Amplitude and curvature development at the switch-points of the beat cycle in bull sperm are augmented by ADP and reduced by ATP that is un-complexed with Mg2+.
Kathleen A. Lesich, Tania G. dePinho, and Charles B. Lindemann
We investigated the roles of ADP, Mg2+, and ATP in the beat cycle of Triton X-100- extracted bull sperm models. We recorded the motility of reactivated sperm models that were firmly attached to a glass surface by their heads with their flagella beating freely. The curvature of the flagellum at ~7 µm from the head tail junction was measured on images that were closest to the point of beat direction reversal. These images corresponded to the principal and reverse bend switch-points. The amplitude of the beat frequency was measured by tracking the excursion of a point on the flagellum 20 µm from the head tail junction relative to the origin on the midline axis of the sperm head when the flagellum is straight. Beat frequencies were hand calculated. The curvature at the switch-point, the amplitude at 20 µm and the beat frequency provided a quantitative basis to evaluate changes of the beat cycle in response to changes in ADP, Mg2+, and ATP concentration. We found that bull sperm exhibit well-developed beat cycles at all concentrations of Mg-ATP between 0.1 mM and 10 mM, but only if the concentrations of Mg2+ and ATP are equal. ATP in excess of the Mg2+ concentration resulted in a reduction of beat amplitude and curvature at the switch-points, and increased beat frequency. A concentration of 4 mM or greater ATP combined with 1 mM Mg2+ effectively terminated the beat cycle by reducing amplitude and curvature to extinction. Increasing ADP from 0 to 4 mM increased the amplitude and curvature developed at the switch-points and offset the reduction in amplitude and curvature produced by high ATP with Mg2+ held at 1 mM. This suggests that ADP has a strong effect on elevating the curvature and amplitude of the beat that is independent of Mg2+ concentration. The rescue of the beat by ADP occurs with a reduction in frequency. Therefore, the beat cycle is restored in spite of a slowing of the dynein cross-bridge cycle. These results can best be understood in the context of the geometric clutch (GC) hypothesis. In the GC hypothesis, switching occurs when the transverse force overcomes the adhesion of the dyneins onto their adjacent doublet. If ADP increases that adhesion by increasing dynein microtubule binding affinity and ATP un-complexed with Mg2+ has the opposite effect, then the balance between these factors will regulate the switch-point for dynein release in the beat cycle. Support: NSF MCB-0918294.