Department of Biology, Molecular Biology Institute and Heart Institute, San Diego State University, San Diego, CA 92182-4614, USA.
J Mol Biol. 2012 Mar 2;416(4):543-57. doi: 10.1016/j.jmb.2011.12.044. Epub 2011 Dec 28.
Myosin isoforms help define muscle-specific contractile and structural properties. Alternative splicing of myosin heavy chain gene transcripts in Drosophila melanogaster yields muscle-specific isoforms and highlights alternative domains that fine-tune myosin function. To gain insight into how native myosin is tuned, we expressed three embryonic myosin isoforms in indirect flight muscles lacking endogenous myosin. These isoforms differ in their relay and/or converter domains. We analyzed isoform-specific ATPase activities, in vitro actin motility and myofibril structure/stability. We find that dorsal acute body wall muscle myosin (EMB-9c11d) shows a significant increase in MgATPase V(max) and actin sliding velocity, as well as abnormal myofibril assembly compared to cardioblast myosin (EMB-11d). These properties differ as a result of alternative exon-9-encoded relay domains that are hypothesized to communicate signals among the ATP-binding pocket, actin-binding site and the converter domain. Further, EMB-11d shows significantly reduced levels of basal Ca- and MgATPase as well as MgATPase V(max) compared to embryonic body wall muscle isoform (EMB) (expressed in a multitude of body wall muscles). EMB-11d also induces increased actin sliding velocity and stabilizes myofibril structure compared to EMB. These differences arise from exon-11-encoded alternative converter domains that are proposed to reposition the lever arm during the power and recovery strokes. We conclude that relay and converter domains of native myosin isoforms fine-tune ATPase activity, actin motility and muscle ultrastructure. This verifies and extends previous studies with chimeric molecules and indicates that interactions of the relay and converter during the contractile cycle are key to myosin-isoform-specific kinetic and mechanical functions.
肌球蛋白异构体有助于定义肌肉特异性的收缩和结构特性。在黑腹果蝇的肌球蛋白重链基因转录本中，通过可变剪接产生了肌肉特异性异构体，并突出了微调肌球蛋白功能的替代结构域。为了深入了解天然肌球蛋白是如何被调节的，我们在缺乏内源性肌球蛋白的间接飞行肌中表达了三种胚胎肌球蛋白异构体。这些异构体在它们的中继和/或转换器结构域上存在差异。我们分析了同工型特异性的 ATP 酶活性、体外肌动蛋白运动和肌原纤维结构/稳定性。我们发现，与心胚肌球蛋白（EMB-11d）相比，背侧急性体壁肌球蛋白（EMB-9c11d）在 MgATPase V(max)和肌动蛋白滑动速度方面表现出显著增加，以及异常的肌原纤维组装。这些特性的差异是由于替代的exon-9 编码的中继结构域引起的，这些结构域被假设在 ATP 结合口袋、肌动蛋白结合位点和转换器结构域之间传递信号。此外，与胚胎体壁肌球蛋白同工型（在多种体壁肌肉中表达）相比，EMB-11d 的基础 Ca 和 MgATPase 以及 MgATPase V(max)水平显著降低。EMB-11d 还诱导了比 EMB 更高的肌动蛋白滑动速度，并稳定了肌原纤维结构。这些差异来自于 exon-11 编码的替代转换器结构域，这些结构域被认为在动力和恢复冲程期间重新定位了杠杆臂。我们得出结论，天然肌球蛋白同工型的中继和转换器结构域微调了 ATP 酶活性、肌动蛋白运动和肌肉超微结构。这验证并扩展了以前用嵌合分子进行的研究，并表明在收缩周期中中继和转换器的相互作用是肌球蛋白同工型特异性动力学和机械功能的关键。
