Altered nucleus-cytoskeleton coupling in dystrophic muscle

项目摘要： 杜氏肌营养不良症（DMD）是最常见和最严重的肌营养不良症， 以骨骼肌的逐渐萎缩和对损伤的明显敏感性为特征。几 相关的过程可能是病理学的基础。细胞核是基因表达的调节器， mechanotransduction hub，在mdx（DMD的鼠模型）肌肉中具有增加的运动。微管 (MTs)充当细胞器运输的“铁路”，包括细胞核。细胞核是 通过LINC（细胞核和细胞骨架的连接体）连接到MT和细胞骨架的其余部分 复杂. MT组织和LINC复合体表达在营养不良的肌肉中均发生改变。我将测试 假设由于疾病驱动的MT网络和LINC复合体改变而导致的核不稳定性， 不适当的肌电域维持（具有过度移动和不适当定位的核），以及受损 核机械转导，进一步驱动肌无力和易受损伤的营养不良 肌肉.在WT和mdx肌肉中，我将测量： 1)用延时显微镜观察核的空间分布和核的运动 2)通过测量运载蛋白的RNA空间分布来维持myocardial结构域， 荧光原位杂交;和核运动和全球转录活性后的差距， 使用激光烧蚀的肌层结构域 3)Yes相关蛋白（机械信号的核中继），ERK 1/2（一个关键的 肌肉生长的标志物）和基于FRET的核应变传感器，作为核应变的终点。 被动牵张、等长收缩和离心收缩后的机械传导 4)牵张阻断后肌纤维结构域的维持和核机械转导 激活通道阻断肌膜信号通路 5)上述参数使用已建立的遗传/药理学操作的MT网络和 LINC复合物，以及先前已显示完全或部分抑制 急救MT网络与损伤易感性 成功完成这项提案将有助于开发新的途径， DMD患者的肌肉骨骼健康，以及潜在的其他营养不良。该提案发生在一个 马里兰州医学院的多学科环境，在专家的支持下， 物理治疗，生理学，分子生物学，生物化学和工程学，这样我就可以获得技能， 细胞和肌肉力学走向一个独立的，终身制的研究教师的位置。

Project Summary:   Duchenne muscular dystrophy (DMD), the most common and severe form of muscular dystrophy, is characterized by progressive wasting of skeletal muscles and marked susceptibility to damage. Several associated processes could underlie the pathology. The nucleus, a regulator of gene expression and a mechanotransduction hub, has increased movement in mdx (murine model of DMD) muscle. Microtubules (MTs) serve as the “railroad tracks” for cellular organelle transport, including the nucleus. The nucleus is connected to MTs and the rest of the cytoskeleton through the LINC (linkers of nucleus and cytoskeleton) complex. Both, MT organization and LINC complex expression are altered in dystrophic muscle. I will test the hypothesis that nuclear instability, due to disease-driven MT network and LINC complex alterations, results in improper myonuclear domain maintenance (with hypermobile and improperly positioned nuclei), and impaired nuclear mechanotransduction, further driving muscle weakness and susceptibility to injury in dystrophic muscle. In WT and mdx muscle I will measure: 1) nuclear spatial distribution & nuclear movement using time-lapse microscopy 2) myonuclear domain maintenance by measuring RNA spatial distribution of cargoed proteins using fluorescence in-situ hybridization; and nuclear movement & global transcriptional activity following gaps in myonuclear domain using laser ablation 3) nuclear localization of Yes- associated protein (a nuclear relay of mechanical signaling), ERK 1/2 (a key marker of muscle growth) and FRET based nuclear strain sensors, as end points of nuclear mechanotransduction, following passive stretch, isometric and eccentric contractions 4) myonuclear domain maintenance and nuclear mechanotransduction, following blockage of stretch activated channels to block sarcolemmal signaling pathways 5) the above parameters using established genetic/pharmacologic manipulations to the MT network & the LINC complex, and following mini- and micro-dystrophins that have previously shown to either fully or partially rescue MT network and susceptibility to injury Successful completion of this proposal will allow for the development of new avenues to improve musculoskeletal health for patients with DMD, and potentially other dystrophies. This proposal takes place in a multi-disciplinary environment at University of Maryland School of Medicine, with support from experts in physical therapy, physiology, molecular biology, biochemistry, and engineering, such that I can gain skills in cellular and muscle mechanics to move towards an independent, tenure-track research faculty position.