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 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 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)

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.

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.

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 Molecular Cloning and Characterization of a Radial Spoke Head Protein of Sea Urchin Sperm Axonemes: Involvement of the Protein in the Regulation of Sperm Motility

1998 ◽  
Vol 9 (2) ◽  
pp. 513-522 ◽  
Author(s):  
Denis Gingras ◽  
Daniel White ◽  
Jérome Garin ◽  
Jacky Cosson ◽  
Philippe Huitorel ◽  
...  
Keyword(s):  
Sea Urchin ◽  
Beat Frequency ◽  
Three Dimensional ◽  
Coiled Coil ◽  
Structural Features ◽  
Flagellar Motility ◽  
Native Form ◽  
Radial Spoke ◽  
Single Polypeptide ◽  
Sea Urchin Sperm

Monoclonal antibodies raised against axonemal proteins of sea urchin spermatozoa have been used to study regulatory mechanisms involved in flagellar motility. Here, we report that one of these antibodies, monoclonal antibody D-316, has an unusual perturbating effect on the motility of sea urchin sperm models; it does not affect the beat frequency, the amplitude of beating or the percentage of motile sperm models, but instead promotes a marked transformation of the flagellar beating pattern which changes from a two-dimensional to a three-dimensional type of movement. On immunoblots of axonemal proteins separated by SDS-PAGE, D-316 recognized a single polypeptide of 90 kDa. This protein was purified following its extraction by exposure of axonemes to a brief heat treatment at 40°C. The protein copurified and coimmunoprecipitated with proteins of 43 and 34 kDa, suggesting that it exists as a complex in its native form. Using D-316 as a probe, a full-length cDNA clone encoding the 90-kDa protein was obtained from a sea urchin cDNA library. The sequence predicts a highly acidic (pI = 4.0) protein of 552 amino acids with a mass of 62,720 Da (p63). Comparison with protein sequences in databases indicated that the protein is related to radial spoke proteins 4 and 6 (RSP4 and RSP6) of Chlamydomonas reinhardtii, which share 37% and 25% similarity, respectively, with p63. However, the sea urchin protein possesses structural features distinct from RSP4 and RSP6, such as the presence of three major acidic stretches which contains 25, 17, and 12 aspartate and glutamate residues of 34-, 22-, and 14-amino acid long stretches, respectively, that are predicted to form α-helical coiled-coil secondary structures. These results suggest a major role for p63 in the maintenance of a planar form of sperm flagellar beating and provide new tools to study the function of radial spoke heads in more evolved species.

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.

scholarly journals Effects of antibodies against tubulin on the movement of reactivated sea urchin sperm flagella.

1980 ◽  
Vol 87 (1) ◽  
pp. 114-123 ◽  
Author(s):  
D J Asai ◽  
C J Brokaw
Keyword(s):  
Affinity Chromatography ◽  
Sea Urchin ◽  
Inhibitory Activity ◽  
Beat Frequency ◽  
Bend Angle ◽  
First Report ◽  
Inhibiting Effect ◽  
Immobilized Substrate ◽  
Sea Urchin Sperm

Antibodies binding to sea urchin flagellar outer-doublet tubulin have been isolated from rabbit sera by tubulin-affinity chromatography employing electrophoretically purified tubulin as the immobilized substrate. This procedure provides "induced" antitubulin antibody from immune sera and "spontaneous" antitubulin antibody from preimmune sera. These antitubulins were characterized in terms of their specificity, ability to bind to sea urchin axonemes, and effects on the motility of reactivated spermatozoa. Induced antitubulin antibody specifically reduced the bend angle and symmetry of the movement of demembranated reactivated spermatozoa without affecting the beat frequency. At identical concentrations, spontaneous antitubulin had no effect on motility. Affinity-purified induced antitubulins from three other rabbits all gave specific bend-angle inhibition, whereas their corresponding spontaneous antitubulins had no effect on the flagellar movement. The effects of antitubulin on microtubule sliding were examined by observing the sliding disintegration of elastase-digested axonemes induced by MgATP2+-. Affinity-purified induced antitubulin antibody, in quantities sufficient to completely paralyze reactivated flagella, did not inhibit microtubule sliding. The amplitude-inhibiting effect of induced antitubulin on reactivated spermatozoa may be caused by action on a mechanism responsible for controlling flagellar bending rather than by interference with the active sliding process. This is the first report of an antitubulin antibody having an inhibitory activity on microtubule-associated movement.

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.

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