scholarly journals PROPERTIES OF FLAGELLAR "RIGOR WAVES" FORMED BY ABRUPT REMOVAL OF ADENOSINE TRIPHOSPHATE FROM ACTIVELY SWIMMING SEA URCHIN SPERM

1974 ◽  
Vol 63 (3) ◽  
pp. 970-985 ◽  
Author(s):  
Barbara H. Gibbons ◽  
I. R. Gibbons
Keyword(s):  
Sea Urchin ◽  
Sperm Suspension ◽  
Complete Relaxation ◽  
Viscous Shear ◽  
Radial Spokes ◽  
Wave Forms ◽  
Cross Bridges ◽  
Triton X ◽  
The Waves ◽  
Sea Urchin Sperm

Sea urchin sperm were demembranated and reactivated with a solution containing 0.04% Triton X-100 and 0.03 mM ATP. The ATP concentration was then lowered abruptly by diluting the sperm suspension 50-fold into reactivating solution containing no ATP. The flagella of the sperm in the diluted suspension were not motile, but they were bent into a variety of stationary rigor wave forms closely resembling the wave forms occurring at different stages of the flagellar bending cycle during normal movement. The form of these rigor waves was unchanged upon storage for several hours in the presence of dithiothreitol and EDTA. Addition of 1 µM ATP induced slow relaxation of the waves, with most of the sperm becoming partially straightened over a period of about 30 min; somewhat higher concentrations gave a more rapid and complete relaxation. Concentrations of ATP above 10 µM induced resumption of normal beating movements. Addition of ITP, GTP, or GDP (up to 1 mM) produced no relaxation of the rigor waves. Digestion with trypsin to an extent sufficient to disrupt the radial spokes and the nexin links caused no change in the rigor wave forms, suggesting that these wave forms could be maintained by the dynein cross-bridges between the outer doublet tubules of the flagellar axoneme. Study of the effects of viscous shear on the rigor wave axonemes has shown that they are resistant to distortion by bending, although they can be twisted relatively easily.

scholarly journals EFFECTS OF TRYPSIN DIGESTION ON FLAGELLAR STRUCTURES AND THEIR RELATIONSHIP TO MOTILITY

1973 ◽  
Vol 58 (3) ◽  
pp. 618-629 ◽  
Author(s):  
Keith E. Summers ◽  
I. R. Gibbons
Keyword(s):  
Electron Microscope ◽  
Trypsin Digestion ◽  
Shearing Force ◽  
Digestion Mixture ◽  
Radial Spokes ◽  
Time Periods ◽  
Dynein Arms ◽  
Cross Bridges ◽  
Sea Urchin Sperm ◽  
Bending Wave

Flagellar axonemes isolated from sea urchin sperm were digested with trypsin for various time periods. The course of digestion was monitored turbidimetrically and was found to take two different courses depending on the presence or absence of ATP in the digestion mixture. It was found that ATP induced active disintegration of the axonemes after slight digestion. Samples of the digested axonemes were examined with the electron microscope to determine the effects of trypsin digestion on the substructures of the axonemes. The rate at which trypsin sensitized the axonemes to ATP paralleled the rate at which it damaged the radial spokes and the nexin links, while the dynein arms were removed much more slowly. The results suggest that inactive dynein arms form cross bridges between the adjacent doublet tubules in digested axonemes, and that when activated by the addition of ATP, they induce an active shearing force between adjacent doublets. The radial spokes and the nexin links are not directly involved in the production of mechanical force, but they may participate in regulating the sliding between tubules to produce a propagated bending wave.

Calcium-dependent bidirectional power stroke of the dynein arms in sea urchin sperm axonemes

1996 ◽  
Vol 109 (12) ◽  
pp. 2833-2842 ◽  
Author(s):  
S. Ishijima ◽  
M. Kubo-Irie ◽  
H. Mohri ◽  
Y. Hamaguchi
Keyword(s):  
Light Microscopy ◽  
Sea Urchin ◽  
Electron Microscopic Examination ◽  
Dark Field ◽  
Power Stroke ◽  
Electron Microscopic ◽  
Calcium Dependent ◽  
Dynein Arms ◽  
Triton X ◽  
Sea Urchin Sperm

Active sliding between doublet microtubules of sea urchin sperm axonemes that were demembranated with Triton X-100 in the presence or absence of calcium was induced with ATP and elastase at various concentrations of Ca2+ to examine the effects of Ca2+ on the direction of the power stroke of the dynein arms. Dark-field light microscopy of microtubule sliding revealed that the sliding from the axonemes demembranated with Triton and millimolar calcium and disintegrated with ATP and elastase showed various patterns of sliding disintegration, including loops of doublet microtubules formed near the head or the basal body. These loops were often thicker than the remaining axonemal bundle. In contrast, only thinner loops were found from the axonemes demembranated with Triton in the absence of calcium and disintegrated with ATP and elastase at high Ca2+ concentrations. Electron microscopic examination of the direction of microtubule sliding showed that the doublet microtubules in the axonemes demembranated in the presence of millimolar calcium moved toward the base of the axonemes by the dynein arms on the adjacent doublet microtubule as well as by their own dynein arms. Doublet microtubules in the axonemes demembranated in the absence of calcium moved toward the base of the axonemes only by their own dynein arms. Similar observations have been obtained from the axonemes from which the outer dynein arms were selectively extracted. From these observations, we can conclude that the dynein arms generate force in both directions and this feature of the dynein arms arises from at least the inner dynein arms.

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.

C/A dynein isolated from sea urchin sperm flagellar axonemes. Enzymatic properties and interaction with microtubules

1994 ◽  
Vol 107 (2) ◽  
pp. 353-361 ◽  
Author(s):  
E. Yokota ◽  
I. Mabuchi
Keyword(s):  
Atpase Activity ◽  
Sea Urchin ◽  
Maximal Inhibition ◽  
Heavy Chains ◽  
Lower Molecular Mass ◽  
Cross Bridges ◽  
Polypeptide Chains ◽  
Brain Tubulin ◽  
Sea Urchin Sperm

C/A dynein is a novel dynein isolated from sea urchin sperm flagellar axonemes. It is composed of C and A heavy chains and some additional lower molecular mass polypeptide chains. The characterization of ATPase activity and the interaction of this dynein with microtubules polymerized from calf brain tubulin were investigated in this study. The ATPase activity of C/A dynein (0.3-0.4 mumol Pi/min per mg) was about one half that of outer arm 21 S dynein (0.6-0.8 mumol Pi/min per mg) at 25 degrees C. Vanadate inhibited the ATPase activity with a half-maximal inhibition at 1 microM. C/A dynein absorbed to the glass surface was able to translocate the microtubules towards its plus end. The velocity of the microtubule movement in the presence of 1 mM ATP was 4.0 to 4.5 microns/s at 22 degrees C. C/A dynein binds to and bundles the microtubules even in the presence of ATP. Cross-bridges were found between adjacent microtubules in the bundle with an axial periodicity of about 24 nm. The ATPase activity of C/A dynein was enhanced up to several-fold by the microtubules at concentration as low as 1 mg/ml. On the other hand, 21 S dynein bound to the microtubules with 24 nm axial periodicity only in the absence of ATP. Its ATPase activity was not activated by the microtubules. From these results, it is concluded that the manner of interaction with microtubules of C/A dynein is different from that of the outer arm dynein.

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.

scholarly journals A quantitative description of flagellar movement in golden hamster spermatozoa

1985 ◽  
Vol 114 (1) ◽  
pp. 463-475
Author(s):  
S. Ishijima ◽  
H. Mohri
Keyword(s):  
Sea Urchin ◽  
Golden Hamster ◽  
Large Amplitude ◽  
Quantitative Description ◽  
Low Frequency ◽  
Before And After ◽  
Epididymal Spermatozoa ◽  
Triton X ◽  
Sea Urchin Sperm

Flagellar movement of golden hamster spermatozoa obtained from the testis and the caput and cauda epididymides was observed by a light microscope while holding them at their heads with a micropipette. Flagellar movement of capacitated spermatozoa and of reactivated spermatozoa demembranated with Triton X-100 was also observed. Testicular and caput epididymal spermatozoa showed weak movement in Tyrode's solution, whereas cauda epididymal spermatozoa showed vigorous movement. The flagellar bends of the cauda epididymal spermatozoa were almost planar. Capacitated spermatozoa moved with waves of a large amplitude. Demembranated spermatozoa reactivated with ATP only had a latent period before the initiation of flagellar movement, and beat at low frequency, whereas demembranated spermatozoa reactivated with both ATP and cAMP began to move immediately at high frequency. Thrust and hydrodynamic power output were calculated using the parameters for the typical waveforms of cauda epididymal spermatozoa before and after capacitation. The possible role of the large amplitude beat in capacitated spermatozoa is discussed. A comparison of the ‘principal’ and ‘reverse’ bends in golden hamster sperm flagella as defined by Woolley (1977) with those in sea urchin sperm flagella suggests that the so-called ‘principal’ bend in golden hamster sperm flagella corresponds to the reverse bend in sea urchin sperm flagella and vice versa.

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.

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