DAPC structural adaption in regulating nanotopography-responsive myotube orientation

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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.