Sliding velocity between outer doublet microtubules of sea-urchin sperm axonemes

1980 ◽  
Vol 44 (1) ◽  
pp. 169-186
Author(s):  
Y. Yano ◽  
T. Miki-Noumura
Keyword(s):  
Polyethylene Glycol ◽  
Atpase Activity ◽  
Sea Urchin ◽  
High Efficiency ◽  
Dark Field ◽  
Video Tape ◽  
Sliding Velocity ◽  
Atp Concentration ◽  
Close Relationship ◽  
Hemicentrotus Pulcherrimus

Using a dark-field microscope equipped with a high-efficiency TV camera including a video tape-recorder, we recorded the sliding movement between outer doublet microtubules of the demembranated axonemes of sea-urchin (Pseudocentrotus depressus and Hemicentrotus pulcherrimus) sperm flagella by adding ATP and trypsin at 25 degrees C. The time and length of the sliding doublet microtubules from axonemes were measured directly from the image on the picture monitor from the video tape. The sliding velocity was almost constant in the range from 0 to 2% polyethylene glycol concentration in the reactivation medium and decreased a little at more than 2%. We prepared various lengths of axoneme fragments by homogenizing whole axonemes and found that the shorter fragments showed similar sliding velocity to that of longer ones at less than 200 microM ATP, but slightly decreased speed at more than 500 microM. ATP. The sliding movement sometimes stopped and the percentage of sliding axonemes was lower below 2 micrograms/ml trypsin. Above 3 micrograms/ml, the process appeared to be more like disintegration than sliding movement, which may be due to excess digestion by trypsin. Sliding speed was therefore measured in a reactivation medium containing 2% polyethylene glycol with the addition of ATP and 2 micrograms/ml trypsin. The velocity increased in proportion to the increase in ATP concentration. Vmax was approximately 14 micrograms/s at 2 mM ATP. In order to compare the Km for the sliding velocity with that of the ATPase activity of the axonemes, we measured ATPase activity of axonemes prepared and assayed under conditions in which sliding movement in the axonemes could be induced. Neither the curve of ATPase activity nor the curve of sliding velocity plotted against ATP concentration obeyed Michaelis-Menten kinetics. The close relationship between ATPase activity and sliding velocity suggested that ‘sliding-movement-coupled ATPase activity’ may well be reflected in the axoneme ATPase reported here.

Change in Na+, K+ -ATPase activity in embryos of the sea urchin, Hemicentrotus pulcherrimus, during early development

2020 ◽  
pp. 391-391
Author(s):  
K. Mitsunaga ◽  
M. Hatoh ◽  
K. Yamazaki ◽  
I. Yasumasu ◽  
K. Yamada ◽  
...  
Keyword(s):  
Atpase Activity ◽  
Early Development ◽  
Sea Urchin ◽  
Hemicentrotus Pulcherrimus

Recovery of sliding ability in arm-depleted flagellar axonemes after recombination with extracted dynein I

1981 ◽  
Vol 48 (1) ◽  
pp. 223-239
Author(s):  
Y. Yano ◽  
T. Miki-Noumura
Keyword(s):  
Electron Microscopy ◽  
Sea Urchin ◽  
Extraction Process ◽  
Sliding Velocity ◽  
Basal Segment ◽  
Edta Solution ◽  
Close Relationship

We compared sliding velocity between outer doublet tubules in demembranated axonemes of sea-urchin (Pseudocentrotus depressus) sperm flagella with that of arm-depleted axonemes recombined with extracted dynein I. The outer arm-depleted axonemes after extraction with 0.5 M NaCl had a velocity of 6.9 +/− I.0 micrometer/s, while the intact axonemes had a velocity of 14.3 +/− I.5 micrometer/s in the presence of I mM ATP and 2 microgram/ml trypsin at 25 degrees C. The sliding velocity was closely related to the number of remaining outer arms following the NaCl-extraction process. When the outer arm-depleted axonemes were recombined with dynein I, the sliding velocity increased to 11.3 +/− 1.3 micrometer/s. Electron microscopy confirmed the recovery of 94% of outer arms in the axonemes. After extraction with Tris-EDTA solution for 10 min, the axonemes lost their sliding ability completely, even in the presence of ATP and trypsin. Such axonemes lacked most of both inner and outer arms, although sometimes the basal segment of the arms appeared to remain. When the exogenous dynein I fraction extracted from other axonemes was added, the axonemes could extrude tubules, and both types of arms reappeared clearly and distinctly in the axonemes. The recombined axonemes with one-fold stoichiometric excess of dynein I recovered 58% of the total number of arms and had a velocity of 7.4 +/− 1.6 micrometer/s. Those with 2-fold stoichiometric excess had a velocity of 11.0 +/− 1.5 micrometer/s, up to 82% of the arms in these axonemes being restored. These results indicated that the exogenous dynein I fraction derived from the outer arms restored sliding ability to arm-depleted axonemes, recombining with th outer doublet tubules as inner and outer arms, and that the sliding velocity had a close relationship to the total number of arms in the axonemes, irrespective of their being inner or outer arms.

scholarly journals Conformational change in the outer doublet microtubules from sea urchin sperm flagella.

1979 ◽  
Vol 81 (2) ◽  
pp. 355-360 ◽  
Author(s):  
T Miki-Noumura ◽  
R Kamiya
Keyword(s):  
Sea Urchin ◽  
Conformational Changes ◽  
Helical Structure ◽  
Dark Field ◽  
Ion Concentration ◽  
Wave Form ◽  
Buffer System ◽  
Left Handed ◽  
Dark Field Microscopy ◽  
Hemicentrotus Pulcherrimus

Dark-field microscopy with a high-powered light source revealed that the outer doublet microtubules (DMTs) from sea urchin (Pseudocentrotus depressus and Hemicentrotus pulcherrimus) sperm flagella assume helically coiled configurations (Miki-Noumura, T., and R. Kamiya. 1976. Exp. Cell Res. 97: 451.). We report here that the DMTs change shape when the pH or Ca-ion concentration is changed. The DMTs assumed a left-handed helical shape with a diameter of 3.7 +/- 0.5 micron and a pitch of 2.8 +/- 0.7 micron at pH 7.4 in the presence of 0.1 mM CaCl2, 1 mM MgSO4, and 10 mM Tris-HCl. When the pH was raised to 8.3, the helical diameter and pitch decreased to 2.1 +/- 0.1 micron and 1.3 +/- 0.3 micron, respectively. This transformation was a rapid and reversible process and was completed within 1 min. Between pH 7.2 and 8.3, the DMTs assumed intermediate shapes. When the Ca-ion concentration was depleted with EGTA, the helical structure became significantly larger in both pitch and diameter. For instance, the diameter was 3.8 +/- 0.4 micron at pH 8.3 in the presence of 1 mM EGTA and 2 mM MgSO4. Using a Ca-buffer system, we obtained results which suggested that this Ca-induced transformation took place at a Ca concentration of approximately 10(-7) M. These results were highly reproducible. The conformational changes in the DMT may play some role in the bending wave form of flagellar movement.

ATP-driven tubule extrusion from axonemes without outer dynein arms of sea-urchin sperm flagella

1980 ◽  
Vol 41 (1) ◽  
pp. 331-340
Author(s):  
H. Hata ◽  
Y. Yano ◽  
T. Mohri ◽  
H. Mohri ◽  
T. Miki-Noumura
Keyword(s):  
Electron Microscopy ◽  
Atpase Activity ◽  
Sea Urchin ◽  
Extrusion Process ◽  
Dynein Arms ◽  
Sea Urchin Sperm ◽  
Hemicentrotus Pulcherrimus

We have prepared axonemes without outer dynein arms from sea-urchin (Pseudocentrotus depressus, Hemicentrotus pulcherrimus) sperm flagella by selective solubilization with NaCl. Electron microscopy revealed that the axonemes gradually lost their outer arms in 0.5 M NaCl during 10 min. Such axonemes retained 42.8 +/− 7.3% of their total axonemal ATPase activity and showed C, A, D and B bands in the dynein region of 4% SDS-gel, while a solubilized fraction of the outer arms consisted almost entirely of A band polypeptide. We have succeeded in causing extrusion of the outer doublets from such axonemes by addition of ATP and trypsin. A bundle of outer doublets was sometimes observed to be extruded first from an axoneme and to show bending motion for a while, subsequently followed by a sliding of separate doublets past each other. The speed of the tubule extrusion process was slower and around 60% of that of intact axonemes having both types of arm. These observations indicate that the inner arms have a function equivalent to that of the outer arms, of sliding on adjacent doublets, although the inner arms seem to be constituted from polypeptide(s) different from that of the outer arms.

scholarly journals Involvement of Huntingtin in Development and Ciliary Beating Regulation of Larvae of the Sea Urchin, Hemicentrotus pulcherrimus

2021 ◽  
Vol 22 (10) ◽  
pp. 5116
Author(s):  
Hideki Katow ◽  
Tomoko Katow ◽  
Hiromi Yoshida ◽  
Masato Kiyomoto
Keyword(s):  
Sea Urchin ◽  
The Body ◽  
Brdu Incorporation ◽  
Wild Type ◽  
Blastula Stage ◽  
Ciliary Beating ◽  
Marker Proteins ◽  
Disease Protein ◽  
Pattern Regulation ◽  
Hemicentrotus Pulcherrimus

The multiple functions of the wild type Huntington’s disease protein of the sea urchin Hemicentrotus pulcherrimus (Hp-Htt) have been examined using the anti-Hp-Htt antibody (Ab) raised against synthetic oligopeptides. According to immunoblotting, Hp-Htt was detected as a single band at around the 350 kDa region at the swimming blastula stage to the prism larva stage. From the 2-arm pluteus stage (2aPL), however, an additional smaller band at the 165 kDa region appeared. Immunohistochemically, Hp-Htt was detected in the nuclei and the nearby cytoplasm of the ectodermal cells from the swimming blastula stage, and the blastocoelar cells from the mid-gastrula stage. The Ab-positive signal was converged to the ciliary band-associated strand (CBAS). There, it was accompanied by several CBAS-marker proteins in the cytoplasm, such as glutamate decarboxylase. Application of Hp-Htt morpholino (Hp-Htt-MO) has resulted in shortened larval arms, accompanied by decreased 5-bromo-2-deoxyuridin (BrdU) incorporation by the ectodermal cells of the larval arms. Hp-Htt-MO also resulted in lowered ciliary beating activity, accompanied by a disordered swirling pattern formation around the body. These Hp-Htt-MO-induced deficiencies took place after the onset of CBAS system formation at the larval arms. Thus, Hp-Htt is involved in cell proliferation and the ciliary beating pattern regulation signaling system in pluteus larvae.

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.

Metabolic importance of Na+/K+-ATPase activity during sea urchin development.

1997 ◽  
Vol 200 (22) ◽  
pp. 2881-2892 ◽  
Author(s):  
P Leong ◽  
D Manahan
Keyword(s):  
Enzyme Activity ◽  
Atpase Activity ◽  
Sea Urchin ◽  
Sodium Pump ◽  
Sea Water ◽  
Strongylocentrotus Purpuratus ◽  
Lytechinus Pictus ◽  
Stimulation Of

Early stages of animal development have high mass-specific rates of metabolism. The biochemical processes that establish metabolic rate and how these processes change during development are not understood. In this study, changes in Na+/K+-ATPase activity (the sodium pump) and rate of oxygen consumption were measured during embryonic and early larval development for two species of sea urchin, Strongylocentrotus purpuratus and Lytechinus pictus. Total (in vitro) Na+/K+-ATPase activity increased during development and could potentially account for up to 77 % of larval oxygen consumption in Strongylocentrotus purpuratus (pluteus stage) and 80 % in Lytechinus pictus (prism stage). The critical issue was addressed of what percentage of total enzyme activity is physiologically active in living embryos and larvae and thus what percentage of metabolism is established by the activity of the sodium pump during development. Early developmental stages of sea urchins are ideal for understanding the in vivo metabolic importance of Na+/K+-ATPase because of their small size and high permeability to radioactive tracers (86Rb+) added to sea water. A comparison of total and in vivo Na+/K+-ATPase activities revealed that approximately half of the total activity was utilized in vivo. The remainder represented a functionally active reserve that was subject to regulation, as verified by stimulation of in vivo Na+/K+-ATPase activity in the presence of the ionophore monensin. In the presence of monensin, in vivo Na+/K+-ATPase activities in embryos of S. purpuratus increased to 94 % of the maximum enzyme activity measured in vitro. Stimulation of in vivo Na+/K+-ATPase activity was also observed in the presence of dissolved alanine, presumably due to the requirement to remove the additional intracellular Na+ that was cotransported with alanine from sea water. The metabolic cost of maintaining the ionic balance was found to be high, with this process alone accounting for 40 % of the metabolic rate of sea urchin larvae (based on the measured fraction of total Na+/K+-ATPase that is physiologically active in larvae of S. purpuratus). Ontogenetic changes in pump activity and environmentally induced regulation of reserve Na+/K+-ATPase activity are important factors that determine a major proportion of the metabolic costs of sea urchin development.

Tension at the Surface of Sea-Urchin Egg: A Critical Examination of Cole's Experiment

1964 ◽  
Vol 41 (4) ◽  
pp. 893-906 ◽  
Author(s):  
MITSUKI YONEDA
Keyword(s):  
Surface Area ◽  
Sea Urchin ◽  
Contact Area ◽  
Surface Force ◽  
Critical Examination ◽  
Compression Method ◽  
Osmotic Swelling ◽  
Technical Errors ◽  
Sea Urchin Egg ◽  
Hemicentrotus Pulcherrimus

1. The compression method for calculating the surface force of the sea-urchin egg, developed by Cole (1932), has been critically repeated using unfertilized eggs of Hemicentrotus pulcherrimus. 2. Estimation of contact area involved in Cole's equation introduces technical errors. 3. The tension recalculated by another equation including surface area as a parameter is found to remain constant irrespective of change in surface area. This is in conflict with the classical belief that the cortex of the egg of sea urchin is elastic. 4. Neither osmotic swelling nor osmotic shrinkage of the egg affects the tension.

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