A protocol for single molecule imaging and tracking of processive myosin motors

Area of Science:

• Biophysics
• Cell Biology
• Molecular Motors

Background:

• Myosin motors are essential for intracellular transport.
• Understanding myosin motor function requires precise biophysical measurements.

Purpose of the Study:

• To describe protocols for single myosin motor tracking and analysis.
• To enable derivation of fundamental biophysical parameters.
• To provide methods adaptable for different motor proteins.

Main Methods:

• Labelling single myosin motors with quantum dots.
• Tracking motors in an in vitro single-molecule motility assay.
• Acquiring and analyzing image stacks to obtain motor trajectories.
• Utilizing an ensemble actin gliding assay for motor viability testing.

Main Results:

• Detailed steps for obtaining single myosin motor trajectories are provided.
• Fundamental biophysical parameters (velocity, run length, step size) can be derived.
• Protocols allow investigation of nucleotide, ion, and buffer effects on motor properties.

Conclusions:

• The described protocols facilitate in-depth analysis of single myosin motor dynamics.
• These methods are versatile and generalizable to other motor proteins.
• The study provides a valuable toolkit for motor protein research.