Kinesin force generation measured using a centrifuge microscope sperm-gliding motility assay

Area of Science:

• Biophysics
• Cell Biology
• Molecular Motors

Background:

• Kinesin motors are crucial for intracellular transport.
• Understanding kinesin force generation is key to cell motility.
• Sperm motility assays provide insights into molecular motor function.

Purpose of the Study:

• To develop a novel centrifuge microscope assay for measuring kinesin force.
• To characterize the force-velocity relationship in kinesin-driven sperm motility.
• To investigate factors influencing force generation and detachment in sperm.

Main Methods:

• Development of a centrifuge microscope sperm-gliding motility assay.
• Measurement of maximum isometric force using the developed assay.
• Estimation of maximum isometric force using a laser trap-based assay.
• Analysis of sperm axoneme behavior under centrifugal force.

Main Results:

• The centrifuge microscope assay yielded an extrapolated maximum isometric force of 0.90 ± 0.14 pN at low kinesin density.
• Sperm detached between 0.40 and 0.75 pN in the centrifuge assay.
• Laser trap assay estimated maximum isometric force at 4.34 ± 1.5 pN.
• Axoneme stiffness values suggest lever arm function and force magnification in the centrifuge assay.

Conclusions:

• The sperm axoneme acts as a lever arm, magnifying centrifugal forces and causing detachment before stall.
• Increased drag on the axoneme due to proximity to the glass surface adds to the forces kinesin must overcome.
• The developed assay provides a new method for studying kinesin motor mechanics in a biological context.