Department of Biochemistry and Molecular Biology, University of Maryland School of Medicine, Baltimore, Maryland, United States of America.
PLoS One. 2011;6(12):e28524. doi: 10.1371/journal.pone.0028524. Epub 2011 Dec 9.
Smyd1b is a member of the Smyd family that plays a key role in sarcomere assembly during myofibrillogenesis. Smyd1b encodes two alternatively spliced isoforms, smyd1b_tv1 and smyd1b_tv2, that are expressed in skeletal and cardiac muscles and play a vital role in myofibrillogenesis in skeletal muscles of zebrafish embryos.
METHODOLOGY/PRINCIPAL FINDINGS: To better understand Smyd1b function in myofibrillogenesis, we analyzed the subcellular localization of Smyd1b_tv1 and Smyd1b_tv2 in transgenic zebrafish expressing a myc-tagged Smyd1b_tv1 or Smyd1b_tv2. The results showed a dynamic change of their subcellular localization during muscle cell differentiation. Smyd1b_tv1 and Smyd1b_tv2 were primarily localized in the cytosol of myoblasts and myotubes at early stage zebrafish embryos. However, in mature myofibers, Smyd1b_tv1, and to a small degree of Smyd1b_tv2, exhibited a sarcomeric localization. Double staining with sarcomeric markers revealed that Smyd1b_tv1was localized on the M-lines. The sarcomeric localization was confirmed in zebrafish embryos expressing the Smyd1b_tv1-GFP or Smyd1b_tv2-GFP fusion proteins. Compared with Smyd1b_tv1, Smyd1b_tv2, however, showed a weak sarcomeric localization. Smyd1b_tv1 differs from Smyd1b_tv2 by a 13 amino acid insertion encoded by exon 5, suggesting that some residues within the 13 aa insertion may be critical for the strong sarcomeric localization of Smyd1b_tv1. Sequence comparison with Smyd1b_tv1 orthologs from other vertebrates revealed several highly conserved residues (Phe223, His224 and Gln226) and two potential phosphorylation sites (Thr221 and Ser225) within the 13 aa insertion. To determine whether these residues are involved in the increased sarcomeric localization of Smyd1b_tv1, we mutated these residues into alanine. Substitution of Phe223 or Ser225 with alanine significantly reduced the sarcomeric localization of Smyd1b_tv1. In contrast, other substitutions had no effect. Moreover, replacing Ser225 with threonine (S225T) retained the strong sarcomeric localization of Smyd1b_tv1.
CONCLUSION/SIGNIFICANCE: Together, these data indicate that Phe223 and Ser225 are required for the M-line localization of Smyd1b_tv1.
Smyd1b 是 Smyd 家族的成员，在肌原纤维发生过程中对肌节组装起着关键作用。Smyd1b 编码两种选择性剪接的异构体，smyd1b_tv1 和 smyd1b_tv2，它们在骨骼肌和心肌中表达，并在斑马鱼胚胎骨骼肌的肌原纤维发生中起着至关重要的作用。
方法/主要发现：为了更好地了解 Smyd1b 在肌原纤维发生中的功能，我们分析了在表达 myc 标记的 Smyd1b_tv1 或 Smyd1b_tv2 的转基因斑马鱼中 Smyd1b_tv1 和 Smyd1b_tv2 的亚细胞定位。结果显示，在早期斑马鱼胚胎的肌肉细胞分化过程中，其亚细胞定位发生了动态变化。Smyd1b_tv1 和 Smyd1b_tv2 主要定位于肌母细胞和肌管的细胞质中。然而，在成熟的肌纤维中，Smyd1b_tv1 以及少量的 Smyd1b_tv2，呈现出肌节定位。用肌节标记物进行双重染色显示，Smyd1b_tv1 定位于 M 线上。在表达 Smyd1b_tv1-GFP 或 Smyd1b_tv2-GFP 融合蛋白的斑马鱼胚胎中证实了肌节定位。与 Smyd1b_tv1 相比，Smyd1b_tv2 表现出较弱的肌节定位。Smyd1b_tv1 与 Smyd1b_tv2 的区别在于外显子 5 编码的 13 个氨基酸插入，这表明 13 个氨基酸插入内的一些残基可能对 Smyd1b_tv1 的强肌节定位至关重要。与来自其他脊椎动物的 Smyd1b_tv1 同源物的序列比较显示，在 13 个氨基酸插入内有几个高度保守的残基（Phe223、His224 和 Gln226）和两个潜在的磷酸化位点（Thr221 和 Ser225）。为了确定这些残基是否参与 Smyd1b_tv1 肌节定位的增加，我们将这些残基突变为丙氨酸。用丙氨酸替换 Phe223 或 Ser225 显著降低了 Smyd1b_tv1 的肌节定位。相比之下，其他替换没有影响。此外，用苏氨酸（S225T）替换 Ser225 保留了 Smyd1b_tv1 的强肌节定位。
结论/意义：综上所述，这些数据表明 Phe223 和 Ser225 是 Smyd1b_tv1 定位于 M 线所必需的。
