Mechanisms and Function of Myonuclear Positioning

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.

Grant Number: 5R01AR068128-10
NIH Institute/Center: NIH
Principal Investigator: MARY BAYLIES