scholarly journals Calcium regulation of microtubule sliding in reactivated sea urchin sperm flagella

2000 ◽  
Vol 113 (5) ◽  
pp. 831-839 ◽  
Author(s):  
H. Bannai ◽  
M. Yoshimura ◽  
K. Takahashi ◽  
C. Shingyoji
Keyword(s):  
Sea Urchin ◽  
Regulatory Mechanism ◽  
Bend Angle ◽  
Sliding Velocity ◽  
Trypsin Treatment ◽  
Central Pair ◽  
Intracellular Ca ◽  
Asymmetric Bending ◽  
Sea Urchin Sperm ◽  
Similar Decrease

The changes in the bending pattern of flagella induced by an increased intracellular Ca(2+) concentration are caused by changes in the pattern and velocity of microtubule sliding. However, the mechanism by which Ca(2+) regulates microtubule sliding in flagella has been unclear. To elucidate it, we studied the effects of Ca(2+) on microtubule sliding in reactivated sea urchin sperm flagella that were beating under imposed head vibration. We found that the maximum microtubule sliding velocity obtainable by imposed vibration, which was about 170–180 rad/second in the presence of 250 microM MgATP and <10(−9) M Ca(2+), was decreased by 10(−6)-10(−5) M Ca(2+) by about 15–20%. Similar decrease of the sliding velocity was observed at 54 and 27 microM MgATP. The Ca(2+)-induced decrease of the sliding velocity was due mainly to a decrease in the reverse bend angle. When the plane of beat was artificially rotated by rotating the plane of vibration of the pipette that held the sperm head, the asymmetric bending pattern also rotated at 10(−5) M Ca(2+) as well as at <10(−9) M Ca(2+). The rotation of the bending pattern was observed at MgATP higher than 54 microM (approximately 100 microM ATP). These results indicate that the Ca(2+)-induced decrease of the sliding velocity is mediated by a rotatable component or components (probably the central pair) at high MgATP, but is not due to specific dynein arms on particular doublets. We further investigated the effects of a mild trypsin treatment and of trifluoperazine on the Ca(2+)-induced decrease in sliding velocity. Axonemes treated for 3 minutes with a low concentration (0.1 microgram/ml) of trypsin beat with a more symmetrical waveform than before the treatment. Also, their microtubule sliding velocity and reverse bend angle were not affected by high Ca(2+) concentrations. Trifluoperazine (25-50 microM) had no effect on the decrease of the sliding velocity in beating flagella at 10(−5) M Ca(2+). However, the flagella that had been ‘quiescent’ at 10(−4) M Ca(2+) resumed asymmetrical beating following an application of 10–50 microM trifluoperazine. In such beating flagella, both the sliding velocity and the reverse bend angle were close to their respective values at 10(−5) M Ca(2+). Trypsin treatment induced a similar recovery of beating in quiescent flagella at 10(-)(4) M Ca(2+), albeit with a more symmetrical waveform. These results provide first evidence that, at least at ATP concentrations higher than approximately 100 microM, 10(−6)-10(−5) M Ca(2+) decreases the maximum sliding velocity of microtubules in beating flagella through a trypsin-sensitive regulatory mechanism which possibly involves the central pair apparatus. They also suggest that calmodulin may be associated with the mechanism underlying flagellar quiescence induced by 10(−4) M Ca(2+).

scholarly journals Effect of beat frequency on the velocity of microtubule sliding in reactivated sea urchin sperm flagella under imposed head vibration.

1995 ◽  
Vol 198 (3) ◽  
pp. 645-653 ◽  
Author(s):  
C Shingyoji ◽  
K Yoshimura ◽  
D Eshel ◽  
K Takahashi ◽  
I R Gibbons
Keyword(s):  
Sea Urchin ◽  
Vibration Frequency ◽  
Beat Frequency ◽  
Distal Region ◽  
Bend Angle ◽  
Vibration Frequencies ◽  
Sliding Velocity ◽  
Flagellar Beat ◽  
Tripneustes Gratilla ◽  
Sea Urchin Sperm

The heads of demembranated spermatozoa of the sea urchin Tripneustes gratilla, reactivated at different concentrations of ATP, were held by suction in the tip of a micropipette and vibrated laterally with respect to the head axis. This imposed vibration resulted in a stable rhythmic beating of the reactivated flagella that was synchronized to the frequency of the micropipette. The reactivated flagella, which in the absence of imposed vibration had an average beat frequency of 39 Hz at 2 mmol l-1 ATP, showed stable beating synchronized to the pipette vibration over a range of 20-70 Hz. Vibration frequencies above 70 Hz caused irregular, asymmetrical beating, while those below 20 Hz induced instability of the beat plane. At ATP concentrations of 10-100 mumol l-1, the range of vibration frequency capable of maintaining stable beating was diminished; an increase in ATP concentration above 2 mmol l-1 had no effect on the range of stable beating. In flagella reactivated at ATP concentrations above 100 mumol l-1, the apparent time-averaged sliding velocity of axonemal microtubules decreased when the imposed frequency was below the undriven flagellar beat frequency, but at higher imposed frequencies it remained constant, with the higher frequency being accompanied by a decrease in bend angle. This maximal sliding velocity at 2 mmol l-1 ATP was close to the sliding velocity in the distal region of live spermatozoa, possibly indicating that it represents an inherent limit in the velocity of active sliding.(ABSTRACT TRUNCATED AT 250 WORDS)

Effect of imposed head vibration on the stability and waveform of flagellar beating in sea urchin spermatozoa

1991 ◽  
Vol 156 (1) ◽  
pp. 63-80 ◽  
Author(s):  
C. Shingyoji ◽  
I. R. Gibbons ◽  
A. Murakami ◽  
K. Takahashi
Keyword(s):  
Sea Urchin ◽  
Beat Frequency ◽  
Distal Region ◽  
Proximal Region ◽  
Bend Angle ◽  
Vibration Frequencies ◽  
Sliding Velocity ◽  
Additional Energy ◽  
The Stability ◽  
Cyclic Changes

The heads of live spermatozoa of the sea urchin Hemicentrotus pulcherrimus were held by suction in the tip of a micropipette mounted on a piezoelectric device and vibrated either laterally or axially with respect to the head axis. Within certain ranges of frequency and amplitude, lateral vibration of the pipette brought about a stable rhythmic beating of the flagella in the plane of vibration, with the beat frequency synchronized to the frequency of vibration [Gibbons et al. (1987), Nature 325, 351–352]. The sperm flagella, with an average natural beat frequency of 48 Hz, showed stable beating synchronized to the pipette vibration over a range of 35–90 Hz when the amplitude of vibration was about 20 microns or greater. Vibration frequencies below this range caused instability of the beat plane, often associated with irregularities in beat frequency. Frequencies above about 90 Hz caused irregular asymmetrical flagellar beating with a marked decrease in amplitude of the propagated bends and a skewing of the flagellar axis towards one side; the flagella often stopped in a cane shape. In flagella that were beating stably under imposed vibration, the wavelength was reduced at higher frequencies and increased at lower frequencies. When the beat frequency was equal to or lower than the natural beat frequency, the apparent time-averaged sliding velocity of axonemal microtubules, obtained as twice the product of frequency and bend angle, decreased with beat frequency in both the proximal and distal regions of the flagella. However, at vibration frequencies above the natural beat frequency, the sliding velocity increased with frequency only in the proximal region of the flagellum and remained essentially unchanged in more distal regions. This apparent limit to the velocity of sliding in the distal region may represent an inherent limit in the intrinsic velocity of active sliding, while the faster sliding observed in the proximal region may be a result of passive sliding or elastic distortion of the microtubules induced by the additional energy supplied by the vibrating pipette. Axial vibration with frequencies either close to or twice the natural beat frequency induced cyclic changes in the waveform, compressing and expanding the bends in the proximal region, but did not affect bends in the distal region or alter the beat frequency.

scholarly journals Properties of an antiserum against native dynein 1 from sea urchin sperm flagella.

1977 ◽  
Vol 73 (1) ◽  
pp. 182-192 ◽  
Author(s):  
K Ogawa ◽  
D J Asai ◽  
C J Brokaw
Keyword(s):  
Atpase Activity ◽  
Sea Urchin ◽  
Strongylocentrotus Purpuratus ◽  
Beat Frequency ◽  
Bend Angle ◽  
Tryptic Fragment ◽  
Effective Inhibition ◽  
Dynein Arms ◽  
Sea Urchin Sperm ◽  
Anthocidaris Crassispina

Effects of an antiserum against native dynein 1 from sperm flagella of the sea urchin Strongylocentrotus purpuratus were compared with effects of an antiserum previously obtained against an ATPase-active tryptic fragment (fragment 1A) of dynein 1 from sperm flagella of the sea urchin, Anthocidaris crassispina. Both antisera precipitate dynein 1 and do not precipitate dynein 2. Only the fragment 1A antiserum precipitates fragment 1A and produces a measurable inhibition of dynein 1 ATPase activity. Both antisera inhibit the movement and the movement-coupled ATP dephosphorylation of reactivated spermatozoa. The inhibition of movement by the antiserum against dynein 1 is much less than by the antiserum against fragment 1A, suggesting that a specific interference with the active ATPase site may be required for effective inhibition of movement. Both antisera reduce the bend angle as well as the beat frequency of reactivated S. purpuratus spermatozoa, suggesting that the bend angle may depend on the activity of the dynein arms which generate active sliding.

scholarly journals Inhibition of movement of trition-demembranated sea-urchin sperm flagella by Mg2+, ATP4-, ADP and P1

1979 ◽  
Vol 38 (1) ◽  
pp. 105-123
Author(s):  
M. Okuno ◽  
C.J. Brokaw
Keyword(s):  
Sea Urchin ◽  
Glass Surface ◽  
Beat Frequency ◽  
Bend Angle ◽  
Flagellar Motility ◽  
Reaction Products ◽  
Inhibitory Effects ◽  
Linear Relationships ◽  
Clinical Patterns ◽  
Sea Urchin Sperm

Three clinical patterns of inhibition of MgATP2—activated flagellar motility have been found by measuring the motility of Triton-demembranated sea-urchin spermatozoa beating with their heads attached to a glass surface. Inhibition of beat frequency by the reaction products, ADP and Pi, is competitive with the normal substrate, MgATP2-, and the inhibitory effects are similar to a reduction in MgATP2- concentration. Inhibition of beat frequency by ATP4- is competitive with MgATP2, but is accompanied by an inhibition of bending, as measured by the angle between the straight regions on either side of a bend, which is not seen when MgATP2- concentration is reduced. Inhibition of beat frequency by Mg2+ is not competitive with MgATP2-, and is accompanied by an increase in bend angle, so that there is no change in the rate of sliding between flagellar tubules. These differences suggest unexpected complexity of dynein ATPase action in flagella. The beat frequencies of both swimming and attached spermatozoa show a linear double reciprocal dependence on MgATP2- concentration, with identical slopes. The calculated sliding velocities between tubules also give linear relationships, but the slopes are different, suggesting that beat frequency may be the more fundamental dependent variable in this system.

Polarity in spontaneous unwinding after prior rotation of the flagellar beat plane in sea-urchin spermatozoa

1991 ◽  
Vol 98 (2) ◽  
pp. 183-189 ◽  
Author(s):  
K. Takahashi ◽  
C. Shingyoji ◽  
J. Katada ◽  
D. Eshel ◽  
I.R. Gibbons
Keyword(s):  
Angular Velocity ◽  
Sea Urchin ◽  
Initial Velocity ◽  
Central Pair ◽  
Counterclockwise Direction ◽  
Flagellar Beat ◽  
Elastic Distortion ◽  
The Difference ◽  
Sea Urchin Sperm

The flagellar beat plane of live and reactivated sea-urchin sperm held by their heads in the tip of a vibrating micropipette will rotate along with the plane of the imposed vibration for up to 10 revolutions in either a clockwise or a counterclockwise direction. Subsequent cessation of the imposed vibration is followed by spontaneous unwinding of the flagellar beat plane. Nearly complete unwinding occurs after prior counterclockwise winding. The unwinding of the beat plane after prior clockwise winding is incomplete, but the number of revolutions that remain unwound affects the response of the flagellar beat plane to a second set of imposed revolutions. The initial angular velocity of spontaneous unwinding is approximately proportional to the number of prior winding cycles, independent of their direction. The maximum initial velocity of unwinding was 27 rad s-1 and 20 rad s-1 for live and reactivated sperm, respectively. These data suggest that the force responsible for unwinding of the beat plane is derived from the elastic distortion of some component in the axonemal structure. The difference in completeness of spontaneous unwinding between the two directions of rotation is consistent with the previously suggested hypothesis that imposed rotation of the beat plane reflects the forced rotation of the central pair within the axoneme.

Modification of flagellar waveform and adenosine triphosphatase activity in reactivated sea-urchin sperm treated with N-ethylmaleimide

1983 ◽  
Vol 60 (1) ◽  
pp. 231-249
Author(s):  
M.P. Cosson ◽  
W.J. Tang ◽  
I.R. Gibbons
Keyword(s):  
Atpase Activity ◽  
Sea Urchin ◽  
Atp Hydrolysis ◽  
Beat Frequency ◽  
Prolonged Treatment ◽  
Bend Angle ◽  
The Asymmetry ◽  
Bend Angles ◽  
The Waves ◽  
Sea Urchin Sperm

Treatment of demembranated sea-urchin sperm for 1–2 min with 10 microM-N-ethylmaleimide (Mal-NEt) at pH 8.0 prior to reactivation with 1 mM-ATP causes the asymmetry of the flagellar waveform to become desensitized to the presence or absence of Ca2+ in the reactivating medium. In such sperm, changes in concentration of free Ca2+ between 10(−7) M and 10(−3) M have no effect on the asymmetry of the waveforms as measured by the turning rate of the sperm in radians per beat cycle, while the beat frequency and the propulsive efficiency of the waves remain unchanged from the values observed in control preparations not treated with MalNEt. A somewhat more prolonged treatment with MalNEt causes a progressive decrease in the bend angles of the flagellar waves, while the beat frequency and the wavelength still remain largely unchanged. Further extension of the treatment with MalNEt causes complete loss of motility. Little ATP-induced sliding of the doublet tubules is observed upon treatment with trypsin of sperm flagella that have been rendered non-motile with MalNEt. However, the preparations of solubilized dynein 1 obtained by 0.6 M-NaCl extraction of axonemes treated with MalNEt appear almost identical to those obtained from untreated axonemes, both in terms of the amount solubilized and in the specific ATPase activities of their latent and Triton-activated forms. These preparations also appear capable of restoring much of the beat frequency of dynein-1-depleted flagella. These results suggest that the observed desensitization to Ca2+ and decrease in bend angle result from the reaction of MalNEt with axonemal polypeptides that are not part of the dynein 1 particle extracted with 0.6 M-NaCl. The rate of ATP hydrolysis by demembranated sperm rendered non-motile with MalNEt remains relatively high, and it decreases about 50% when the flagella are broken by brief homogenization. This ‘homogenizer-sensitive’ ATPase activity appears to be derived from some flagellar regulatory mechanism, which controls the ATPase activity of intact non-motile axonemes.

scholarly journals Modulation of the asymmetry of sea urchin sperm flagellar bending by calmodulin.

1985 ◽  
Vol 100 (6) ◽  
pp. 1875-1883 ◽  
Author(s):  
C J Brokaw ◽  
S M Nagayama
Keyword(s):  
Sea Urchin ◽  
Gel Electrophoresis ◽  
Binding Activity ◽  
Calmodulin Binding ◽  
Bending Waves ◽  
Affinity Resin ◽  
The Asymmetry ◽  
Triton X ◽  
Asymmetric Bending ◽  
Sea Urchin Sperm

Sea urchin spermatozoa demembranated with Triton X-100 in the presence of EGTA, termed potentially asymmetric, generate asymmetric bending waves in reactivation solutions containing EGTA. After they are converted to the potentially symmetric condition by extraction with Triton and millimolar Ca++, they generate symmetric bending waves in reactivation solutions containing EGTA. In the presence of EGTA, their asymmetry can be restored by addition of brain calmodulin or the concentrated supernatant obtained from extraction with Triton and millimolar Ca++. These extracts contain calmodulin, as assayed by gel electrophoresis, radioimmunoassay, activation of brain phosphodiesterase, and Ca++-dependent binding of asymmetry-restoring activity to a trifluorophenothiazine-affinity resin. Conversion to the potentially symmetric condition can also be achieved with trifluoperazine substituted for Triton during the exposure to millimolar Ca++, which suggests that the calmodulin-binding activity of Triton is important for this conversion. These observations suggest that the conversion to the potentially symmetric condition is the result of removal of some of the axonemal calmodulin and provide additional evidence for axonemal calmodulin as a mediator of the effect of Ca++ on the asymmetry of flagellar bending.

scholarly journals The axonemal axis and Ca2+-induced asymmetry of active microtubule sliding in sea urchin sperm tails.

1986 ◽  
Vol 102 (6) ◽  
pp. 2042-2052 ◽  
Author(s):  
W S Sale
Keyword(s):  
Sea Urchin ◽  
Pair Interaction ◽  
Central Pair ◽  
Sperm Tail ◽  
Lytechinus Pictus ◽  
Motile Cells ◽  
Definition Of ◽  
Triton X ◽  
Explicit Discussion ◽  
Sea Urchin Sperm

Structural studies of stationary principal bends and of definitive patterns of spontaneous microtubule sliding disruption permitted description of the bending axis in sea urchin sperm tail axonemes. Lytechinus pictus sperm were demembranated in a buffer containing Triton X-100 and EGTA. Subsequent resuspension in a reactivation buffer containing 0.4 mM CaCl2 and 1.0 mM MgATP2- resulted in quiescent, rather than motile, cells and each sperm tail axoneme took on an extreme, basal principal bend of 5.2 rad. Thereafter, such flagellar axonemes began to disrupt spontaneously into two subsets of microtubules by active sliding requiring ATP. Darkfield light microscopy demonstrated that subset "1" is composed of microtubules from the inside edge of the principal bend. Subset "2" is composed of microtubules from the outside edge of the principal bend and always scatters less light in darkfield than subset 1. Subset 2, which always slides in the proximal direction, relative to subset 1, results in a basal loop of microtubules, and the subset 2 loop is restricted to the bend plane during sliding disruption. Electron microscopy revealed that doublets 8, 9, 1, 2, 3 and the central pair comprise subset 1, and doublets 4, 5, the bridge, 6, and 7 comprise subset 2. The microtubules of isolated subset 2 are maintained in a circular arc in the absence of spoke-central pair interaction. Longitudinal sections show that the bending plane bisects the central pair. We therefore conclude that the bend plane passes through doublet 1 and the 5-6 bridge and that doublet 1 is at the inside edge of the principal bend. Experimental definition of the axis permits explicit discussion of the location of active axonemal components which result in Ca2+-induced stationary basal bends and explicit description of components responsible for alternating basal principal and reverse bends.

Sliding Velocity Between Outer Doublet Microtubules of Sea-Urchin Sperm Axonemes

1980 ◽  
Vol 38 ◽  
pp. 600-603 ◽  
Author(s):  
Y. Yano ◽  
T. Miki-Noumura
Keyword(s):  
Sea Urchin ◽  
Beat Frequency ◽  
Sliding Velocity ◽  
Bending Waves ◽  
Flagellar Beat ◽  
Cylindrical Form ◽  
Activity Form ◽  
Dynein Arms ◽  
The Relationship ◽  
Sea Urchin Sperm

The flagellar axonemes have a cylindrical form, which consists of nine doublet microtubules surrounding a central pair of single microtubules. Each doublet tubule has two parallel rows of projections, called outer and inner arms. Sliding movement between doublet microtubules was first reported by Summers and Gibbons, who observed that doublet tubules were extruded from trypsin-treated axonemes of sea-urchin sperm flagella on addition of ATP. Their observation indicated that the bending movement of flagella results basically from these active sliding movements between the adjacent doublet tubules in the axonemes. Experimental evidence suggests that the dynein arms projecting from the doublet tubules interact with the adjacent tubules and by hydrolysing ATP, produce the mechanical force to slide. According to Gibbons and Gibbons the outer arms were removed from the doublet tubules by extracting the demembranated sea-urchin sperm with 0.5 M KCl or NaCl, while the inner arms and other axonemal structures remained apparently intact. Although the form of their bending waves was not significantly altered, the KCl-extracted sperm had only about half the flagellar beat frequency of the demembranated sperm. The 21S latent ATPase activity form of dynein 1 restored up to 90% of the outer arms on the doublet tubules and increased the beat frequency of KCl-extracted sperm from 14 Hz to 25 Hz. We found that the NaCl-extracted axonemes of sea-urchin sperm had the ability to extrude outer doublet tubules on addition of ATP and trypsin, in a similar manner to that of the intact axonemes. We attempted to compare the sliding velocity of the outer doublet tubules in the arm-depleted axonemes and in the arm-recombined axonemes, with that in the intact axonemes, in order to find the relationship between the sliding velocity and the number of arms in these axonemes.

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