The polarized total internal reflection fluorescence microscopy (polTIRFM) processive motility assay for myosin V

Area of Science:

• Biophysics
• Molecular Motors
• Microscopy Techniques

Background:

• Single-molecule studies are crucial for understanding molecular mechanisms.
• Polarized total internal reflection fluorescence microscopy (polTIRFM) offers insights into molecular orientation.
• Myosin V is a key motor protein involved in intracellular transport.

Purpose of the Study:

• To describe a processive motility assay for investigating myosin V in vitro.
• To demonstrate the application of polTIRFM for determining single-molecule angular orientation and dynamics.
• To provide a generalizable protocol for single-molecule experiments involving angular changes.

Main Methods:

• Utilizing polarized total internal reflection fluorescence microscopy (polTIRFM).
• Employing a processive motility assay with biotin-Alexa actin filaments and myosin V.
• Tracking the orientation of a rhodamine-calmodulin (CaM) probe bound to myosin V as it moves along actin.

Main Results:

• The study details a method to determine the 3D angular orientation and wobble of a fluorescent probe attached to a macromolecule.
• The motility of myosin V was investigated in vitro using this technique.
• The protocol ensures proper calmodulin (CaM) levels for accurate myosin V function.

Conclusions:

• The described polTIRFM-based motility assay is effective for studying myosin V's in vitro movement.
• This technique provides detailed information on single-molecule orientation and dynamics.
• The methodology is adaptable for other single-molecule studies where angular dynamics are mechanistically important.