An optimized approach to study nanoscale sarcomere structure utilizing super-resolution microscopy with nanobodies

PLoS One. 2024 Apr 30;19(4):e0300348. doi: 10.1371/journal.pone.0300348. eCollection 2024.

Abstract

The sarcomere is the fundamental contractile unit in skeletal muscle, and the regularity of its structure is critical for function. Emerging data demonstrates that nanoscale changes to the regularity of sarcomere structure can affect the overall function of the protein dense ~2μm sarcomere. Further, sarcomere structure is implicated in many clinical conditions of muscle weakness. However, our understanding of how sarcomere structure changes in disease, especially at the nanoscale, has been limited in part due to the inability to robustly detect and measure at sub-sarcomere resolution. We optimized several methodological steps and developed a robust pipeline to analyze sarcomere structure using structured illumination super-resolution microscopy in conjunction with commercially-available and fluorescently-conjugated Variable Heavy-Chain only fragment secondary antibodies (nanobodies), and achieved a significant increase in resolution of z-disc width (353nm vs. 62nm) compared to confocal microscopy. The combination of these methods provides a unique approach to probe sarcomere protein localization at the nanoscale and may prove advantageous for analysis of other cellular structures.

Publication types

  • Research Support, N.I.H., Extramural
  • Research Support, Non-U.S. Gov't

MeSH terms

  • Animals
  • Mice
  • Microscopy, Confocal / methods
  • Microscopy, Fluorescence / methods
  • Sarcomeres* / metabolism
  • Sarcomeres* / ultrastructure
  • Single-Domain Antibodies* / chemistry

Substances

  • Single-Domain Antibodies

Grants and funding

This work was supported by National Institutes of Health grant 5R01AR079220 to Karyn A Esser and National Institutes of Health grant 1R01AR079449 to Daniel Kopinke. The funders had no role in the study design, data collection and analysis, decision to publish, or preparation of the manuscript.