DAPC structural adaption in regulating nanotopography-responsive myotube orientation

项目摘要 Dystrophin-Associated-Protein-Complex（DAPC）是一种重要的跨膜蛋白复合物， 在结构上、机械上和功能上连接细胞骨架和细胞外基质， 调节肌肉和非肌肉细胞的机械和信号枢纽。影响DAPC的突变 这些成分与广泛的疾病如肌营养不良症有关。虽然有 关于DAPC组成的丰富知识，DAPC如何感知和适应的机制 生物化学，机械和地形线索在细胞微环境，从而调节细胞 表型和功能仍然未知。我们最近发现， 脊/槽并用基质胶或层粘连蛋白功能化的细胞微环境提供了工程化的细胞微环境， 使来自未患病的人诱导多能干细胞（hiPSC）的肌管几乎对齐 垂直于脊，而肌管来自受影响较小和受影响较小的Duchenne 肌营养不良症（一种由肌营养不良蛋白突变引起的遗传性疾病，通常导致患者死亡， 由于心脏和呼吸衰竭，年龄在20- 30岁）的细胞在排列和 方向，提供敏感的表型生物标志物来区分这些细胞，这可以作为 用于高通量疗法开发的表型读出。我们的初步数据表明， 纳米地形响应肌管方向是由DAPC调节;然而，如何的细节， nanotopography通过DAPC调节肌管取向尚不清楚。我们假设 垂直肌管取向是由各向异性DAPC对肌管的结构适应引起的。 纳米地形以保持其稳定性。我们希望超分辨率单分子定位 显微镜（SMLM）将使我们能够检查DAPC纳米结构及其在生物体内的结构适应性。 对纳米形貌的反应，并验证我们的假设。该项目的成功完成将揭示 DAPC前所未有的纳米级细节，DAPC结构对纳米形貌的适应，以及 纳米地形响应肌管取向的潜在机制，目前都是未知的。 本研究为今后联合收割机SMLM与生物材料工程相结合， 阐明更多的机制细节背后的DAPC介导的力传递，机械传感，和 在正常肌肉功能和各种肌肉和神经肌肉中的机械化学转导 紊乱验证DAPC在调节纳米地形响应性肌管取向中的作用 可以将其作为与DAPC缺陷相关的许多其他肌营养不良症的表型生物标志物 件.这项研究还将揭示新的工程方法来调节细胞行为和细胞命运 通过DAPC的地形控制。

Project Summary The Dystrophin-Associated-Protein-Complex (DAPC) is an important transmembrane protein complex that structurally, mechanically, and functionally links cytoskeleton and the extracellular matrix and serves as both mechanical and signaling hubs in regulating muscle and non-muscle cells. Mutations affecting DAPC components are associated with a wide range of diseases such as muscular dystrophies. Although there is a wealth of knowledge on the DAPC composition, the mechanisms underlying how the DAPC senses and adapts to biochemical, mechanical, and topographic cues in cell microenvironments and consequently regulates cell phenotypes and functions remain unknown. We recently discovered that substrates patterned with submicron ridges/grooves and functionalized with Matrigel or laminin present an engineered cell microenvironment to allow myotubes derived from non-diseased human induced pluripotent stem cells (hiPSCs) to align nearly perpendicular to the ridges, while myotubes derived from less-affected and severely-affected Duchenne Muscular Dystrophy (a genetic disorder resulting from mutations in dystrophin and often leading to death at an age of 20-30s due to cardiac and respiratory failure) cells exhibit prominent differences in alignment and orientation, providing a sensitive phenotypic biomarker to distinguish these cells, which may serve as a phenotypic readout for high throughput therapy development. Our preliminary data suggest that this nanotopography-responsive myotube orientation is regulated by the DAPC; however, details of how nanotopography regulates myotube orientation through the DAPC are unclear. We hypothesize that the perpendicular myotube orientation is caused by structural adaption of the anisotropic DAPC on the nanotopography to remain its stability. We expect that super-resolution Single Molecule Localization Microscopy (SMLM) will enable us to examine the DAPC nanoarchitecture and its structural adaption in response to nanotopography and verify our hypothesis. Successful accomplishment of this project will reveal unprecedented nanoscale details of the DAPC, DAPC structural adaption on nanotopography, and the mechanism underlying the nanotopography-responsive myotube orientation, which are all unknown currently. This project will lay the foundation for future studies that combine SMLM and biomaterials engineering to elucidate more mechanistic details underlying DAPC-mediated force transmission, mechanosensing, and mechanochemical transduction in normal muscle function and in various muscular and neuromuscular disorders. Verification of the role of the DAPC in regulating nanotopography-responsive myotube orientation may extend it as a phenotypic biomarker for many other muscular dystrophies associated with defective DAPC components. This study will also reveal novel engineering approaches to regulate cell behavior and cell fate through topographic control of the DAPC.