Mechanisms and Function of Myonuclear Positioning

项目摘要/摘要 我们的长期目标是显著影响肌肉生物学的基础知识，并提供新的 疾病治疗的方法。横纹肌纤维是大的多核细胞，具有高度的 包含细胞器的有组织的细胞结构，这些细胞器定位于最佳肌肉功能。这一定位是 肌核的位置尤其明显，位于肌节上方的肌节周围。 肌纤维，并被放置在最大限度地增加其核间距离。我们的目标是确定 负责肌核运动和定位的机制。位于中央的肌核已经 几十年来一直被用作肌肉疾病的标志。然而，关于这种机制仍有许多需要了解的地方。 正常控制肌核运动及肌核位置异常对病因学的影响 和/或肌肉疾病的进展。以我们在资助期内公布的结果为基础(例如Metzger等人， 2012年；Folker等，2012,2014；Schulman等，2013,2014；Azevedo等，2016；Manhart等，2018,2020； Rosen等人，2019)，我们的具体目标是首先机械地解决肌腱和运动神经元是如何发出信号的 以微调肌核的位置。我们在肌腱上发现了两条信号通路。 调节核定位的连接点。同样，我们将剖析运动神经元对核能的贡献 放置。其次，我们将研究为什么当肌核错位时，肌肉不能发挥最佳功能。 肌肉生理学将通过量化ATP和ROS来测试线粒体功能 并通过电生理方法测试神经肌肉通讯。我们还将调查 我们确定的特定代谢和信号蛋白对肌肉功能的输入被错误地调节为 肌核错位的结果。第三，我们将推进对肌核定位的剖析 机制从飞行到人类系统。我们将使用人体3D肌肉培养，与 运动神经元在肌纤维发育过程中定义并扰乱肌核的运动和定位。我们的 方法学利用了我们开发的尖端的活体时间推移成像方法 也将应用于人类3D培养，以跟踪肌核运动和细胞骨架 动力学。我们将利用果蝇和人类培养中可用的遗传资源来操纵基因， 流程，以及我们分析的细胞类型。这些基因实验将得到生化和细胞技术的支持 生物学方法。这项提案中概述的工作将共同揭示这一鲜为人知的问题， 但也是肌肉生物学的重要领域。这项研究的结果将使我们能够突出基因和 这些机制是与不同人类肌肉疾病相关的变化的候选者。

PROJECT SUMMARY/ABSTRACT Our long-term goal is to significantly impact the fundamental knowledge of muscle biology and provide new approaches for disease treatment. Striated muscle fibers are large multinucleated cells and possess a highly organized cytoarchitecture containing organelles positioned for optimal muscle function. This positioning is particularly evident in the placement of myonuclei, which reside above the sarcomere at the periphery of the myofiber and are positioned to maximize their internuclear distance. Our objective is the identification of mechanisms responsible for myonuclear movement and positioning. Centrally located myonuclei have been used for decades as a hallmark of muscle disease. However, much remains to be learned about the mechanisms that control myonuclear movement normally and the contribution of aberrant myonuclear position to the etiology and/or progression of muscle disease. Building on our published results over funding period (e.g. Metzger et al., 2012; Folker et al., 2012, 2014; Schulman et al., 2013, 2014; Azevedo et al., 2016; Manhart et al., 2018, 2020; Rosen et al., 2019), our specific aims are to first address mechanistically how tendon and motoneurons signal to the myofiber to fine-tune myonuclear positioning. We identified two signaling pathways at the myotendinous junction that regulate nuclear positioning. Likewise, we will dissect the contribution of the motoneuron to nuclear placement. Secondly, we will examine why muscles fail to function optimally when myonuclei are mispositioned. Muscle physiology will be assayed through testing mitochondrial function via quantification of ATP and ROS levels and by testing neuromuscular communication via electrophysiological approaches. We will also investigate the input to muscle function of specific metabolic and signaling proteins that we identified are misregulated as a result of mispositioned myonuclei. Thirdly, we will push forward our dissection of the myonuclear positioning mechanisms from fly to the human system. We will employ human 3D muscle cultures that are co-cultured with motoneurons to define and then perturb myonuclear movement and positioning as the myofibers develop. Our methodologies take advantage of cutting edge, in vivo time lapse imaging approaches that we have developed in Drosophila and will also apply to human 3D cultures to follow myonuclear movement and cytoskeletal dynamics. We will employ the genetic resources available in Drosophila and human cultures to manipulate genes, processes, and cell types for our analyses. These genetic experiments will be supported by biochemical and cell biological approaches. Together the work outlined in this proposal will shed new light on this little understood, but important area of muscle biology. The results of this research will permit us to highlight genes and mechanisms that are candidates for changes associated with different human muscle diseases.