Probing myosin structural conformation in vivo by second-harmonic generation microscopy.

Area of Science:

• Biophysics
• Molecular Biology
• Cellular Dynamics

Background:

• Understanding complex biological processes necessitates knowledge of molecular structures and in vivo dynamics.
• Myosin molecules, acting as molecular motors in muscle sarcomeres, exemplify this need for studying chemomechanical action.

Purpose of the Study:

• To develop and validate a label-free imaging method for assessing protein conformation in vivo.
• To investigate the structural states of actomyosin motors using second-harmonic generation (SHG).

Main Methods:

• Utilized order-based contrast enhancement by SHG for functional imaging of muscle cells.
• Employed SHG polarization anisotropy (SPA) measurements to probe myosin conformation.
• Reconstructed SHG emitters at an atomic scale for molecular interpretation of SPA data.

Main Results:

• SPA measurements demonstrated high sensitivity to myosin conformation across different physiological states (relaxed, rigor, isometric contraction).
• Distinct SPA values were observed for each probed state, indicating structural changes in actomyosin motors.
• Successfully discriminated between attached and detached myosin heads in isometrically contracting muscle fibers.

Conclusions:

• SPA measurements provide a molecular interpretation of myosin conformations in vivo.
• Isometrically contracting muscle maintains force through a steady-state commitment of approximately 30% of myosin heads.
• This label-free imaging approach using SPA and molecular modeling offers a foundation for new in vivo diagnostic tools for molecular structures and dynamics.