart using a custom C-terminal antibody. However, alternative anti-Myo18A antibodies detected a larger than expected protein, and RNA-Seq analysis indicated that a novel Myo18A transcript is expressed in mouse ventricular myocytes (and human heart). Cloning and sequencing revealed that this cardiac isoform, denoted Myo18A, lacks the PDZ-containing N terminus of Myo18A but includes an alternative N-terminal extension and a long serine-rich C terminus. EGFP-tagged Myo18A expressed in ventricular myocytes localized to the level of A-bands in sarcomeres, and Myo18a knockout embryos at day 10.5 exhibited disorganized sarcomeres with wavy thick filaments. We additionally generated myeloid-restricted Myo18a knockout mice to investigate the role of Myo18A in nonmuscle cells, exemplified by macrophages, which express more Myo18A than Myo18A, but no defects in cell shape, motility, or Golgi shape were detected. In summary, we have identified a previously unrecognized sarcomere component, a large novel isoform (denoted Myo18A) of Myo18A. Thus, both members of class XVIII myosins are critical components of cardiac sarcomeres.