Mechanics of the eukaryotic flagellar axoneme: Evidence for structural distortion during bending.

The sliding doublet mechanism is the established explanation that allows us to understand the process of ciliary and flagellar bending. In this study, we apply the principles of the sliding doublet mechanism to analyze the mechanics of the counterbend phenomenon in sea urchin sperm flagella. When a passive, vanadate-treated, flagellum is forced into a bend with a glass microprobe, the portion of the flagellum distal to the probe exhibits a bend of opposite curvature (counterbend) to the imposed bend. This phenomenon was shown to be caused by the induction of inter-doublet shear and is dependent on the presence of an inter-doublet shear resistance. Here we report that in sea urchin flagella there is systematically less shear induced in the distal flagellum than is predicted by the sliding doublet mechanism, if we follow the assumption that the diameter of the flagellum is uniform. To account for the reduced shear that is observed, the likeliest and most direct interpretation is that the portion of the axoneme that is forced to bend undergoes substantial compression of the axoneme in the bending plane. A compression of 30-50 nm would be sufficient to account for the shear reduction from a bend of 2 radians. A compression of this magnitude would require considerable flexibility in the axoneme structure. This would necessitate that the radial spokes and/or the central pair apparatus are easily compressed by transverse stress. © 2016 Wiley Periodicals, Inc.

Ultrastructural evidence that motility changes caused by variations in ATP, Mg2+ , and ADP correlate to conformational changes in reactivated bull sperm axonemes.

We report the results of an ultrastructural study of Triton X-100-extracted, Mg-adenosine triphosphate (ATP)-reactivated bull sperm. We utilized a rapid fixation method to look for differences in the flagellar apparatus that correlate to the state of motility of reactivated sperm models. In a companion article, we examined the motility characteristics induced in bull sperm models by varying the concentration ratio of ATP and Mg(2+) and the stabilizing effect of adenosine diphosphate (ADP) on coordinated beating. Based on the results of that report, we selected four dissimilar states that appeared to represent extremes. One reactivation condition produces vigorous motility similar to live sperm, another produces large amplitude, low frequency beating while the remaining two conditions produce small amplitude vibrations of the flagellum with little coordinated beating. Morphometric analysis of transmission electron micrographs of sperm from these four treatment conditions revealed statistically significant differences between the samples in regard to axoneme diameter, inter-microtubule doublet spacing, and outer dense fiber (ODF) spacing. Our results show that Mg(2+) decreases the axoneme diameter and reduces interdoublet spacing, while ATP, uncomplexed with Mg(2+) , had the opposite effect. We also provide supporting evidence that this may be due to Mg(2+) increasing, and ATP decreasing, the interdoublet adhesion of dynein. We also found that 4 mM ADP significantly increases the separation between the ODFs and the space between the ODFs and the central axoneme within the middle piece. We present a hypothetical explanation that is consistent with our results to explain how ATP, ADP, and Mg(2+) act to regulate the beat cycle. © 2014 Wiley Periodicals, Inc.

The physiological role of ADP and Mg2+ in maintaining a stable beat cycle in bull sperm.

Sperm flagella derive their motive power from the motor protein dynein. In this study, we show that maintenance of the flagellar beat cycle in detergent-extracted bull sperm models is highly dependent on the ratio of Mg(2+) to adenosine triphosphate (ATP). An excess of either ATP un-complexed with Mg(2+) , or an excess of Mg(2+) without an equivalent concentration of ATP, results in the loss of beat amplitude and a reduced curvature development in the beat cycle. In addition, we find that adenosine diphosphate (ADP) can stabilize the beat cycle and permit rhythmic beating across a broader range of ATP and Mg(2+) concentrations. We provide evidence that suggests that when ATP is un-complexed with Mg(2+) , it disrupts the beat cycle by reducing dynein adhesion and thereby, reduces the transmission of dynein-generated force between the doublets. Excess Mg(2+) does not act by the same mechanism and induces a condition where the flagellum is more resistant to bending. This is consistent with the idea that high Mg(2+) stabilizes rigor bridges, and ATP reduces the microtubule binding affinity of dynein. Our results may explain how intact sperm are able to sustain coordinated flagellar beating under a wide range of metabolic conditions, as intact sperm produce ADP in direct proportion to their consumption of ATP.

The effects of Ca2+ and ADP on dynein switching during the beat cycle of reactivated bull sperm models.

Calcium regulation of flagellar motility is the basis for chemotaxis, phototaxis, and hyperactivation responses in eukaryotic flagellates and spermatozoa. Ca2+ is the internal messenger for these responses, but the coupling between Ca2+ and the motor mechanism that generates the flagellar beat is incompletely understood. We examined the effects of Ca2+ on the flagellar curvature at the switch-points of the beat cycle in bull sperm. The sperm were detergent extracted and reactivated with 0.1 mM adenosine triphosphate (ATP). With their heads immobilized and their tails beating freely it is possible to calculate the bending torque and the transverse force acting on the flagellum at the switch-points. An increase in the free Ca2+ concentration (pCa 8 to pCa 4) significantly decreased the development of torque and t-force in the principal bending direction, while having negligible effect on the reverse bend. The action of Ca2+ was more pronounced when the sperm were also treated with 4 mM adenosine diphosphate (ADP); it was sufficient to change the direction of bending that reaches the greater curvature. We also observed that the curvature of the distal half of the flagellum became locked in one direction in the presence of Ca2+ . This indicates that a subset of the dynein becomes continuously activated by Ca2+ and fails to switch with the beat cycle. Our evidence suggests this subset of dyneins is localized to doublets #1-4 of the axoneme.