Resolution of the competitive inhibitory effects of lithium and AMPPNP on the beat frequency of ATP-reactivated, demembranated, sea urchin sperm flagella

1985 ◽  
Vol 6 (4) ◽  
pp. 507-512 ◽  
Author(s):  
E. F. Pate ◽  
C. J. Brokaw
Keyword(s):  
Sea Urchin ◽  
Beat Frequency ◽  
Inhibitory Effects ◽  
Sea Urchin Sperm

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.

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.

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 FLAGELLAR MOVEMENT AND ADENOSINE TRIPHOSPHATASE ACTIVITY IN SEA URCHIN SPERM EXTRACTED WITH TRITON X-100

1972 ◽  
Vol 54 (1) ◽  
pp. 75-97 ◽  
Author(s):  
Barbara H. Gibbons ◽  
I. R. Gibbons
Keyword(s):  
Atpase Activity ◽  
Sea Urchin ◽  
Adenosine Triphosphatase ◽  
Beat Frequency ◽  
Average Frequency ◽  
Mitochondrial Atpase ◽  
The Difference ◽  
Live Sperm ◽  
Triton X ◽  
Sea Urchin Sperm

Extraction with 0 04% (w/v) Triton X-100 removes the flagellar membrane from sea urchin sperm while leaving the motile apparatus apparently intact When reactivated in a suitable medium containing exogenous adenosine triphosphate (ATP), nearly 100% of the sperm are motile and they swim in a manner resembling that of live sperm. Under standard conditions, with 1 mM ATP at 25°C, the reactivated sperm had an average frequency of 32 beats/sec and progressed forward a distance of 2.4 µm/beat; comparable figures for live sperm in seawater were 46 beats/sec and 3 9 µm/beat. The adenosine triphosphatase (ATPase) activity of the reactivated sperm was measured with a pH-stat in the presence of oligomycin to inhibit residual mitochondrial ATPase. The motile sperm had an ATPase activity of 0.16 µmole Pi/(min x mg protein), while sperm that had been rendered non-motile by homogenizing had an activity of 0 045 µmole Pi/(min x mg protein). The difference between the ATPase activities of the motile and nonmotile sperm was tentatively interpreted as the amount of activity coupled to movement, and under optimal conditions it amounted to about 72% of the total ATPase activity Under some conditions the movement-coupled ATPase activity was proportional to the beat frequency, but it was possibly also affected by other wave parameters. The coupled ATPase activity decreased to almost zero when movement was prevented by raising the viscosity, or by changing the pH or salt concentration. The motility of reactivated sperm was wholly dependent on the presence of ATP; other nucleotides gave very low phosphatase activity and no movement. The requirement for a divalent cation was best satisfied with Mg++, although some motility was also obtained with Mn++ and Ca++. The coupled ATPase activity had a Michaelis constant (Km) of 0.15 mM. The beat frequency of the reactivated sperm varied with the ATP concentration, with an effective "Km" of 0.2 mM.

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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