Calaxin is required for asymmetric bend initiation and propagation in sperm flagella

Abstract

Regulation of waveform asymmetry in flagella is critical for changes in direction when sperm are swimming, as seen during the chemotaxis of sperm towards eggs. Ca²⁺ is an important regulator of asymmetry in flagellar waveforms. A calcium sensor protein, calaxin, is associated with the outer arm dynein and plays a key role in the regulation of flagellar motility in a Ca²⁺-dependent manner. However, the underlying mechanism of regulating asymmetric waves by means of Ca²⁺ and calaxin remains unclear. To clarify the calaxin-dependent mechanism for generating Ca²⁺-dependent asymmetric flagellar waveforms, we analyzed the initial step of flagellar bend formation and propagation in the sperm of the ascidian Ciona intestinalis. Our experiment used demembranated sperm cells, which were then reactivated by UV flash photolysis of caged ATP under both high and low Ca²⁺ concentrations. Here, we show that initial bends in the flagella are formed at the base of the sperm and propagate towards the tip during waveform generation. However, the direction of the initial bend differed between asymmetric and symmetric waves. When a calaxin inhibitor (repaglinide) was applied, it resulted in the failure of asymmetric wave formation and propagation. This was because repaglinide had no effect on initial bend formation, but it significantly inhibited the generation of the subsequent bend in the reverse direction. Switching of dynein sliding activity by mechanical feedback is crucial for flagellar oscillation. Our results suggest that the Ca²⁺/calaxin mechanism plays an important role in the switching of dynein activity from microtubule sliding in the principal bend into the suppressed sliding in the reverse bend, thereby allowing the sperm to successfully change direction.

Keywords: caged ATP; calcium ion; cilia; dynein; sperm motility.

FIGURE 1

Estimation of the concentration of ATP released from caged ATP. (A) Beat frequency of demembranated Ciona sperm incubated with 1 mM caged ATP and reactivated by a 150 ms UV flash. N = 11 (pCa10), and N = 12 (pCa5). (B) Beat frequency of demembranated and reactivated Ciona sperm by various concentrations of Mg-ATP in the absence of caged ATP. Closed and open circles show Ca²⁺ concentrations in the reactivation solutions at pCa10 and pCa5, respectively. N = 13–28 from three different experiments. Values are expressed as mean ± S.D.

FIGURE 2

Generation of symmetric and asymmetric flagellar waveforms in Ciona sperm by photolytic release of caged ATP. Sperm cells were demembranated and incubated in the reactivation solution with caged ATP at low (pCa10; (A)) or high (pCa5; (B)) Ca²⁺ concentrations. Release of ATP was triggered by UV flash. Upper panel: sequential images of sperm flagellar waveforms at 15 ms-intervals from 100 ms after the UV flash. Arrow heads and arrows indicate the initial bend and the second bend, respectively. Scale bar, 20 μm. Middle panel: overwritten images of demembranated sperm at 5 ms intervals during the periods of time (105–250 ms) after release of ATP. Arrow heads and arrows indicate the initial bend and the second bend, respectively. Lower panel: changes of flagellar curvature occurring 105–250 ms after the UV flash are plotted against the distance from the base of flagellum. Ten waveforms produced at 50 ms are overwritten. Symmetric and asymmetric waveforms were generated in low calcium concentrations (pCa10, (A)) and high calcium concentrations (pCa10, (B)), respectively. One typical sperm example from the all experiments at least three times with three different specimens was shown.

FIGURE 3

Effect of a calaxin inhibitor on the formation and propagation of asymmetry waveforms induced by photolytic release of caged ATP at high calcium concentrations. Sperm cells were demembranated and incubated in the reactivation solution with caged ATP at low (pCa10; (A)) or high (pCa5; (B)) Ca²⁺ concentrations with 150 μM repaglinide. Release of ATP was triggered by UV flash. Upper panel: sequential images of sperm flagellar waveforms at 15 ms-intervals from 100 ms after the UV flash. Arrow heads and arrows indicate the initial bend and the second bend, respectively. Scale bar, 20 μm. Middle panel: overwritten images of demembranated sperm at 5 ms intervals during the periods of time (105–250 ms) after release of ATP. Arrow heads and arrows indicate the initial bend and the second bend, respectively. Lower panel: changes of flagellar curvature occurring 105–250 ms after the UV flash are plotted against the distance from the base of flagellum. Ten waveforms produced at 50 ms are overwritten. One typical sperm example from the all experiments at least three times with three different specimens was shown.

FIGURE 4

Preference for the direction of initial bend in the generation of symmetric and asymmetric flagellar waveforms. The relative frequencies of principal (P) and reverse (R) bends are shown for the initial bends formed after activation of demembranated Ciona sperm. Sperm was reactivated by photolysis of caged ATP in low (pCa10) or high (pCa5) Ca²⁺ concentrations in the presence of 0.5% DMSO (control) or 150 μM repaglinide. In both control and repaglinide-treated sperm, the formation of flagellar bends started with R-bends at pCa10, whereas it started with P-bends at pCa5. The number in brackets represents the number of observed spermatozoa in three different experiments.

FIGURE 5

Pseudocolor maps showing spatiotemporal changes of the flagellar curvature. Left panels: the flagellar curvature of demembranated Ciona sperm was plotted against the distance from the base and time after the UV flash by pseudocolor mapping. Right panels: the flagellar curvature at 5 μm from the base was plotted against time after the UV flash. Sperm was reactivated through photolysis of caged ATP in low pCa10; (A, C) or high pCa5; (B, D) Ca²⁺ concentrations in the presence of 0.5% DMSO control; (A, B) or 150 μM repaglinide (C, D).

FIGURE 6

A model for the formation and propagation of the first bend and second bend in symmetric and asymmetric sperm flagellar waves. (A) Schematic representation for the R- and P-sliding in relation to the number of active dynein on doublet microtubule. Based on the studies of sea urchin and mammalian sperm. The formation of P-bends and R-bends is induced by the activation of dyneins on doublet 7 and 3, respectively. (B) A putative mechanism for initial bend formation and its propagation under low and high Ca²⁺ conditions are shown. Left, formation of a symmetric wave under low Ca²⁺ conditions. The first bend is formed by R-sliding. In turn, this R-bend induces switching of active dynein to that on the opposite side across the axoneme, resulting in P-sliding to form a P-bend. Right, generation of an asymmetric wave under high Ca²⁺ conditions. P-bend is first formed by P-sliding. In turn, this P-bend induces the switching of active dynein to that on the opposite side across the axoneme and generates suppressed R-sliding to form a R-bend with smaller curvature, resulting in the propagation of an asymmetric wave. A calaxin inhibitor, repaglinide, suppresses the generation of R-bend, possibly by inhibiting the mechanical transmission from P- to R-sliding.