The load dependence of kinesin's mechanical cycle

Area of Science:

• Molecular Motor Proteins
• Cellular Transport Mechanisms
• Biophysics

Background:

• Kinesin is a dimeric motor protein essential for intracellular organelle transport along microtubules.
• Kinesin utilizes ATP hydrolysis to generate mechanical force, moving towards the microtubule plus-end.
• Previous studies indicated kinesin stalls under opposing loads of approximately 5 pN.

Purpose of the Study:

• To investigate the behavior of individual kinesin molecules under super-stall loads (>5 pN) and forward loads.
• To test theoretical predictions of kinesin direction reversal under high external forces.
• To elucidate the impact of load on kinesin's mechanical cycle and microtubule interactions.

Main Methods:

• Utilized an in vitro motility assay combined with a high-resolution optical trapping microscope.
• Applied super-stall and forward loads to individual kinesin molecules.
• Measured kinesin's velocity, directionality, and dissociation rates under varying load conditions and ATP concentrations.

Main Results:

• Kinesin did not reverse direction, even under loads up to 13 pN, suggesting an irreversible mechanical transition.
• Forward loads significantly accelerated the kinesin mechanical cycle across a range of ATP concentrations.
• Kinesin's dissociation rate from microtubules increased with applied load, supporting load-dependent kinetic pathways.

Conclusions:

• Kinesin's mechanical cycle exhibits robustness against high opposing loads, preventing backward movement.
• Forward loads can enhance kinesin's motor activity, offering potential for optimizing intracellular transport.
• Load-dependent partitioning between coordinated and independent head pathways influences kinesin's interaction with microtubules.