Mechanisms and Function of Myonuclear Positioning

描述（由申请人提供）：我们的长期目标是显著影响肌肉生物学知识，并为疾病治疗提供新方法。横纹肌纤维是一种大的多核细胞，具有高度组织化的细胞结构，其中包含最佳肌肉功能的细胞器。这种定位在肌核的位置上尤其明显，它位于肌纤维周围的肌节上方，并被定位以最大化其核间距离。我们的目标是确定负责核运动和定位的机制。几十年来，位于中心的肌核一直被用作肌肉疾病的标志。然而，关于控制肌核正常运动的机制，或异常肌核位置对肌肉疾病的病因和/或进展的贡献知之甚少。基于我们最近发表的研究结果（Metzger et al., Nature, 2012; Folker et al., Development, 2012），我们的具体目标是描述参与肌核定位的新基因，解决肌腱和运动神经元如何在肌肉功能过程中微调肌核定位，并研究为什么肌核定位错误时肌肉不能发挥最佳功能。该建议将确定由异常核放置引起的生理变化，为检查/治疗肌肉疾病提供新的生物标志物/治疗靶点。最后，这些数据将阐明在发育和生长过程中肌纤维细胞结构的组织是如何实现的。我们的调查将主要在果蝇中进行；然而，我们将在哺乳动物肌肉培养中测试我们的范式。我们的方法利用了我们在果蝇身上开发的最前沿的体内延时成像技术来跟踪核运动和细胞骨架动力学。我们将利用果蝇中可用的遗传资源来操纵基因，过程和细胞类型以进行我们的分析。这些基因实验将得到生化和细胞生物学方法的支持。肌肉生理学将通过量化ATP和ROS水平来分析线粒体输出，包括使用一种新型的ROS传感器用于后者，以及神经肌肉通信，重要的是肌肉细胞输出，通过电生理学方法来研究。基因组学方法，特别是RNAseq，将揭示肌核位置不当导致的肌肉转录组变化。总之，本提案中概述的工作将为肌肉生物学这个鲜为人知但重要的领域提供新的启示。这项研究的结果将使我们能够突出与不同人类肌肉疾病相关的候选变化的基因和机制。

DESCRIPTION (provided by applicant): Our long-term goal is to significantly impact the 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, little is known about the mechanisms that control myonuclear movement normally, or the contribution of aberrant myonuclear position to the etiology and/or progression of muscle disease. Building on our recently published results (Metzger et al., Nature, 2012; Folker et al., Development, 2012), our specific aims in this proposal are to characterize new genes involved in myonuclear positioning, address how tendon and motorneurons fine-tune myonuclear positioning during muscle function, and investigate why muscles fail to function optimally when myonuclei are mispositioned. This proposal will identify physiological changes that result from aberrant nuclear placement, providing new biomarkers/therapeutic targets to examine/treat muscle disease. Lastly, these data will shed light on how the organization of the muscle fiber cytoarchitecture is achieved during development and growth. Our investigation will be primarily carried out in Drosophila; however, we will test our paradigm in mammalian muscle cultures. Our methodologies take advantage of cutting edge, in vivo time lapse imaging that we have developed in Drosophila to follow myonuclear movement and cytoskeletal dynamics. We will employ the genetic resources available in Drosophila to manipulate genes, processes, and cell types for our analyses. These genetic experiments will be supported by biochemical and cell biological approaches. Muscle physiology will be investigated by assaying mitochondrial output via quantification of ATP and ROS levels, including using a novel ROS sensor for the latter, and neuromuscular communication, and importantly muscle cellular output, via electrophysiological approaches. Genomic approaches, specifically RNAseq, will reveal changes in the muscle transcriptome as a result of improper myonuclear position. 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.