Highly-Parallel Microfluidics-Based Force Spectroscopy on Single Cytoskeletal Motors

Area of Science:

• Molecular Biology
• Biophysics
• Cellular Mechanics

Background:

• Cytoskeletal motors are essential molecular machines converting chemical energy into mechanical work for cellular functions.
• Optical trapping is a key technique for studying molecular motors under load, but its throughput is limited.
• Existing force spectroscopy methods are time-consuming, measuring one motor at a time.

Purpose of the Study:

• To introduce a novel, highly-parallel microfluidics-based method for studying cytoskeletal motors.
• To achieve a significant improvement in throughput for collecting force-dependent motility parameters.
• To enable parallelized single-molecule force studies on molecular motors.

Main Methods:

• Developed a microfluidics platform for high-throughput single-molecule force measurements.
• Utilized tunable hydrodynamic forces applied to DNA-tethered beads attached to kinesin-1 motors.
• Employed a large field of view to simultaneously track hundreds of individual kinesin-1 motor parameters under varying loads.

Main Results:

• Achieved a two-orders-of-magnitude improvement in throughput for force-dependent motility parameter collection.
• Successfully tracked velocities, run lengths, and interaction times of hundreds of kinesin-1 motors concurrently.
• Demonstrated that long DNA tethers minimize unwanted vertical forces on motors.

Conclusions:

• The developed microfluidics method offers a powerful new tool for high-throughput single-molecule force studies.
• This approach significantly accelerates the collection of crucial data on molecular motor mechanics.
• The methodology is adaptable for investigating other molecular systems and motor proteins.