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.

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.

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)

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 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.

Rotating the plane of imposed vibration can rotate the plane of flagellar beating in sea-urchin sperm without twisting the axoneme

1991 ◽  
Vol 98 (2) ◽  
pp. 175-181 ◽  
Author(s):  
C. Shingyoji ◽  
J. Katada ◽  
K. Takahashi ◽  
I.R. Gibbons
Keyword(s):  
Detailed Analysis ◽  
Sea Urchin ◽  
Regulatory Structure ◽  
Polystyrene Beads ◽  
Bending Waves ◽  
Flagellar Beat ◽  
Vibration Rotation ◽  
Sea Urchin Sperm

When the head of a sea-urchin sperm is held in the tip of a micropipette and vibrated laterally, the flagellum beats in phase with the imposed vibration. Rotation of the plane of pipette vibration around the head axis induces a corresponding rotation of the plane of beating, in both live and reactivated sperm. Detailed analysis of the waveforms occurring at different stages of this rotation shows that the characteristic asymmetry of the flagellar bending waves rotates along with the plane of beat. The positions of small polystyrene beads attached as markers on the axonemes of demembranated sperm flagella appear unaffected by the rotation of the beat plane and asymmetry. The imposed rotation of the waveform is thus the result of a rotation of the coordinated pattern of sliding among the doublet tubules of the axoneme, and is not accompanied by a twisting of the whole axonemal structure. These data indicate that neither the plane of flagellar beat nor the direction of beat asymmetry is tightly dependent upon a structural or chemical specialization of particular members of the nine outer doublet microtubules, but that both are the result of some regulatory structure that can be forced to rotate relative to the outer structure of the axoneme.

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 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.

The Outer Dynein Arms from Sea Urchin Sperm Flagella

1980 ◽  
Vol 38 ◽  
pp. 608-611
Author(s):  
W. S. Sale ◽  
W.-J. Y. Tang ◽  
I. R. Gibbons
Keyword(s):  
Sea Urchin ◽  
Energy Transduction ◽  
Cyclic Process ◽  
Cross Bridge ◽  
Bending Waves ◽  
Composition And Structure ◽  
Dynein Arms ◽  
Cilia And Flagella ◽  
Hydrolysis Of ◽  
Sea Urchin Sperm

A wide variety of experimental evidence now indicates that the bending waves of cilia and flagella are the result of coordinated, localized slid¬ing movements between doublet microtubules of the axoneme (Satir, 1968; Summers and Gibbons, 1971; Shingyoji, et al. 1977; Sale and Satir, 1977). These sliding movements are thought to occur as the consequence of a mecha¬nism in which the dynein arms on each doublet microtubule make and break cross-bridge attachments to the B-subfiber of the adjacent doublet micro¬tubule in a cyclic process involving the binding and hydrolysis of ATP (Summers and Gibbons, 1973; Gibbons and Gibbons, 1973; 1974). The structu¬ral and chemical steps of this cross-bridge cycle are only beginning to be understood (Warner, 1978; Penningroth and Witman, 1979; Sale and Gibbons, 1979; Okuno, 1979). Further understanding of the nature of the events of energy transduction will require knowledge of the composition and structure of the dynein arms.

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.

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