In Caenorhabditis elegans, unc-89 encodes a set of giant multi-domain proteins (up 8081 residues) localized to the M-lines of muscle sarcomeres and required for normal sarcomere organization and whole-animal locomotion. Multiple UNC-89 isoforms contain two protein kinase domains. There is conservation in arrangement of domains between UNC-89 and its two mammalian homologs, obscurin and SPEG: kinase, a non-domain region of 647–742 residues, Ig domain, Fn3 domain and a second kinase domain. In all three proteins, this non-domain “interkinase region” has low sequence complexity, has high proline content, and lacks predicted secondary structure. We report that a major portion of this interkinase (571 residues out of 647 residues) when examined by single molecule force spectroscopy in vitro displays the properties of a random coil and acts as an entropic spring. We used CRISPR/Cas9 to create nematodes carrying an in-frame deletion of the same 571-residue portion of the interkinase. These animals display severe disorganization of all portions of the sarcomere in body wall muscle. Super-resolution microscopy reveals extra, short-A-bands lying close to the outer muscle cell membrane and between normally spaced A-bands. Nematodes with this in-frame deletion show defective locomotion and muscle force generation. We designed our CRISPR-generatedin-frame deletion to contain an HA tag at the N terminus of the large UNC-89 isoforms. This HA tag results in normal organization of body wall muscle, but approximately half the normal levels of the giant UNC-89 isoforms, dis-organization of pharyngeal muscle, small body size, and reduced muscle force, likely due to poor nutritional uptake.

在秀丽隐杆线虫中，unc-89编码一组巨大的多域蛋白（最多 8081 个残基），定位于肌肉肌节的 M 线，是正常肌节组织和整个动物运动所必需的。多个 UNC-89 异构体包含两个蛋白激酶结构域。UNC-89 与其两个哺乳动物同源物 obscurin 和 SPEG 之间的结构域排列存在保守性：激酶，647-742 个残基的非结构域区域，Ig 结构域，Fn3 结构域和第二个激酶结构域。在所有三种蛋白质中，这个非结构域“激酶间区”序列复杂性低，脯氨酸含量高，并且缺乏预测的二级结构。我们报告说，当通过单分子力光谱检查时，这种激酶的主要部分（647 个残基中的 571 个残基）体外显示随机线圈的特性并充当熵弹簧。我们使用 CRISPR/Cas9 来创建线虫，该线虫携带对激酶间相同的 571 个残基部分进行框内删除。这些动物在体壁肌肉中显示出肌节所有部分的严重紊乱。超分辨率显微镜显示靠近外部肌肉细胞膜和正常间隔的 A 带之间有额外的短 A 带。具有这种帧内删除的线虫显示出有缺陷的运动和肌肉力量产生。我们设计了我们的 CRISPR-generatedin-frame 删除以在大型 UNC-89 同种型的 N 末端包含一个 HA 标签。这个 HA 标签导致体壁肌肉组织正常，但大约是巨大 UNC-89 同种型正常水平的一半，咽肌组织紊乱，体型小，