scholarly journals Effects of viscosity and ATP concentration on the movement of reactivated sea-urchin sperm flagella

1975 ◽  
Vol 62 (3) ◽  
pp. 701-719 ◽  
Author(s):  
C. J. Brokaw
Keyword(s):  
Sea Urchin ◽  
Beat Frequency ◽  
Methyl Cellulose ◽  
Intrinsic Rate ◽  
Quantitative Agreement ◽  
Experimental Results ◽  
The Other ◽  
Bend Angle ◽  
Atp Concentration ◽  
Flagellar Beat

Spermatozoa from the sea urchin, Lytechinus pictus, can be demembranated with solutions containing Triton X-100 and 5mM-CaCl2 and reactivated in ATP solutions containing low concentrations (10(−9)M) Of free Ca2+ ion to give symmetrical bending wave movements, even at very low ATP concentrations. At ATP concentrations of 0.01-0.02 mM the reactivated spermatozoa have beat frequencies near 1 Hz, nearly normal bend angles, and wave-lengths about 50% longer than normal. 2. The effects of increased viscosity, obtained by addition of methyl cellulose to the reactivation solutions, on bend angle and beat frequency decrease with decreasing ATP concentration, and become almost undetectable at 0.01 mM ATP. On the other hand, the effect of increased viscosity on wavelength shows relatively little change with ATP concentration, although it is noticeably reduced at 0.01 mM ATP. 3. These observations suggest that the beat frequency at low ATP concentrations is determined by an intrinsic rate-limiting process, in contrast to the viscocity-limited behaviour at high beat frequencies. Quantitative agreement with the experimental results is obtained with a model in which ATP concentration and viscosity each determine the rates of one step in a two-step reaction cycle which determines the beat frequency. 4. Two other models which can qualitatively explain the effects of ATP concentration and viscosity on flagellar beat frequency fail to show quantitative agreement with the experimental results. In one of these models, ATP concentration determines the maximum rate of shear between filaments. In the other, ATP concentration determines a time delay which is required to bring the active moment into phase with the elastic moments which would be expected to dominate the bending resistance at low beat frequencies.

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)

scholarly journals CO2-inhibition of the amplitude of bending of triton-demembranated sea urcin sperm flagella

1977 ◽  
Vol 71 (1) ◽  
pp. 229-240
Author(s):  
C. J. Brokaw
Keyword(s):  
Feedback Control ◽  
Sea Urchin ◽  
Critical Level ◽  
Control Mechanism ◽  
Beat Frequency ◽  
Bend Angle ◽  
Direct Inhibition ◽  
Flagellar Beat ◽  
Frequency Observation ◽  
Small Inhibition

Demembranated sea urchin spermatozoa were reactivated in solutions containing KHCO3 and observed in a covered well slide. Although KHCO3 itself causes a small inhibition of flagellar beat frequency, the results confirm previous observations of a direct inhibition of flagellar bend angle by CO2 with no effect of CO2 on frequency. Observation of the effect of pH on the inhibition of bend angle in solutions containing KHCO3 indicates that a given concentration of OH- has a similar effect to the same concentration of HCO3-, as would be expected if CO2- inhibition results from reaction of CO2 with protein-NH3+ groups to form carbamates. CO2 may interfere with a control mechanism which selectively suppresses dynein cross-bridge activity in order to generate rhythmic bending. This control mechanism may incorporate a feedback control involving a measure of flagellar amplitude, which fails to operate successfully when the amplitude is reduced below a critical level.

scholarly journals Transient behavior of sea urchin sperm flagella following an abrupt change in beat frequency

1990 ◽  
Vol 152 (1) ◽  
pp. 441-451 ◽  
Author(s):  
D. Eshel ◽  
C. Shingyoji ◽  
K. Yoshimura ◽  
B. H. Gibbons ◽  
I. R. Gibbons ◽  
...  
Keyword(s):  
Sea Urchin ◽  
Vibration Frequency ◽  
High Speed ◽  
Beat Frequency ◽  
Transient Behavior ◽  
Bend Angle ◽  
Initiation Rate ◽  
Flagellar Beat ◽  
Final Length ◽  
Sea Urchin Sperm

Within the approximate range of 30–80 Hz, the flagellar beat frequency of a sea urchin sperm held by its head in the tip of a micropipet is governed by the vibration frequency of the micropipet. We have imposed abrupt changes in flagellar beat frequency by changing the vibration frequency of the micropipet within this range and used a high-speed video system to analyze the flagellar wave parameters during the first few cycles following the change. Our results demonstrate that the various flagellar beat parameters differ in the time they take to adjust to the new conditions. The initiation rate of new bends at the base is directly governed by the frequency of the vibration and changes immediately to the new frequency. The length and the propagation velocity of the developed bends become adjusted to the new conditions within approximately 1 beat cycle, whereas the bend angles take more than 4 beat cycles to attain their new steady-state value. Bends initiated shortly before the change in frequency occurs attain a final length and angle that depends on the relative durations of growth at the old and new frequencies. Our results suggest that the flagellar wavelength and bend angle are regulated by different mechanisms with the second not being directly dependent on bend initiation.

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.

scholarly journals Movement of the Flagella of Polytoma Uvella

1963 ◽  
Vol 40 (1) ◽  
pp. 149-156
Author(s):  
C. J. BROKAW
Keyword(s):  
Beat Frequency ◽  
Methyl Cellulose ◽  
Dark Field ◽  
Maximum Efficiency ◽  
Sinusoidal Wave ◽  
Low Frequencies ◽  
Atp Concentration ◽  
Wave Patterns ◽  
Internal Viscosity ◽  
Photographic Records

1. Dark-field photographic records of the wave patterns of moving flagella have been made using multiple-flash exposures at flash rates of up to 50 per sec. Patterns obtained from ATP-reactivated isolated flagella show reduced amplitude of bending, but are otherwise similar to those obtained from flagella under normal conditions. The co-ordination required to produce propagated waves of active bending appears to be preserved after isolation and reactivation. 2. Addition of methyl cellulose to the medium to increase the viscosity reduces the frequency of beat much more than the amplitude. This behaviour can be partially explained by an analysis of the equations for sinusoidal wave movement of flagella which shows that maximum efficiency of forward swimming will be obtained if the amplitude of beat is maintained greater than ½π times the wavelength, and variations in available power or viscosity are compensated by changes in beat frequency. 3. Wave patterns at low frequencies in low ATP concentrations are unlike those obtained when the frequency is reduced by increased viscosity. The effect of ATP concentration on beat frequency is not explained by an effect on the power available for beating or by an effect on the ‘internal viscosity‘ of the flagella.

The polyglutamylated lateral chain of alpha-tubulin plays a key role in flagellar motility

1996 ◽  
Vol 109 (6) ◽  
pp. 1545-1553 ◽  
Author(s):  
C. Gagnon ◽  
D. White ◽  
J. Cosson ◽  
P. Huitorel ◽  
B. Edde ◽  
...  
Keyword(s):  
Sea Urchin ◽  
Beat Frequency ◽  
Cortical Microtubule ◽  
Flagellar Motility ◽  
Microtubule Network ◽  
Alpha Tubulin ◽  
Oxyrrhis Marina ◽  
Terminal Domain ◽  
Flagellar Beat ◽  
Sea Urchin Sperm

To investigate whether a specific isotype of tubulin is involved in flagellar motility, we have developed and screened a panel of monoclonal antibodies (mAb) generated against sea urchin sperm axonemal proteins. Antibodies were selected for their ability to block the motility of permeabilized sperm models. The antitubulin mAb B3 completely inhibited, at low concentrations, the flagellar motility of permeabilized sperm models from four sea urchin species. On immunoblots, B3 recognized predominantly alpha-tubulin in sea urchin sperm axonemes and equally well brain alpha- and beta-tubulins. Subtilisin cleavage of tubulin removed the B3 epitope, indicating that it was restricted to the last 13 amino acid residues of the C-terminal domain of alpha-tubulin. In enzyme-linked immunosorbant assays, B3 reacted with glutamylated alpha-tubulin peptides from sea urchin or mouse brain but did not bind to the unmodified corresponding peptide, indicating that it recognized polyglutamylated motifs in the C-terminal domain of alpha-tubulin. On the other hand, other tubulin antibodies directed against various epitopes of the C-terminal domain, with the exception of the antipolyglutamylated mAb GT335, had no effect on motility while having binding properties similar to that of B3. B3 and GT335 acted by decreasing the beating amplitude without affecting the flagellar beat frequency. B3 and GT335 were also capable of inhibiting the motility of flagella of Oxyrrhis marina, a 400,000,000 year old species of dinoflagellate, and those of human sperm models. Localization of the antigens recognized by B3 and GT335 by immunofluorescence techniques revealed their presence along the whole axoneme of sea urchin spermatozoa and flagella of O. marina, except for the distal tip and the cortical microtubule network of the dinoflagellate. Taken together, the data reported here indicate that the polyglutamylated lateral chain of alpha-tubulin plays a dynamic role in a dynein-based motility process.

The Effect of Partial Extraction of Dynein Arms on the Movement of Reactivated Sea-Urchin Sperm

1973 ◽  
Vol 13 (2) ◽  
pp. 337-357 ◽  
Author(s):  
BARBARA H. GIBBONS ◽  
I. R. GIBBONS
Keyword(s):  
Sea Urchin ◽  
Room Temperature ◽  
Beat Frequency ◽  
Wave Form ◽  
Bending Waves ◽  
Flagellar Beat ◽  
Elastic Forces ◽  
Dynein Arms ◽  
Triton X ◽  
Sea Urchin Sperm

Sea-urchin sperm were extracted with o.5 M KCl for 45 s at room temperature in the presence of Triton X-100, and then transferred to reactivating solution containing 1 mM ATP. The flagellar beat frequency of these KCl-extracted sperm (16 beats/s) was only about half that of control Triton-extracted sperm that had not been exposed to 0.5 M KCl (31 beats/s), although the form of their bending waves was not significantly altered. Examination by electron microscopy showed that the extraction with 0.5 M KCl removed the majority of the outer arms from the doublet tubules, leaving the inner arms apparently intact. By varying the duration of the KCl-extraction, it was shown that the rate of decrease in beat frequency paralleled the rate of disappearance of the arms. Prolonging the extraction time beyond 45 s at room temperature, or 4 min at o °C, had little further effect on beat frequency. ATPase measurements suggested that 6o-65% of the dynein in the original axonemes had been solubilized when the extraction with KCl was permitted to go to completion. These results indicate that the generation and propagation of flagellar bending waves of essentially typical form are not prevented by the removal of the outer row of dynein arms from the doublet tubules. In terms of the sliding filament model of flagellar bending, the results suggest that the rate of sliding between tubules under these conditions is proportional to the number of dynein arms present. The lack of significant change in wave form implies that the total amount of sliding that occurs during each bending cycle is not affected by the reduced number of dynein arms, but is regulated independently in some manner by the elastic forces generated by other structures in the bent axoneme.

scholarly journals Effects of adenylyl imidodiphosphate, a nonhydrolyzable adenosine triphosphate analog, on reactivated and rigor wave sea urchin sperm.

1978 ◽  
Vol 79 (3) ◽  
pp. 827-832 ◽  
Author(s):  
S M Penningroth ◽  
G B Witman
Keyword(s):  
Adenosine Triphosphate ◽  
Sea Urchin ◽  
Beat Frequency ◽  
Atp Binding ◽  
Flagellar Motility ◽  
Lytechinus Pictus ◽  
Cross Bridge ◽  
Atp Concentration ◽  
Sea Urchin Sperm

A nonhydrolyzable ATP analog, adenylyl imidodiphosphate (AMP-PNP), has been used to study the role of ATP binding in flagellar motility. Sea urchin sperm of Lytechinus pictus were demembranated, reactivated, and locked in "rigor waves" by a modification of the method of Gibbons and Gibbons (11). Rigor wave sperm relaxed within 2 min after addition of 4 micrometer ATP, and reactivated upon addition of 10-12 micrometer ATP. The beat frequency of the reactivated sperm varied with ATP concentration according to Michaelis-Menten kinetics ("Km" = 0.24 mM; "Vmax" = 44 Hz) and was competitively inhibited by AMP-PNP (Ki" approximately to 8.1 mM). Rigor wave sperm were completely relaxed (straightened) within 2 min by AMP-PNP at concentrations of 2-4 mM. The possibilities that relaxation in AMP-PNP was a result of ATP contamination, AMP-PNP hydrolysis, or lowering of the free Mg++ concentration were conclusively ruled out. The results suggest that dynein cross-bridge release is dependent upon ATP binding but not hydrolysis.

Sign in / Sign up

Export Citation Format

Share Document