KINESIN FORCE GENERATION MEASURED USING A CENTRIFUGE MICROSCOPE SPERM-GLIDING MOTILITY ASSAY

To measure force generation and characterize the relationship between force and velocity in kinesin-driven motility we have developed a cent rifuge microscope sperm-gliding motility assay. The average (extrapola ted) value of maximum isometric force at low kinesin density was 0.90 +/- 0.14 pN. Furthermore, in the experiments at low kinesin density, s perm pulled off before stall at forces between 0.40 and 0.75 pN. To fu rther characterize our kinesin-demembranated sperm assay we estimated maximum isometric force using a laser trap-based assay. At low kinesin density, 4.34 +/- 1.5 pN was the maximum force, Using values of axone me stiffness available from other studies, we concluded that, in our c entrifuge microscope-based assay, a sperm axoneme functions as a lever arm, magnifying the centrifugal force and leading to pull-off before stall, In addition, drag of the distal portion of the axoneme is incre ased by the centrifugal force (because the axoneme is rotated into clo ser proximity to the glass surface) and represents an additional force that the kinesin motor must overcome.