Measurement of the Force Produced by an Intact Bull Sperm Flagellum in Isometric Arrest and Estimation of the Dynein Stall Force

Abstract

The force generated by a detergent-extracted reactivated bull sperm flagellum during an isometric stall was measured with a force-calibrated glass microprobe. The average isometric stall force from 48 individual measurements was 2.5 +/- 0.7 x 10(-5) dyne (2.5 +/- 0.7 x 10(-10) N). The force measurements were obtained by positioning a calibrated microprobe in the beat path of sperm cells that were stuck by their heads to a glass microscope slide. The average position of the contact point of the flagellum with the probe was 15 microm from the head-tail junction. This average lever arm length multiplied by the measured force yields an estimate of the active bending moment (torque) of 3.9 x 10(-8) dyne x cm (3.9 x 10(-15) N x m). The force was sustained and was for the most part uniform, despite the fact that the flagellum beyond the point of contact with the probe usually continued beating. It appears that the dynein motors in the basal portion of the flagellum continue to pull in an isometric stall for as long as the motion of the flagellum is blocked. If dynein motors in the flagellum distal to the contact point with the probe were contributing force to the displacement of the probe, then the flagellar segment immediately past the point of contact would have to show a net curvature in the direction of the probe displacement. No such curvature bias was observed in the R-bend arrests, and only a small positive curvature bias was measured in the P-bend arrests. Our analysis of the data suggests that more than 90% of the sustained force component is generated by the part of the flagellum between the probe and the flagellar base. Based on this premise, the isometric stall force per dynein head is estimated to be 5.0 x 10(-7) dyne (5 pN). This equals approximately 1.0 x 10(-6) dyne (10 pN) per intact dynein arm. These values are close to the isometric stall force of isolated dynein. This suggests that all of the dynein heads between the base and the probe, on the active side of the axoneme, are contributing to the force exerted against the probe.

Does axonemal dynein push, pull, or oscillate?

“…The elasticity of the B-link is sufficient to support this mechanism up to a limit of about 3.8 pN, at which point rotation of the B-link would produce zero displacement (the motor is stalled). Measurements of the dynein force by laser trap [Shingyoji et al, 1998;Sakakibara et al, 1999;Hirakawa et al, 2000] and estimates from whole flagella [Schmitz et al, 2000] suggest that the maximum force per dynein is approximately 5 pN per head. Therefore, most of the force could be generated by lateral displacement of the B-links.…”

The geometric clutch at 20: stripping gears or gaining traction?

“…At the most basic level, if t-force is responsible for dynein switching, then preventing the development of t-force should interrupt the beat cycle. We tested this directly in bull sperm and showed that preventing the development of curvature by mechanically blocking the formation of a basal bend arrests the beat in an active, force-generating stall (Holcomb-Wygle et al 1999, Schmitz et al 2000. Shortening a bull sperm flagellum by clipping it to a length of 15 mm (30% of the total length) or less also stalls the beat at either endpoint of the beat cycle (Holcomb-Wygle et al 1999), as was predicted by the GC computer model (Lindemann & Kanous 1995).…”

Thin-foil magnetic force system for high-numerical-aperture microscopy

“…Among the mechanical probe techniques, glass fibers or microneedles [13][14][15] have been used to measure the effects of forces on the movement of chromosomes and the force exerted by myosin on actin. 16 More recently, atomic force microscopy has emerged as a suitable method to obtain subnanometer spatial resolution 17 with piconewton force sensitivity 18 using techniques relying on the deformation of a cantilever spring element.…”