Language：English   Publisher：CELL PRESS  

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：JASRI  

SAXS signals from the DNA of salmon spermatozoa in an aqueous solution were analyzed using a rheometer-device, which controlled the rate of shearing during SAXS observations. When shearing rate > 1000 s−1 was applied to the DNA suspension, DNA strands were not aligned as observed with other biological filaments, but a stable diffraction peak approximately ranging 1.7–1.8 [nm−1] of Q-values were observed. We compared our observation with simulated SAXS signals.

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Language：Japanese   Publishing type：Research paper (scientific journal)   Publisher：Chuo Uiversity  

In this study, we designed a novel optics to scan laser light on the front focal plane of condenser lens, i.e., aperture plane, that is, with light sources placed optically at an optically infinite distance from specimens. Diatom standard specimens were used for the observation in both the phase contrast image and the dark field microscopy, and we could reduce speckle noise under applicable magnitude. Although we could not solve the problem of biased brightness of illumination depending on specimen angles, the new optics presented here is expected to be useful for the observation of fine structures with high image brightness.

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：ELIFE SCIENCES PUBLICATIONS LTD  

Microtubules (MTs) are hollow cylinders made of tubulin, a GTPase responsible for essential functions during cell growth and division, and thus, key target for anti-tumor drugs. In MTs, GTP hydrolysis triggers structural changes in the lattice, which are responsible for interaction with regulatory factors. The stabilizing GTP-cap is a hallmark of MTs and the mechanism of the chemical-structural link between the GTP hydrolysis site and the MT lattice is a matter of debate. We have analyzed the structure of tubulin and MTs assembled in the presence of fluoride salts that mimic the GTP-bound and GDP•Pi transition states. Our results challenge current models because tubulin does not change axial length upon GTP hydrolysis. Moreover, analysis of the structure of MTs assembled in the presence of several nucleotide analogues and of taxol allows us to propose that previously described lattice expansion could be a post-hydrolysis stage involved in Pi release.

DOI： 10.7554/eLife.50155

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Language：Japanese   Publisher：(一社)日本細胞生物学会  

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Language：English   Publisher：The Biophysical Society of Japan General Incorporated Association  

DOI： 10.2142/biophys.54.S164_5

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Language：English   Publisher：The Biophysical Society of Japan General Incorporated Association  

DOI： 10.2142/biophys.53.S138_4

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Language：English   Publisher：The Biophysical Society of Japan General Incorporated Association  

DOI： 10.2142/biophys.50.S56_6

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Language：English   Publisher：CELL PRESS  

DOI： 10.1016/j.bpj.2008.12.2680

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Language：English   Publisher：The Biophysical Society of Japan General Incorporated Association  

DOI： 10.2142/biophys.49.S56_4

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Publishing type：Research paper (scientific journal)  

DOI： 10.2108/zsj.25.560

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：AMER INST PHYSICS  

With conventional light microscopy, precision in the measurement of the displacement of a specimen depends on the signal-to-noise ratio when we measure the light intensity of magnified images. This implies that, for the improvement of precision, getting brighter images and reducing background light noise are both inevitably required. For this purpose, we developed a new optics for laser dark-field illumination. For the microscopy, we used a laser beam and a pair of axicons (conical lenses) to get an optimal condition for dark-field observations. The optics was applied to measuring two dimensional microbead displacements with subnanometer precision. The bandwidth of our detection system overall was 10 kHz. Over most of this bandwidth, the observed noise level was as small as 0.1 nm/radicalHz.

DOI： 10.1063/1.2839914

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Publishing type：Research paper (international conference proceedings)  

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Language：English   Publisher：The Biophysical Society of Japan General Incorporated Association  

DOI： 10.2142/biophys.48.S106_6

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Language：English   Publisher：The Biophysical Society of Japan General Incorporated Association  

DOI： 10.2142/biophys.48.S180_3

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Language：English   Publisher：The Biophysical Society of Japan General Incorporated Association  

DOI： 10.2142/biophys.47.S154_3

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Language：English   Publisher：ZOOLOGICAL SOC JAPAN  

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Language：English   Publisher：The Biophysical Society of Japan General Incorporated Association  

DOI： 10.2142/biophys.46.S360_3

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Language：English   Publisher：ZOOLOGICAL SOC JAPAN  

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Language：English   Publisher：ZOOLOGICAL SOC JAPAN  

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Language：English   Publisher：ZOOLOGICAL SOC JAPAN  

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Language：Japanese   Publisher：The Biophysical Society of Japan General Incorporated Association  

DOI： 10.2142/biophys.44.S239_4

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Fragmented flagellar axonemes of sand dollar spermatozoa were reactivated by rapid photolysis of caged ATP. After a time lag of 10 ms, axonemes treated with protease started sliding disintegration. Axonemes without protease digestion started nanometer-scale high-frequency oscillation after a similar time lag. Force development in the sliding disintegration was measured with a flexible glass needle and its time course was corresponded well to that of the dynein-ADP intermediate production estimated using kinetic rates previously reported. However, with a high concentration (~80 μM) of vanadate, which binds to the dynein-ADP intermediate and forms a stable complex of dynein-ADP-vanadate, the time course of force development in sliding disintegration was not affected at all. In the case of high frequency oscillation, the time lag to start the oscillation, the initial amplitude, and the initial frequency were not affected by vanadate, though the oscillation once started was damped more quickly at higher concentrations of vanadate. These results suggest that during the initial turnover of ATP hydrolysis, force generation of dynein is not blocked by vanadate. A vanadate-insensitive dynein-ADP is postulated as a force-generating intermediate.

DOI： 10.1016/S0006-3495(99)76999-7

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Authorship：Lead author   Language：Japanese  

File： THECELL_SKamimura2023Mar.pdf

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Total pages：3, 480, 4, 2p   Language：Japanese  

ASIN

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Total pages：3, 248, 2p   Language：Japanese  

ASIN

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Total pages：728   Responsible for pages：60   Language：Japanese   Book type：Scholarly book

http://www.tkd-pbl.com/book/b182504.html

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Total pages：360   Responsible for pages：360   Language：Japanese   Book type：Scholarly book

http://www.tkd-pbl.com/book/b182504.html

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Language：Japanese   Book type：Scholarly book

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Total pages：1620   Responsible for pages：40   Language：Japanese   Book type：Scholarly book

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Total pages：403   Language：English   Book type：Scholarly book

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Language：English   Publisher：WILEY-BLACKWELL  

Microtubules are key components of the cytoskeleton in eukaryotic cells. The dynamics between assembled microtubules and free tubulin dimers in the cytoplasm is closely related to the active shape changes of microtubule networks. One of the most fundamental questions is the association of microtubule dynamics with the molecular conformation of tubulin within microtubules. To address this issue, we applied a new technique for the rapid shear-flow alignment of biological filaments, enabling us to acquire the structural periodicity data of microtubules by X-ray fiber diffraction under various physiological conditions. We classified microtubules into three main groups on the basis of distinct axial tubulin periodicities and mean microtubule diameters that varied depending on GTP hydrolysis and the content of paclitaxel, a microtubule stabilizer. Paclitaxel induced rapid changes in tubulin axial repeats in a cooperative manner. This is the first demonstration of dynamic changes of axial tubulin repeats within native microtubules without fixation. We also found extraordinary features of negative thermal expansion of axial tubulin repeats in both paclitaxel-stabilized and GMPCPP-containing microtubules. Our results suggest that even in assembled microtubules, both GTP- and GDP-tubulin dimers can undergo dynamic conversion between at least two different states: short and long configurations. (c) 2016 Wiley Periodicals, Inc.

DOI： 10.1002/cm.21283

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Language：English   Publisher：WILEY  

Microtubules are key components of the cytoskeleton in eukaryotic cells. The dynamics between assembled microtubules and free tubulin dimers in the cytoplasm is closely related to the active shape changes of microtubule networks. One of the most fundamental questions is the association of microtubule dynamics with the molecular conformation of tubulin within microtubules. To address this issue, we applied a new technique for the rapid shear-flow alignment of biological filaments, enabling us to acquire the structural periodicity data of microtubules by X-ray fiber diffraction under various physiological conditions. We classified microtubules into three main groups on the basis of distinct axial tubulin periodicities and mean microtubule diameters that varied depending on GTP hydrolysis and the content of paclitaxel, a microtubule stabilizer. Paclitaxel induced rapid changes in tubulin axial repeats in a cooperative manner. This is the first demonstration of dynamic changes of axial tubulin repeats within native microtubules without fixation. We also found extraordinary features of negative thermal expansion of axial tubulin repeats in both paclitaxel-stabilized and GMPCPP-containing microtubules. Our results suggest that even in assembled microtubules, both GTP- and GDP-tubulin dimers can undergo dynamic conversion between at least two different states: short and long configurations. (c) 2016 Wiley Periodicals, Inc.

DOI： 10.1002/cm.21283

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Language：English   Publisher：環境バイオテクノロジー学会  

We could generate very small air-nanobubbles, having diameters smaller than 100 nm, using a household hand mixer. The bubbles were stable in water containing 1% ethanol more than one month and show zeta potential of -30 to -40 mV. The addition of NaCl to nonobubble-water caused changes in the diameter and the number of bubbles, larger in size and less in numbers.

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Language：English   Publisher：CELL PRESS  

The high homology of its axonemal components with humans and a large repertoire of axonemal mutants make Chlamydomonas a useful model system for experiments on the structure and function of eukaryotic cilia and flagella. Using this organism, we explored the spatial arrangement of axonemal components under physiological conditions by small-angle x-ray fiber diffraction. Axonemes were oriented in physiological solution by continuous shear flow and exposed to intense and stable x rays generated in the synchrotron radiation facility SPring-8, BL45XU. We compared diffraction patterns from axonemes isolated from wild-type and mutant strains lacking the whole outer arm (oda1), radial spoke (pf14), central apparatus (pf18), or the a-chain of the outer arm dynein (oda11). Diffraction of the axonemes showed a series of well-defined meridional/layer-line and equatorial reflections. Diffraction patterns from mutant axonemes exhibited a systematic loss/attenuation of meridional/layer-line reflections, making it possible to determine the origin of various reflections. The 1/24 and 1/12 nm(-1) meridional reflections of oda1 and oda11 were much weaker than those of the wild-type, suggesting that the outer dynein arms are the main contributor to these reflections. The weaker 1/32 and 1/13.7 nm(-1) meridional reflections from pf14 compared with the wild-type suggest that these reflections come mainly from the radial spokes. The limited contribution of the central pair apparatus to the diffraction patterns was confirmed by the similarity between the patterns of the wild-type and pf18. The equatorial reflections were complex, but a comparison with electron micrograph-based models allowed the density of each axonemal component to be estimated. Addition of ATP to rigor-state axonemes also resulted in subtle changes in equatorial intensity profiles, which could report nucleotide-dependent structural changes of the dynein arms. The first detailed description of axonemal reflections presented here serves as a landmark for further x-ray diffraction studies to monitor the action of constituent proteins in functional axonemes.

DOI： 10.1016/j.bpj.2015.04.039

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Language：English   Publisher：CELL PRESS  

The high homology of its axonemal components with humans and a large repertoire of axonemal mutants make Chlamydomonas a useful model system for experiments on the structure and function of eukaryotic cilia and flagella. Using this organism, we explored the spatial arrangement of axonemal components under physiological conditions by small-angle x-ray fiber diffraction. Axonemes were oriented in physiological solution by continuous shear flow and exposed to intense and stable x rays generated in the synchrotron radiation facility SPring-8, BL45XU. We compared diffraction patterns from axonemes isolated from wild-type and mutant strains lacking the whole outer arm (oda1), radial spoke (pf14), central apparatus (pf18), or the a-chain of the outer arm dynein (oda11). Diffraction of the axonemes showed a series of well-defined meridional/layer-line and equatorial reflections. Diffraction patterns from mutant axonemes exhibited a systematic loss/attenuation of meridional/layer-line reflections, making it possible to determine the origin of various reflections. The 1/24 and 1/12 nm(-1) meridional reflections of oda1 and oda11 were much weaker than those of the wild-type, suggesting that the outer dynein arms are the main contributor to these reflections. The weaker 1/32 and 1/13.7 nm(-1) meridional reflections from pf14 compared with the wild-type suggest that these reflections come mainly from the radial spokes. The limited contribution of the central pair apparatus to the diffraction patterns was confirmed by the similarity between the patterns of the wild-type and pf18. The equatorial reflections were complex, but a comparison with electron micrograph-based models allowed the density of each axonemal component to be estimated. Addition of ATP to rigor-state axonemes also resulted in subtle changes in equatorial intensity profiles, which could report nucleotide-dependent structural changes of the dynein arms. The first detailed description of axonemal reflections presented here serves as a landmark for further x-ray diffraction studies to monitor the action of constituent proteins in functional axonemes.

DOI： 10.1016/j.bpj.2015.04.039

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Language：English   Publisher：WILEY-BLACKWELL  

Ciliobrevin has recently been found to be a membrane-permeable inhibitor that is specific to AAA+ molecular motors such as cytoplasmic dyneins. In this study, we investigated how ciliobrevin inhibited the motility of sperm from sea urchins: Hemicentrotus pulcherrimus, Pseudocentrotus depressus, and Anthocidaris crassispina. After application of 100 M of ciliobrevin A to live spermatozoa, swimming speed decreased gradually and flagellar motion stopped almost completely within 5 to 10 min. This inhibition was reversible and the frequency of flagellar beating was reduced in a concentration-dependent manner. Ciliobrevin had similar inhibitory effects on the flagellar beating of demembranated and reactivated sperm and the sliding disintegration of trypsin-treated axonemes. We also analyzed the curvature and shear angle of the beating flagella and found that the proximal region of the sperm flagellum was less sensitive to ciliobrevin compared with more distal regions, where bending motions were blocked completely. Interestingly, the shear angle analysis of flagellar motility showed that ciliobrevin induced highly asymmetric bends in the proximal region of the flagellum. These results suggest that there is heterogeneity in the inhibitory thresholds of dynein motors, which depend on the regions along the flagellar shaft (proximal or distal) and on the sites of doublets in the flagellar cross-section (doublet numbers). We expect that it will be possible to map the functional differences in dynein subtypes along and/or around the cross-sections of flagellar axonemes by analyzing the inhibitory effects of ciliobrevin. (c) 2015 Wiley Periodicals, Inc.

DOI： 10.1002/cm.21218

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Language：English   Publisher：WILEY-BLACKWELL  

Ciliobrevin has recently been found to be a membrane-permeable inhibitor that is specific to AAA+ molecular motors such as cytoplasmic dyneins. In this study, we investigated how ciliobrevin inhibited the motility of sperm from sea urchins: Hemicentrotus pulcherrimus, Pseudocentrotus depressus, and Anthocidaris crassispina. After application of 100 M of ciliobrevin A to live spermatozoa, swimming speed decreased gradually and flagellar motion stopped almost completely within 5 to 10 min. This inhibition was reversible and the frequency of flagellar beating was reduced in a concentration-dependent manner. Ciliobrevin had similar inhibitory effects on the flagellar beating of demembranated and reactivated sperm and the sliding disintegration of trypsin-treated axonemes. We also analyzed the curvature and shear angle of the beating flagella and found that the proximal region of the sperm flagellum was less sensitive to ciliobrevin compared with more distal regions, where bending motions were blocked completely. Interestingly, the shear angle analysis of flagellar motility showed that ciliobrevin induced highly asymmetric bends in the proximal region of the flagellum. These results suggest that there is heterogeneity in the inhibitory thresholds of dynein motors, which depend on the regions along the flagellar shaft (proximal or distal) and on the sites of doublets in the flagellar cross-section (doublet numbers). We expect that it will be possible to map the functional differences in dynein subtypes along and/or around the cross-sections of flagellar axonemes by analyzing the inhibitory effects of ciliobrevin. (c) 2015 Wiley Periodicals, Inc.

DOI： 10.1002/cm.21218

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Language：English   Publisher：COMPANY OF BIOLOGISTS LTD  

During their chemotactic swimming toward eggs, sperm cells detect their species-specific chemoattractant and sense concentration gradients by unknown mechanisms. After sensing the attractant, sperm cells commonly demonstrate a series of responses involving different swimming patterns by changing flagellar beats, gradually approaching a swimming path toward the eggs, which is the source of chemoattractants. Shiba et al. observed a rapid increase in intracellular Ca2+ concentrations in Ciona spermatozoa after sensing chemoattractants; however, the biochemical processes occurring inside the sperm cells are unclear. In the present study, we focused on the timing and sensing mechanism of chemical signal detection in Ciona. One of the most crucial problems to be solved is defining the initial epoch of chemotactic responses. We adopted a high rate of video recording (600 Hz) for detailed analysis of sperm motion and a novel method for detecting subtle signs of beat forms and moving paths of sperm heads. From these analyses, we estimated a virtual sensing point of the attractant before initiation of motility responses and found that the time delay from sensing to motility responses was almost constant. To evaluate the efficiency of this constant delay model, we performed computer simulation of chemotactic behaviors of Ciona spermatozoa.

DOI： 10.1242/bio.20137351

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Language：English   Publisher：COMPANY OF BIOLOGISTS LTD  

During their chemotactic swimming toward eggs, sperm cells detect their species-specific chemoattractant and sense concentration gradients by unknown mechanisms. After sensing the attractant, sperm cells commonly demonstrate a series of responses involving different swimming patterns by changing flagellar beats, gradually approaching a swimming path toward the eggs, which is the source of chemoattractants. Shiba et al. observed a rapid increase in intracellular Ca2+ concentrations in Ciona spermatozoa after sensing chemoattractants; however, the biochemical processes occurring inside the sperm cells are unclear. In the present study, we focused on the timing and sensing mechanism of chemical signal detection in Ciona. One of the most crucial problems to be solved is defining the initial epoch of chemotactic responses. We adopted a high rate of video recording (600 Hz) for detailed analysis of sperm motion and a novel method for detecting subtle signs of beat forms and moving paths of sperm heads. From these analyses, we estimated a virtual sensing point of the attractant before initiation of motility responses and found that the time delay from sensing to motility responses was almost constant. To evaluate the efficiency of this constant delay model, we performed computer simulation of chemotactic behaviors of Ciona spermatozoa.

DOI： 10.1242/bio.20137351

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Language：English   Publisher：SPRINGER/PLENUM PUBLISHERS  

Diving beetle larvae use their mandibles in two ways: capturing prey and sucking their body fluid. Catching and consuming the prey's most nutritious body part leads to the highest feeding efficiency. To test this, Dytiscus sharpi sharpi larvae were given tadpoles (Rana ornativentris) as food and their feeding behaviors were observed. Dytiscus larvae preferred to catch tadpoles by the abdomens rather than by other parts. Tadpoles soon became immobilized and in most cases the beetle larvae started eating abdomens first. Beetle larvae tried to change biting site to tadpole's abdomen when the tadpole was initially caught by the head or tail. More food was absorbed from the abdomen than the head or tail suggesting that the feeding behavior of beetle larva is optimized to obtain nutrition efficiently from the tadpole abdomen.

DOI： 10.1007/s10905-014-9475-z

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Language：Japanese  

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Language：Japanese   Publishing type：Article, review, commentary, editorial, etc. (other)  

From gut-inhabiting bacteria to sea-dwelling algae, microorganisms display a penchant for|rn|coordinated movement. Nonlinear dynamics and fluid mechanics can help explain the curious behavior.

DOI： 10.1063/PT.3.1715

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Language：English   Publisher：ACADEMIC PRESS INC ELSEVIER SCIENCE  

We report the first X-ray diffraction patterns recorded from single axonemes of eukaryotic flagella with a diameter of only <0.2 mu m, by using the technique of cryomicrodiffraction. A spermatozoon isolated from the testis of a fruit fly, Drosophila melanogaster, either intact or demembranated, was mounted straight in a glass capillary, quickly frozen and its 800-mu m segment was irradiated end-on with intense synchrotron radiation X-ray microbeams (diameter, similar to 2 mu m) at 74 K. Well-defined diffraction patterns were recorded, consisting of a large number of isolated reflection spots, extending up to 1/5 nm(-1). These reflections showed a tendency to peak every 20 degrees, i.e., the patterns had features of an 18-fold rotational symmetry as expected from the 9-fold rotational symmetry of axonemal structure. This means that the axonemes remain untwisted, even after the manual mounting procedure. The diffraction patterns were compared with the results of model calculations based on a published electron micrograph of the Drosophila axoneme. The comparison provided information about the native state of axoneme, including estimates of axonemal diameter, interdoublet spacing, and masses of axonemal components relative to those of microtubules (e.g., radial spokes, dynein arms, and proteins associated with accessory singlet microtubules). When combined with the genetic resource of Drosophila, the technique presented here will serve as a powerful tool for studying the structure-function relationship of eukaryotic flagella in general. (c) 2012 Elsevier Inc. All rights reserved.

DOI： 10.1016/j.jsb.2012.03.011

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Language：English   Publisher：ACADEMIC PRESS INC ELSEVIER SCIENCE  

We report the first X-ray diffraction patterns recorded from single axonemes of eukaryotic flagella with a diameter of only <0.2 mu m, by using the technique of cryomicrodiffraction. A spermatozoon isolated from the testis of a fruit fly, Drosophila melanogaster, either intact or demembranated, was mounted straight in a glass capillary, quickly frozen and its 800-mu m segment was irradiated end-on with intense synchrotron radiation X-ray microbeams (diameter, similar to 2 mu m) at 74 K. Well-defined diffraction patterns were recorded, consisting of a large number of isolated reflection spots, extending up to 1/5 nm(-1). These reflections showed a tendency to peak every 20 degrees, i.e., the patterns had features of an 18-fold rotational symmetry as expected from the 9-fold rotational symmetry of axonemal structure. This means that the axonemes remain untwisted, even after the manual mounting procedure. The diffraction patterns were compared with the results of model calculations based on a published electron micrograph of the Drosophila axoneme. The comparison provided information about the native state of axoneme, including estimates of axonemal diameter, interdoublet spacing, and masses of axonemal components relative to those of microtubules (e.g., radial spokes, dynein arms, and proteins associated with accessory singlet microtubules). When combined with the genetic resource of Drosophila, the technique presented here will serve as a powerful tool for studying the structure-function relationship of eukaryotic flagella in general. (c) 2012 Elsevier Inc. All rights reserved.

DOI： 10.1016/j.jsb.2012.03.011

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Language：English   Publisher：ACADEMIC PRESS INC ELSEVIER SCIENCE  

Although eukaryotic flagella and cilia all share the basic 9 + 2 microtubule-organization of their internal axonemes, and are capable of generating bending-motion, the waveforms, amplitudes, and velocities of the bending-motions are quite diverse. To explore the structural basis of this functional diversity of flagella and cilia, we here compare the axonemal structure of three different organisms with widely divergent bending-motions by electron cryo-tomography. We reconstruct the 3D structure of the axoneme of Tetrahymena cilia, and compare it with the axoneme of the flagellum of sea urchin sperm, as well as with the axoneme of Chlamydomonas flagella, which we analyzed previously. This comparative structural analysis defines the diversity of molecular architectures in these organisms, and forms the basis for future correlation with their different bending-motions. (C) 2012 Elsevier Inc. All rights reserved.

DOI： 10.1016/j.jsb.2012.02.012

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Language：English   Publisher：ACADEMIC PRESS INC ELSEVIER SCIENCE  

Although eukaryotic flagella and cilia all share the basic 9 + 2 microtubule-organization of their internal axonemes, and are capable of generating bending-motion, the waveforms, amplitudes, and velocities of the bending-motions are quite diverse. To explore the structural basis of this functional diversity of flagella and cilia, we here compare the axonemal structure of three different organisms with widely divergent bending-motions by electron cryo-tomography. We reconstruct the 3D structure of the axoneme of Tetrahymena cilia, and compare it with the axoneme of the flagellum of sea urchin sperm, as well as with the axoneme of Chlamydomonas flagella, which we analyzed previously. This comparative structural analysis defines the diversity of molecular architectures in these organisms, and forms the basis for future correlation with their different bending-motions. (C) 2012 Elsevier Inc. All rights reserved.

DOI： 10.1016/j.jsb.2012.02.012

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Language：Japanese   Publishing type：Article, review, commentary, editorial, etc. (other)   Publisher：バイオメカニズム学会  

DOI： 10.3951/sobim.34.176

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Other Link： https://jlc.jst.go.jp/DN/JALC/00355928752?from=CiNii

Language：Japanese   Publishing type：Article, review, commentary, editorial, etc. (other)   Publisher：エアロ・アクアバイオメカニズム研究会編  

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Language：English   Publisher：CELL PRESS  

In sea urchin spermatozoa, the energy source powering flagellar motion is provided as ATP produced by mitochondria located at the proximal ends of flagella. However, the bottleneck structure between the sperm head and the flagellar tail seems to restrict the free entry of ATP from mitochondria into the tail region. To test this possibility, we investigated the diffusion properties in sperm cells using fluorescence recovery after photobleaching. We found that the rate of fluorescence recovery in the head region was similar to 10% of that observed in the flagellar tail regions. We also found that, even within the tail region, rates varied depending on location, i.e., rates were slower at the more distal regions. Using computational analysis, the rate heterogeneity was shown to be caused mainly by the geometry of the sperm structure, even if little or no difference in diffusion rates through the neck region was assumed. Therefore, we concluded that materials such as ATP would generally diffuse freely between the heads and the flagella of sperm cells. We believe these findings regarding the diffusion properties inside spermatozoa provide further insights into material transportation and chemical signaling inside eukaryotic cilia and flagella.

DOI： 10.1016/j.bpj.2009.12.4314

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Language：English   Publisher：CELL PRESS  

In sea urchin spermatozoa, the energy source powering flagellar motion is provided as ATP produced by mitochondria located at the proximal ends of flagella. However, the bottleneck structure between the sperm head and the flagellar tail seems to restrict the free entry of ATP from mitochondria into the tail region. To test this possibility, we investigated the diffusion properties in sperm cells using fluorescence recovery after photobleaching. We found that the rate of fluorescence recovery in the head region was similar to 10% of that observed in the flagellar tail regions. We also found that, even within the tail region, rates varied depending on location, i.e., rates were slower at the more distal regions. Using computational analysis, the rate heterogeneity was shown to be caused mainly by the geometry of the sperm structure, even if little or no difference in diffusion rates through the neck region was assumed. Therefore, we concluded that materials such as ATP would generally diffuse freely between the heads and the flagella of sperm cells. We believe these findings regarding the diffusion properties inside spermatozoa provide further insights into material transportation and chemical signaling inside eukaryotic cilia and flagella.

DOI： 10.1016/j.bpj.2009.12.4314

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Language：English   Publisher：Zoological Society of Japan  

In sea urchin spermatozoa, the energy required for flagellar motility depends only on the diffusional supply from proximal mitochondria, and thus the diffusion rate inside flagella is one of the most crucial factors limiting the practical size and design of the motile machinery. To determine the diffusion rates of materials inside sperm cells, FRAP (fluorescence recovery after photobleaching) analysis of incorporated fluorescent probes is one of the most powerful approaches. However, the only practically possible method until now was to use the ester forms of fluorescence, and our choice was limited to those of relatively small molecular masses, such as fluorescein derivatives. In this report, we show that a modified single-cell electroporation technique can be applied as a new microinjection method for sperm cells of the sea urchin. The method was applied to FRAP analysis to determine the rate of intraflagellar diffusion.

DOI： 10.2108/zsj.27.279

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Language：English   Publisher：ZOOLOGICAL SOC JAPAN  

In sea urchin spermatozoa, the energy required for flagellar motility depends only on the diffusional supply from proximal mitochondria, and thus the diffusion rate inside flagella is one of the most crucial factors limiting the practical size and design of the motile machinery. To determine the diffusion rates of materials inside sperm cells, FRAP (fluorescence recovery after photobleaching) analysis of incorporated fluorescent probes is one of the most powerful approaches. However, the only practically possible method until now was to use the ester forms of fluorescence, and our choice was limited to those of relatively small molecular masses, such as fluorescein derivatives. In this report, we show that a modified single-cell electroporation technique can be applied as a new microinjection method for sperm cells of the sea urchin. The method was applied to FRAP analysis to determine the rate of intraflagellar diffusion.

DOI： 10.2108/zsj.27.279

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Language：English   Publishing type：Research paper, summary (international conference)   Publisher：CELL PRESS  

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X-ray fiber diffraction is one of the most useful methods for examining the structural details of live biological filaments under physiological conditions. To investigate biologically active or labile materials, it is crucial to finish fiber alignment within seconds before diffraction analysis. However, the conventional methods, e.g., magnetic field alignment and low-speed centrifugations, are time-consuming and not very useful for such purposes. Here, we introduce a new alignment method using a rheometer with two parallel disks, which was applied to observe fiber diffractions of axonemes, tobacco mosaic tobamovirus, and microtubules. We found that fibers were aligned within 5 s by giving high shearflow (1000-5000 s(-1)) to the medium and that methylcellulose contained in the medium (similar to 1%) was essential to the accomplishment of uniform orientation with a small angular deviation (<5 degrees). The new alignment method enabled us to execute structure analyses of axonemes by small-angle x-ray diffraction. Since this method was also useful for the quick alignment of purified microtubules, as well as tobacco mosaic tobamovirus, we expect that we can apply it to the structural analysis of many other biological filaments.

DOI： 10.1016/j.bpj.2009.09.041

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X-ray fiber diffraction is one of the most useful methods for examining the structural details of live biological filaments under physiological conditions. To investigate biologically active or labile materials, it is crucial to finish fiber alignment within seconds before diffraction analysis. However, the conventional methods, e.g., magnetic field alignment and low-speed centrifugations, are time-consuming and not very useful for such purposes. Here, we introduce a new alignment method using a rheometer with two parallel disks, which was applied to observe fiber diffractions of axonemes, tobacco mosaic tobamovirus, and microtubules. We found that fibers were aligned within 5 s by giving high shearflow (1000-5000 s(-1)) to the medium and that methylcellulose contained in the medium (similar to 1%) was essential to the accomplishment of uniform orientation with a small angular deviation (<5 degrees). The new alignment method enabled us to execute structure analyses of axonemes by small-angle x-ray diffraction. Since this method was also useful for the quick alignment of purified microtubules, as well as tobacco mosaic tobamovirus, we expect that we can apply it to the structural analysis of many other biological filaments.

DOI： 10.1016/j.bpj.2009.09.041

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For the conservation of the diving beetle Dytiscus sharpi (Wehncke) (Coleoptera: Dytiscidae), which is included on the Red Data List of Japan, it is critical to understand its ecological background. In the present study, we focused on its feeding behavior and nutritional needs under laboratory breeding conditions. First, we made a list of the possible candidates of prey in the same habitats where we caught D. sharpi. We found that the tadpoles of Rana ornativentris (Werner) were the major species present from March to April, when the beetle larvae appeared. Second, under our laboratory conditions, we investigated the size preference of beetle larvae preying on R. ornativentris tadpoles. We found a significant positive correlation between the developing stage of the larvae and the preferred prey size, i.e., the first and third instars preferred smaller and larger prey, respectively, but second instars did not show any size preference. The size of full-grown adult beetles was almost the same as that of wild insects found in the field, indicating that R. ornativentris tadpoles provide almost complete nutrition for larval growth. Finally, we investigated how the size and number of R. ornativentris tadpoles were correlated with the developing stage of beetle larvae. We suggest that it is crucial for Dytiscus larvae to have access to tadpoles of the proper size and amounts, depending on their growth stage. © 2009 Coleopterists Society.

DOI： 10.1649/1152.1

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Language：English   Publisher：COLEOPTERISTS SOC  

For the conservation of the diving beetle Dytiscus sharpi (Wehncke) (Coleoptera: Dytiscidae), which is included on the Red Data List of Japan, it is critical to understand its ecological background. In the present study, we focused on its feeding behavior and nutritional needs under laboratory breeding conditions. First, we made a list of the possible candidates of prey in the same habitats where we caught D. sharpi. We found that the tadpoles of Rana ornativentris (Werner) were the major species present from March to April, when the beetle larvae appeared. Second, under our laboratory conditions, we investigated the size preference of beetle larvae preying on R. ornativentris tadpoles. We found a significant positive correlation between the developing stage of the larvae and the preferred prey size, i.e., the first and third instars preferred smaller and larger prey, respectively, but second instars did not show any size preference. The size of full-grown adult beetles was almost the same as that of wild insects found in the field, indicating that R. ornativentris tadpoles provide almost complete nutrition for larval growth. Finally, we investigated how the size and number of R. ornativentris tadpoles were correlated with the developing stage of beetle larvae. We suggest that it is crucial for Dytiscus larvae to have access to tadpoles of the proper size and amounts, depending on their growth stage. © 2009 Coleopterists Society.

DOI： 10.1649/1152.1

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Language：English   Publisher：ELSEVIER ACADEMIC PRESS INC  

Eukaryotic cilia and flagella are highly ordered and precisely assembled cellular organelles. Here, to understand the mechanism of the orderly undulations of cilia and flagella, we shall draw a blueprint of their core structures and supporting scaffolds, that is, axonemes, and we shall describe the dynamic structural changes of components of the organelles. Small-angle X-ray scattering and diffraction are among the principal tools used to study protein polymers. These methods are now well established as indispensable tools that complement electron microscopy, providing information on the structure and dynamics of biological materials at atomic resolution in near-physiological environments. For instance, X-ray diffraction studies of skeletal muscles have contributed greatly to our understanding of the structure and molecular mechanisms of muscles. However, owing to the minute size and low diffracting power of axonemes, few attempts at X-ray diffraction of axonemes have been reported. The advent of third-generation synchrotron radiation facilities now makes these attempts feasible, because we now have stable and intense X-rays that enable us to obtain diffractions from the axonemes. In this chapter, we provide a concise practical guide to this new avenue for structural analysis of axonemes.

DOI： 10.1016/S0091-679X(08)91005-0

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Language：English   Publisher：ELSEVIER ACADEMIC PRESS INC  

Eukaryotic cilia and flagella are highly ordered and precisely assembled cellular organelles. Here, to understand the mechanism of the orderly undulations of cilia and flagella, we shall draw a blueprint of their core structures and supporting scaffolds, that is, axonemes, and we shall describe the dynamic structural changes of components of the organelles. Small-angle X-ray scattering and diffraction are among the principal tools used to study protein polymers. These methods are now well established as indispensable tools that complement electron microscopy, providing information on the structure and dynamics of biological materials at atomic resolution in near-physiological environments. For instance, X-ray diffraction studies of skeletal muscles have contributed greatly to our understanding of the structure and molecular mechanisms of muscles. However, owing to the minute size and low diffracting power of axonemes, few attempts at X-ray diffraction of axonemes have been reported. The advent of third-generation synchrotron radiation facilities now makes these attempts feasible, because we now have stable and intense X-rays that enable us to obtain diffractions from the axonemes. In this chapter, we provide a concise practical guide to this new avenue for structural analysis of axonemes.

DOI： 10.1016/S0091-679X(08)91005-0

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Language：English   Publisher：COMPANY BIOLOGISTS LTD  

In sea-urchin spermatozoa, energy required for flagellar motility is provided by ATP diffusion from mitochondria located at the proximal ends of the flagella along with the creatine shuttle system. However, no direct analysis of the diffusion rates inside flagella has been carried out thus far. Using a FRAP (fluorescence recovery after photobleaching) technique, we determined the diffusion coefficients of fluorescein-derivatives (calcein, carboxyfluorescein and Oregon Green) to be 63-64 mu m(2) s(-1). Although these values are about one third of the estimates that were previously used for theoretical calculations, we concluded that the rate of ATP diffusion inside spermatozoa was high enough to support the continuous motility of sea-urchin sperm flagella if the creatine shuttle system is working. We also investigated the diffusion rate through the 'neck' region between the head and tail. When the head region of a calcein-loaded spermatozoon was photobleached, slow recovery of head fluorescence along with the decrease of fluorescence signal in the tail region was observed. It suggests that small molecules such as calcein (M(r), 622.54) can move beyond the boundary between the head and the flagellum. We expect that these findings regarding the diffusion properties inside spermatozoa will provide us with more general insights into the energy equilibrium and material transportation by passive diffusion inside eukaryotic cilia and flagella.

DOI： 10.1242/jeb.021923

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In sea-urchin spermatozoa, energy required for flagellar motility is provided by ATP diffusion from mitochondria located at the proximal ends of the flagella along with the creatine shuttle system. However, no direct analysis of the diffusion rates inside flagella has been carried out thus far. Using a FRAP (fluorescence recovery after photobleaching) technique, we determined the diffusion coefficients of fluorescein-derivatives (calcein, carboxyfluorescein and Oregon Green) to be 63-64μm2s-1. Although these values are about one third of the estimates that were previously used for theoretical calculations, we concluded that the rate of ATP diffusion inside spermatozoa was high enough to support the continuous motility of sea-urchin sperm flagella if the creatine shuttle system is working. We also investigated the diffusion rate through the 'neck' region between the head and tail. When the head region of a calcein-loaded spermatozoon was photobleached, slow recovery of head fluorescence along with the decrease of fluorescence signal in the tail region was observed. It suggests that small molecules such as calcein (M r, 622.54) can move beyond the boundary between the head and the flagellum. We expect that these findings regarding the diffusion properties inside spermatozoa will provide us with more general insights into the energy equilibrium and material transportation by passive diffusion inside eukaryotic cilia and flagella.

DOI： 10.1242/jeb.021923

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Language：English   Publisher：ZOOLOGICAL SOC JAPAN  

The effects of temperature on the mating behavior, gonad development, germ cell maturation, and egg spawning of the predaceous diving beetle Dytiscus sharpi (Coleoptera; Dytiscidae), were investigated. By field observations, we found that mating behavior started in October and occurred more frequently from November to December. Under our laboratory breeding conditions, we observed almost the same seasonal variation in mating behavior. We found that temperatures lower than 20 degrees C were required to trigger mating behavior. We also found the same temperature threshold triggered gonadogenesis as well as spermatogenesis. Furthermore, for females, exposure to lower temperatures (<8 degrees C) during the winter was required for egg maturation and spawning in spring; that is, there was a second threshold for successful female reproduction. We conclude that the termination of summer reproductive diapause of D. sharpi is regulated in a temperature-dependent manner, thus effecting the adaptation of D. sharpi to southern warm habitats.

DOI： 10.2108/zsj.24.1115

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The effects of temperature on the mating behavior, gonad development, germ cell maturation, and egg spawning of the predaceous diving beetle Dytiscus sharpi (Coleoptera; Dytiscidae), were investigated. By field observations, we found that mating behavior started in October and occurred more frequently from November to December. Under our laboratory breeding conditions, we observed almost the same seasonal variation in mating behavior. We found that temperatures lower than 20 degrees C were required to trigger mating behavior. We also found the same temperature threshold triggered gonadogenesis as well as spermatogenesis. Furthermore, for females, exposure to lower temperatures (<8 degrees C) during the winter was required for egg maturation and spawning in spring; that is, there was a second threshold for successful female reproduction. We conclude that the termination of summer reproductive diapause of D. sharpi is regulated in a temperature-dependent manner, thus effecting the adaptation of D. sharpi to southern warm habitats.

DOI： 10.2108/zsj.24.1115

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Language：Japanese   Publishing type：Article, review, commentary, editorial, etc. (other)   Publisher：羊土社  

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Language：English   Publishing type：Research paper, summary (international conference)   Publisher：BIOPHYSICAL SOCIETY  

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Language：Japanese   Publishing type：Article, review, commentary, editorial, etc. (other)   Publisher：エヌ・ティー・エス  

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Language：Japanese   Publishing type：Article, review, commentary, editorial, etc. (other)   Publisher：東京大学出版会  

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In the present study, we demonstrated that the mouse embryonic stem cells were differentiated into ciliated epithelial cells, with characteristics of normal ciliated cells. These cells expressed ciliary marker proteins, such as beta-tubulin IV and hepatocyte nuclear factor-3/forkhead homolog 4 (HFH-4), and processed microtubules were arranged in the 9 + 2 structure, which is the same specific alignment observed in normal ciliary microtubules. The cilia of these cells were beating at a frequency of 17-20 Hz. The differentiated embryoid bodies (EBs) containing these ciliated cells expressed respiratory marker genes such as thyroid transcription factor-1 and surfactant protein-C. For the induction of ciliated cells, culture of EBs in serum-free medium during the initial 2 days of the attachment was indispensable. When EBs were treated with bone morphogenetic proteins, the expression of HFH-4 was decreased, and the ciliated cells were scarcely differentiated. Previous methods for inducing ciliated cells in vitro from embryonic or adult tissues involved an air-liquid interface. The system used in this study more closely mimics the normal development of ciliated cells; thus, an added advantage of the system is as a tool for studying the differentiation mechanism of normal ciliated epithelial cells.

DOI： 10.1634/stemcells.2005-0464

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Language：English   Publisher：WILEY  

In the present study, we demonstrated that the mouse embryonic stem cells were differentiated into ciliated epithelial cells, with characteristics of normal ciliated cells. These cells expressed ciliary marker proteins, such as beta-tubulin IV and hepatocyte nuclear factor-3/forkhead homolog 4 (HFH-4), and processed microtubules were arranged in the 9 + 2 structure, which is the same specific alignment observed in normal ciliary microtubules. The cilia of these cells were beating at a frequency of 17-20 Hz. The differentiated embryoid bodies (EBs) containing these ciliated cells expressed respiratory marker genes such as thyroid transcription factor-1 and surfactant protein-C. For the induction of ciliated cells, culture of EBs in serum-free medium during the initial 2 days of the attachment was indispensable. When EBs were treated with bone morphogenetic proteins, the expression of HFH-4 was decreased, and the ciliated cells were scarcely differentiated. Previous methods for inducing ciliated cells in vitro from embryonic or adult tissues involved an air-liquid interface. The system used in this study more closely mimics the normal development of ciliated cells; thus, an added advantage of the system is as a tool for studying the differentiation mechanism of normal ciliated epithelial cells.

DOI： 10.1634/stemcells.2005-0464

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Language：English   Publisher：COMPANY OF BIOLOGISTS LTD  

Increased intracellular pH ([pH](i)) activates dynein in sea urchin and mammalian sperm and induces activation of flagellar motility. It is thought that cAMP-dependent protein phosphorylation is associated with motility activation through increasing [pH](i), but little attention has been given to the cAMP-independent phosphorylation also induced by the [pH](i) increase. The present study demonstrates that the increase in [pH](i) in starfish sperm induces the phosphorylation of axonemal proteins and activation of flagellar motility independently of cAMP. Flagellar motility of intact sperm was activated when the [pH](i) was raised by addition of NH4Cl. Histidine, which is known to activate motility of starfish sperm, also raised the [pH](i) during the motility activation. In addition, motility of demembranated sperm flagella was activated in a pH-dependent manner without cAMP. These results indicate that in starfish sperm it is the increase in [pH](i) that induces activation of flagellar motility. Moreover, phosphorylation of axonemal proteins (of molecular mass 25, 32 and 45 kDa) was observed during the pH-dependent and cAMP-independent motility activation of demembranated sperm. This suggests that the increase in [pH](i) regulates flagellar motility via cAMP-independent phosphorylation of axonemal proteins.

DOI： 10.1242/jeb.01906

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Language：English   Publishing type：Research paper, summary (international conference)   Publisher：ZOOLOGICAL SOC JAPAN  

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Language：English   Publisher：The Company of Biologists Limited, Cambridge  

DOI： 10.1242/jeb.01906

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Language：English   Publishing type：Research paper, summary (international conference)   Publisher：ELSEVIER SCIENCE BV  

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Language：English   Publisher：COLEOPTERISTS SOC  

For conservation purposes and to supply rare insects for laboratory use, a system for artificial breeding is crucial. However, in the case of carnivorous freshwater insects such as diving beetles, constant conditions in aquariums are difficult to maintain due to their high rate of food consumption. Furthermore, surface rippling caused by the pumping system for water circulation hinders the respiration of small larvae. We developed a new open aquarium system without water circulation that was successfully applied to the rearing of larvae of diving beetles, Dytiscus sharpi (Wehncke) (Coleoptera: Dytiscidae). In comparison to conventional methods, a high proportion of larvae developed into adult insects. The size of reared adults was almost the same as those of field-collected adults. The new method could be applied to the conservation and breeding of other rare species, such as water beetles and water bugs.

DOI： 10.1649/591

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Language：English   Publisher：COLEOPTERISTS SOC  

For conservation purposes and to supply rare insects for laboratory use, a system for artificial breeding is crucial. However, in the case of carnivorous freshwater insects such as diving beetles, constant conditions in aquariums are difficult to maintain due to their high rate of food consumption. Furthermore, surface rippling caused by the pumping system for water circulation hinders the respiration of small larvae. We developed a new open aquarium system without water circulation that was successfully applied to the rearing of larvae of diving beetles, Dytiscus sharpi (Wehncke) (Coleoptera: Dytiscidae). In comparison to conventional methods, a high proportion of larvae developed into adult insects. The size of reared adults was almost the same as those of field-collected adults. The new method could be applied to the conservation and breeding of other rare species, such as water beetles and water bugs.

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Language：English   Publisher：BIOPHYSICAL SOCIETY  

The 9+2 configuration of axonemes is one of the most conserved structures of eukaryotic organelles. Evidence so far has confirmed that bending of cilia and flagella is the result of active sliding of microtubules induced by dynein arms. If the conformational change of dynein motors, which would be a key step of force generation, is occurring in a three-dimensional manner, we can easily expect that the microtubule sliding should contain some transverse component, i.e., a motion in a direction at a right angle to the longitudinal axis of axonemes. Using a modified technique of atomic force microscopy, we found such transverse motion is actually occurring in an oscillatory manner when the axonemes of sea-urchin sperm flagella were adhered onto glass substrates. The motion was adenosine triphosphate-dependent and the observed frequency of oscillation was similar to that of oscillatory sliding of microtubules that had been shown to reflect the physiological activity of dynein arms (S. Kamimura and R. Kamiya. 1989. Nature. 340: 476-478; 1992. J. Cell Biol. 116: 1443-1454). Maximal amplitude of the diameter oscillation was around 10 nm, which was within a range of morphological change observed with electron microscopy (F.D. Warner. 1978. J. Cell Biol. 77: R19-R26; N. C. Zanetti, D. R. Mitchell, and F. D. Warner. 1979. J. Cell Biol. 80: 573-588).

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Language：English   Publisher：BIOPHYSICAL SOCIETY  

The 9+2 configuration of axonemes is one of the most conserved structures of eukaryotic organelles. Evidence so far has confirmed that bending of cilia and flagella is the result of active sliding of microtubules induced by dynein arms. If the conformational change of dynein motors, which would be a key step of force generation, is occurring in a three-dimensional manner, we can easily expect that the microtubule sliding should contain some transverse component, i.e., a motion in a direction at a right angle to the longitudinal axis of axonemes. Using a modified technique of atomic force microscopy, we found such transverse motion is actually occurring in an oscillatory manner when the axonemes of sea-urchin sperm flagella were adhered onto glass substrates. The motion was adenosine triphosphate-dependent and the observed frequency of oscillation was similar to that of oscillatory sliding of microtubules that had been shown to reflect the physiological activity of dynein arms (S. Kamimura and R. Kamiya. 1989. Nature. 340: 476-478; 1992. J. Cell Biol. 116: 1443-1454). Maximal amplitude of the diameter oscillation was around 10 nm, which was within a range of morphological change observed with electron microscopy (F.D. Warner. 1978. J. Cell Biol. 77: R19-R26; N. C. Zanetti, D. R. Mitchell, and F. D. Warner. 1979. J. Cell Biol. 80: 573-588).

DOI： 10.1016/S0006-3495(04)74110-7

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Language：English   Publisher：UNIV CHICAGO PRESS  

DOI： 10.1086/378903

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Language：English   Publisher：UNIV CHICAGO PRESS  

DOI： 10.1086/378903

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Odors play important roles in the communication of house mice. They release behaviors and prime changes of the physiological conditions of other individuals. In our previous study, we showed that sperm motility was lowered in the subordinate mice comparing with dominant mice. Our hypothesis is that the lowered sperm motility was due to some primer effects by odor substances derived from dominant mice. To test the hypothesis, we destroyed the vomeronasal organ (VNO) of male mice (VNX male) at 5 weeks of age and paired them with intact male mice (Experimental Group). As control group males, intact male mice were kept in pairs (Control Group). At 15 weeks of age, the sperm motility and weights of reproductive organs, and social dominance was analyzed. The subordinate VNX males were found to have high sperm motility comparable to the dominant males. It was suggested that there is male-to-male primer effects, mediated by VNO, that suppress sperm motility of the subordinate mice.

DOI： 10.2108/zsj.20.1355

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Language：English   Publisher：ZOOLOGICAL SOC JAPAN  

Odors play important roles in the communication of house mice. They release behaviors and prime changes of the physiological conditions of other individuals. In our previous study, we showed that sperm motility was lowered in the subordinate mice comparing with dominant mice. Our hypothesis is that the lowered sperm motility was due to some primer effects by odor substances derived from dominant mice. To test the hypothesis, we destroyed the vomeronasal organ (VNO) of male mice (VNX male) at 5 weeks of age and paired them with intact male mice (Experimental Group). As control group males, intact male mice were kept in pairs (Control Group). At 15 weeks of age, the sperm motility and weights of reproductive organs, and social dominance was analyzed. The subordinate VNX males were found to have high sperm motility comparable to the dominant males. It was suggested that there is male-to-male primer effects, mediated by VNO, that suppress sperm motility of the subordinate mice.

DOI： 10.2108/zsj.20.1355

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Language：English   Publisher：PERGAMON-ELSEVIER SCIENCE LTD  

It has been shown that sperm motility and other parameters related to the reproductive activity varied depending on the social status of male mice. In order to clarify whether such variation is derived from inborn difference or depends on any conditions during maturation, we investigated developmental change of sperm motility, reproductive organs, and body size of MY male mice. We also investigated how the establishment of social dominance of male mice during maturation was correlated with sperm motility. It was found that sperm motility was significantly higher during puberty than in adulthood, although there already existed relatively large statistical variance. The correlation between sperm motility and the social status was revealed to start after 10 weeks of age. It was suggested that a certain inborn difference of sperm motility became enlarged due to environmental factors experienced by male mice during maturation. (C) 2003 Elsevier Inc. All rights reserved.

DOI： 10.1016/j.physbeh.2003.07.001

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Language：English   Publisher：PERGAMON-ELSEVIER SCIENCE LTD  

It has been shown that sperm motility and other parameters related to the reproductive activity varied depending on the social status of male mice. In order to clarify whether such variation is derived from inborn difference or depends on any conditions during maturation, we investigated developmental change of sperm motility, reproductive organs, and body size of MY male mice. We also investigated how the establishment of social dominance of male mice during maturation was correlated with sperm motility. It was found that sperm motility was significantly higher during puberty than in adulthood, although there already existed relatively large statistical variance. The correlation between sperm motility and the social status was revealed to start after 10 weeks of age. It was suggested that a certain inborn difference of sperm motility became enlarged due to environmental factors experienced by male mice during maturation. (C) 2003 Elsevier Inc. All rights reserved.

DOI： 10.1016/j.physbeh.2003.07.001

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Language：Japanese   Publisher：日本機械学会  

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Language：English   Publisher：ELSEVIER SCIENCE BV  

We have devised a novel method to visualize the fluorescence spectrum of a single fluorescent molecule using prism-based spectroscopy. Equiping a total internal reflection microscope with a newly designed wedge prism, we obtained a spectral image of a single rhodamine red molecule attached to an essential light chain of myosin. We also obtained a spectral image of single-pair fluorescence resonance energy transfer between rhodamine red and Cy5 in a double-labeled myosin motor domain. This method could become a useful tool to investigate the dynamic processes of biomolecules at the single-molecule level. (C) 2002 Published by Elsevier Science B.V. on behalf of the Federation of European Biochemical Societies.

DOI： 10.1016/S0014-5793(02)02269-X

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Language：English   Publisher：ELSEVIER SCIENCE BV  

We have devised a novel method to visualize the fluorescence spectrum of a single fluorescent molecule using prism-based spectroscopy. Equiping a total internal reflection microscope with a newly designed wedge prism, we obtained a spectral image of a single rhodamine red molecule attached to an essential light chain of myosin. We also obtained a spectral image of single-pair fluorescence resonance energy transfer between rhodamine red and Cy5 in a double-labeled myosin motor domain. This method could become a useful tool to investigate the dynamic processes of biomolecules at the single-molecule level. (C) 2002 Published by Elsevier Science B.V. on behalf of the Federation of European Biochemical Societies.

DOI： 10.1016/S0014-5793(02)02269-X

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Language：English   Publisher：KLUWER ACADEMIC/PLENUM PUBL  

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Language：English   Publisher：KLUWER ACADEMIC/PLENUM PUBL  

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Language：English   Publisher：PERGAMON-ELSEVIER SCIENCE LTD  

In mammals, sperm activity is known to be varied largely according to individuals though physiological reasons have not been clarified yet. In our previous study [Koyama S, Kamimura S. Lowered sperm motility in mice of subordinate social status. Physiol Behav 1999;65:665-669.], we showed that sperm motility was higher in the dominant mice than the subordinate mice, by which it was suggested that social factors could affect sperm activity in mammals. In the present study, we investigated how the observed influence of social dominance would be modified by the existence of females. From 5 to 15 weeks of age, male mice were pair housed and were kept under three different housing conditions: (1) with females; (2) with bedding soiled by females; and (3) control group. The social dominance of the paired males was determined by resident-intruder tests that were carried out from 8 to 15 weeks of age. At the end of 15 weeks of age, sperm activity, weights of organs, level of serum testosterone and corticosterone were determined. It was revealed that sperm density was higher and weight of preputial glands was heavier in dominants than in subordinates when they were kept with females or female bedding, In the subordinates, however, there were no differences among the three housing conditions; that is, there were no female effects on the subordinates. On the other hand, sperm motility was high in the dominants of control group, low in the subordinates, and lower in the dominants that were kept with females. The dominants of the males that were kept with females showed high aggressiveness, and there were negative correlationships to be seen between aggressiveness and sperm motility. It was suggested that: (1) Female odor promotes spermatogenesis of the dominants, but it does not promote that of the subordinates. (2) Sperm motility is more affected by social dominance than by female odor. (3) Excessive aggressiveness has negative influence on sperm motility. (C) 2000 Elsevier Science Inc. All rights reserved.

DOI： 10.1016/S0031-9384(00)00361-9

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Language：English   Publisher：PERGAMON-ELSEVIER SCIENCE LTD  

In mammals, sperm activity is known to be varied largely according to individuals though physiological reasons have not been clarified yet. In our previous study [Koyama S, Kamimura S. Lowered sperm motility in mice of subordinate social status. Physiol Behav 1999;65:665-669.], we showed that sperm motility was higher in the dominant mice than the subordinate mice, by which it was suggested that social factors could affect sperm activity in mammals. In the present study, we investigated how the observed influence of social dominance would be modified by the existence of females. From 5 to 15 weeks of age, male mice were pair housed and were kept under three different housing conditions: (1) with females; (2) with bedding soiled by females; and (3) control group. The social dominance of the paired males was determined by resident-intruder tests that were carried out from 8 to 15 weeks of age. At the end of 15 weeks of age, sperm activity, weights of organs, level of serum testosterone and corticosterone were determined. It was revealed that sperm density was higher and weight of preputial glands was heavier in dominants than in subordinates when they were kept with females or female bedding, In the subordinates, however, there were no differences among the three housing conditions; that is, there were no female effects on the subordinates. On the other hand, sperm motility was high in the dominants of control group, low in the subordinates, and lower in the dominants that were kept with females. The dominants of the males that were kept with females showed high aggressiveness, and there were negative correlationships to be seen between aggressiveness and sperm motility. It was suggested that: (1) Female odor promotes spermatogenesis of the dominants, but it does not promote that of the subordinates. (2) Sperm motility is more affected by social dominance than by female odor. (3) Excessive aggressiveness has negative influence on sperm motility. (C) 2000 Elsevier Science Inc. All rights reserved.

DOI： 10.1016/S0031-9384(00)00361-9

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Language：English   Publisher：The Biophysical Society  

DOI： 10.1016/S0006-3495(99)76999-7

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DOI： 10.1016/S0006-3495(99)76999-7

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Language：Japanese   Publishing type：Article, review, commentary, editorial, etc. (other)   Publisher：日本光生物学協会編  

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Language：English   Publisher：Zoological Society of Japan  

CiNii Books

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Other Link： http://id.nii.ac.jp/1141/00034671/

Language：Japanese   Publisher：The Biophysical Society of Japan General Incorporated Association  

CiNii Books

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Language：English   Publisher：COMPANY OF BIOLOGISTS LTD  

Sea-urchin sperm flagella in a state of rigor were reactivated by rapid photolysis of caged ATP, After a time lag of 11-17 ms, all bends in the axonemes present during rigor began to be propagated towards the tip as if their propagation had not been interrupted. This result suggests that the site-specific activity of dyneins along the length of the axoneme is preserved even during rigor states when ATP is absent and that regulation of the activity can be restarted immediately with a new cycle of ATP turnover. During the starting transient, pre-existing rigor waves in the distal region were propagated without a change in the maximal shear angle until they disappeared at the tip. This was more evident when the rapid reactivation was triggered in high-viscosity solution, in which only the form of new bends was greatly affected by viscous load. After reactivation, the velocity of microtubule sliding increased and reached a plateau within 28 ms. This time course reflects the rate of force generation by dynein in situ.

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Language：English   Publisher：COMPANY OF BIOLOGISTS LTD  

Sea-urchin sperm flagella in a state of rigor were reactivated by rapid photolysis of caged ATP, After a time lag of 11-17 ms, all bends in the axonemes present during rigor began to be propagated towards the tip as if their propagation had not been interrupted. This result suggests that the site-specific activity of dyneins along the length of the axoneme is preserved even during rigor states when ATP is absent and that regulation of the activity can be restarted immediately with a new cycle of ATP turnover. During the starting transient, pre-existing rigor waves in the distal region were propagated without a change in the maximal shear angle until they disappeared at the tip. This was more evident when the rapid reactivation was triggered in high-viscosity solution, in which only the form of new bends was greatly affected by viscous load. After reactivation, the velocity of microtubule sliding increased and reached a plateau within 28 ms. This time course reflects the rate of force generation by dynein in situ.

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Language：Japanese   Publishing type：Article, review, commentary, editorial, etc. (other)   Publisher：日本生物物理学会編  

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Language：English   Publisher：Zoological Society of Japan  

CiNii Books

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Other Link： http://id.nii.ac.jp/1141/00033854/

Language：English   Publisher：PERGAMON-ELSEVIER SCIENCE LTD  

The correlation between social status and sperm motility of mice was investigated. From 5 to 15 weeks of age, mice were kept under two housing conditions, i.e., in pairs or in isolation. The social dominance in the paired mice was determined with the resident-intruder tests, which were carried out from 8 to 15 weeks of age. At the end of 15 weeks of age, sperm activity, weights of reproductive organs, and serum testosterone were determined. It was revealed that the sperm motility of dominant mice was significantly higher than that of the subordinates. The sperm motility of the isolated mice was also significantly higher than the subordinates. It was suggested that the subordinate social status lowered sperm motility. (C) 1999 Elsevier Science Inc.

DOI： 10.1016/S0031-9384(98)00205-4

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Language：English   Publisher：PERGAMON-ELSEVIER SCIENCE LTD  

The correlation between social status and sperm motility of mice was investigated. From 5 to 15 weeks of age, mice were kept under two housing conditions, i.e., in pairs or in isolation. The social dominance in the paired mice was determined with the resident-intruder tests, which were carried out from 8 to 15 weeks of age. At the end of 15 weeks of age, sperm activity, weights of reproductive organs, and serum testosterone were determined. It was revealed that the sperm motility of dominant mice was significantly higher than that of the subordinates. The sperm motility of the isolated mice was also significantly higher than the subordinates. It was suggested that the subordinate social status lowered sperm motility. (C) 1999 Elsevier Science Inc.

DOI： 10.1016/S0031-9384(98)00205-4

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Language：Japanese   Publishing type：Article, review, commentary, editorial, etc. (other)   Publisher：日学選書B 日本学術会議事務局・日本動物学会関東支部編集、財団法人 日本学術協力財団発行  

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Language：Japanese   Publishing type：Article, review, commentary, editorial, etc. (other)   Publisher：日本生物物理学会編  

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Language：Japanese   Publishing type：Article, review, commentary, editorial, etc. (other)   Publisher：遺伝  

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Language：Japanese   Publishing type：Article, review, commentary, editorial, etc. (other)  

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Language：English   Publisher：WILEY-LISS  

Flagellar axonemes of sea urchin sperm display high frequency (200-400 Hz) vibration with nanometer scale amplitudes in the presence of ATP [Kamimura and Kamiya, 1992: J. Cell Biol. 116:1443 -1454]. To investigate how various axonemal components affect the vibration, we examined vibration in wild-type and mutant axonemes of Chlamydomonas. At 1 mM ATP, wild-type axonemes underwent vibration at 100-650 Hz with amplitudes of 4-40 nm. This vibration was similar to, but less regular than, that in sea urchin sperm. Axonemes of the mutants ida1 and ida4 lacking part of the inner arm dynein underwent vibrations indistinguishable from that of wild-type. The mutant oda 1 lacking the entire outer arm underwent vibration at about half the wild-type frequency. Unexpectedly, the paralyzed mutants pf18 lacking the central pair and pf14 lacking the radial spokes displayed vibration with significantly higher frequencies and smaller amplitudes than those in the wild-type vibration. These results indicate that the high-frequency vibration is common to many kinds of mutant axonemes that lack various axonemal substructures, but that its manner is sensitive to the presence of outer arm dynein and the central pair/radial spoke system. Simultaneous measurements of amplitude and frequency in wild-type and mutant axonemes suggest that the velocity of microtubule sliding in vibrating axonemes is lower than the velocity of sliding under load-free conditions. The velocity is particularly low in pf18. A possible mechanism is proposed to explain the lower sliding velocity and vibration amplitude in the pf18 axoneme, based on an assumption that central pair/radial spoke system may work to regulate the switching of two antagonizing forces within the axoneme. (C) 1994 Wiley-Liss, Inc.

DOI： 10.1002/cm.970290209

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Language：English   Publisher：WILEY-LISS  

Flagellar axonemes of sea urchin sperm display high frequency (200-400 Hz) vibration with nanometer scale amplitudes in the presence of ATP [Kamimura and Kamiya, 1992: J. Cell Biol. 116:1443 -1454]. To investigate how various axonemal components affect the vibration, we examined vibration in wild-type and mutant axonemes of Chlamydomonas. At 1 mM ATP, wild-type axonemes underwent vibration at 100-650 Hz with amplitudes of 4-40 nm. This vibration was similar to, but less regular than, that in sea urchin sperm. Axonemes of the mutants ida1 and ida4 lacking part of the inner arm dynein underwent vibrations indistinguishable from that of wild-type. The mutant oda 1 lacking the entire outer arm underwent vibration at about half the wild-type frequency. Unexpectedly, the paralyzed mutants pf18 lacking the central pair and pf14 lacking the radial spokes displayed vibration with significantly higher frequencies and smaller amplitudes than those in the wild-type vibration. These results indicate that the high-frequency vibration is common to many kinds of mutant axonemes that lack various axonemal substructures, but that its manner is sensitive to the presence of outer arm dynein and the central pair/radial spoke system. Simultaneous measurements of amplitude and frequency in wild-type and mutant axonemes suggest that the velocity of microtubule sliding in vibrating axonemes is lower than the velocity of sliding under load-free conditions. The velocity is particularly low in pf18. A possible mechanism is proposed to explain the lower sliding velocity and vibration amplitude in the pf18 axoneme, based on an assumption that central pair/radial spoke system may work to regulate the switching of two antagonizing forces within the axoneme. (C) 1994 Wiley-Liss, Inc.

DOI： 10.1002/cm.970290209

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Language：Japanese   Publishing type：Article, review, commentary, editorial, etc. (other)   Publisher：丸善社  

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Language：English   Publisher：Rockefeller University Press  

DOI： 10.1083/jcb.118.4.865

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Language：English   Publisher：ROCKEFELLER UNIV PRESS  

Microtubules are built of tubulin subunits assembled into hollow cylinders which consist of parallel protofilaments. Thus, motor molecules interacting with a microtubule could do so either with one or several tubulin subunits. This makes it difficult to determine the structural requirements for the interaction. One way to approach the problem is to alter the surface lattice. This can be done in several ways. Protofilaments can be exposed on their inside (C-tubules or "sheets"), they can be made antiparallel (zinc sheets), or they can be rolled up (duplex tubules). We have exploited this polymorphism to study how the motor protein kinesin attached to a glass surface interacts and moves the various tubulin assemblies.
Microtubules glide over the surface along straight paths and with uniform velocities. In the case of C-tubules, approximately 40% glide similarly to microtubules, but a major fraction do not glide at all. This indicates (a) that a full cylindrical closure is not necessary for movement, and (b) that the inside surface of microtubules does not support gliding. With zinc sheets, up to 70% of the polymers move, but the movement is discontinuous, has a reduced speed, and follows along a curved path. Since zinc sheets have protofilaments alternating in orientation and polarity, this result suggests that in principle a single protofilament can produce movement, even when its neighbors cannot. Duplex microtubules do not move because they are covered with protofilaments coiled inside out, thus preventing the interaction with kinesin. The data can be explained by assuming that the outside of one protofilament represents the minimal track for kinesin, but smooth gliding requires several parallel protofilaments. Finally, we followed the motion of kinesin-coated microbeads on sea-urchin sperm flagella, from the flagellar outer doublet microtubules to the singlet microtubule tips extending from the A-tubules. No change in behavior was detected during the transition. This indicates that even if these microtubules differ in surface lattice, this does not affect the motility.

DOI： 10.1083/jcb.118.4.865

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Language：English   Publisher：ROCKEFELLER UNIV PRESS  

Flagellar axonemes of sea urchin sperm display high-frequency (approximately 300 Hz) vibration with nanometer-scale amplitudes in the presence of ATP (Kamimura, S., and R. Kamiya. 1989. Nature (Lond.). 340:476-478). The vibration appears to represent normal mechanochemical interaction between dynein and microtubules because the dependence of the frequency on MgATP concentration is similar to that of the axonemal motility, and because it is inhibited by micromolar concentrations of vanadate. In this study a two-dimensional photo-sensor was used to characterize this phenomenon in detail. Several new features were revealed. First, the vibration was found to be due to a back-and-forth movement of the doublet microtubules along the axonemal length. Two beads attached to different parts of the same axoneme vibrated in unison, i.e., synchronized exactly in phase. This suggested that the outer doublet can be regarded as a stiff rod in vibrating axonemes. Second, evidence was obtained that the amplitude of the vibration reflected the number of active dynein arms. Third, under certain conditions, the vibration amplitude took stepwise values of 8 X N + 4 nm (N = 0, 1, 2, 3, or 4), indicating that the amplitude of microtubule sliding was limited by the size of tubulin dimer (8 nm) or monomer (4 nm). To explain this phenomenon, a model is presented based on an assumption that the force production by dynein is turned off when dynein is subjected to tensile force; i.e., dynein is assumed to be equipped with a feedback mechanism necessary for oscillation.

DOI： 10.1083/jcb.116.6.1443

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Language：English  

Flagellar axonemes of sea urchin sperm display high-frequency (~300 Hz) vibration with nanometer-scale amplitudes in the presence of ATP (Kamimura, S., and R. Kamiya. 1989. Nature (Lond.). 340: 476-478). The vibration appears to represent normal mechanochemical interaction between dynein and microtubules because the dependence of the frequency on MgATP concentration is similar to that of the axonemal motility, and because it is inhibited by micromolar concentrations of vanadate. In this study a two-dimensional photo- sensor was used to characterize this phenomenon in detail. Several new features were revealed. First, the vibration was found to be due to a back- and-forth movement of the doublet microtubules along the axonemal length. Two beads attached to different parts of the same axoneme vibrated in unison, i.e., synchronized exactly in phase. This suggested that the outer doublet can be regarded as a stiff rod in vibrating axonemes. Second, evidence was obtained that the amplitude of the vibration reflected the number of active dynein arms. Third, under certain conditions, the vibration amplitude took stepwise values of 8 x N + 4 nm (N = 0, 1, 2, 3, or 4), indicating that the amplitude of microtubule sliding was limited by the size of tubulin dimer (8 nm) or monomer (4 nm). To explain this phenomenon, a model is presented based on an assumption that the force production by dynein is turned off when dynein is subjected to tensile force
i.e., dynein is assumed to be equipped with a feedback mechanism necessary for oscillation.

DOI： 10.1083/jcb.116.6.1443

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Language：Japanese   Publishing type：Article, review, commentary, editorial, etc. (other)   Publisher：日本機械学会編，ｵｰﾑ社  

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Language：Japanese   Publishing type：Article, review, commentary, editorial, etc. (other)   Publisher：生物物理  

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Language：English   Publisher：Nature Publishing Group  

DOI： 10.1038/340476a0

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Language：English   Publisher：MACMILLAN MAGAZINES LTD  

THE movement of cilia and flagella is based on the interaction between dynein arms and microtubules coupled with ATP hydrolysis. Although it is established that dynein arms cause adjacent microtubules to slide, little is known about the elementary process underlying the force production. To look more closely at the mechano-chemical conversion mechanism, we recently developed an optical method for measuring a nanometre-scale displacement with a time-reslution better than 1 ms. We now report the detection of high frequency ( ∼ 300 Hz) vibration of sub-nanometre ampli-tude in non-beating flagellar axonemes. This vibration could reflect the movement of individual activated dynein arms.

DOI： 10.1038/340476a0

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Language：Japanese   Publishing type：Article, review, commentary, editorial, etc. (other)   Publisher：化学と工業  

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Language：Japanese   Publishing type：Article, review, commentary, editorial, etc. (other)   Publisher：パリティ  

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Language：Japanese   Publishing type：Article, review, commentary, editorial, etc. (other)   Publisher：生体の科学  

DOI： 10.11477/mf.2425905198

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Language：English  

A novel method has been developed to measure nanometric displacement under a conventional optical microscope. The magnified image of a pinhole was divided into two parts using a prism-shaped mirror. The difference of light intensity between the divided images was determined, which was proportional to displacement of the pinhole. Using a 5-Mm diam pinhole, the accuracy to determine displacement was -1 nm. Instead of a pinhole, polystyrene microbeads were used in the new method. Displacement of the microbeads was also measured with nanometric accuracy. This technique could be used to probe nanometric phenomena using optical microscopes. © 1987 Optical Society of America.

DOI： 10.1364/AO.26.003425

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Language：English   Publisher：OPTICAL SOC AMER  

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Language：Japanese   Publishing type：Article, review, commentary, editorial, etc. (other)   Publisher：生物物理  

DOI： 10.2142/biophys.27.a180

CiNii Books

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Other Link： https://jlc.jst.go.jp/DN/JALC/00364705832?from=CiNii

Language：English   Publisher：ACADEMIC PRESS INC JNL-COMP SUBSCRIPTIONS  

DOI： 10.1016/0014-4827(86)90571-9

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Language：English   Publisher：ACADEMIC PRESS INC JNL-COMP SUBSCRIPTIONS  

The turbidity of axonemes during active sliding of microtubules was analysed using the stopped-flow-light-scattering method with high time resolution. Flagella of sea-urchin spermatozoa were demembranated and used after a brief treatment with trypsin. The turbidity of the suspension of flagellar axonemes during ATP-induced disintegration was measured and its time course fitted to a single exponential function which yielded the rate of disintegration, R(l/sec). R coincided well with the velocity of microtubule sliding, V(μfx186-1 sec) as determined by cinematomicrographic analysis [13], i.e., R = 0.22 × V, r = 0.9973. It indicates that turbidimetry is a useful method with which to learn the sliding velocity of microtubules. From the dependency of R on temperature, Q10 of the sliding velocity was estimated to be 2.0-2.3 at 43-820 μM of MgATP. © 1986.

DOI： 10.1016/0014-4827(86)90571-9

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Language：English   Publisher：JAPANESE BIOCHEMICAL SOC  

Using sea urchin (Hemicentrotus pulcherimus) sperm flagella, ATP hydrolysis coupled to sliding movement of microtubules was investigated. Flagellar axonemes were pretreated with trypsin and the microtubules induced to slide by addition of ATP (50-1,000 μM) at 0-20°C. Motion-dependent hydrolysis of ATP was observed immediately after the addition of ATP, the rate of which was higher than that of steady state hydrolysis in axonemes without trypsin-treatment, or after complete disintegration. The rate of hydrolysis of ATP divided by the sliding velocity of microtubules reflects the ATP consumption necessary per unit distance of microtubule sliding. This parameter varied according to the experimental conditions in that it increased when the ATP concentration or temperature was decreased. Our results suggest that there is not a strict stoichiometric relationship between ATP hydrolysis and sliding distance in the dynein-tubulin system, indicating that the mechanochemical coupling is different from that in beating axonemes. © 1985, by the Japanese Biochemical Society.

DOI： 10.1093/oxfordjournals.jbchem.a135206

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Language：English   Publisher：JAPANESE BIOCHEMICAL SOC  

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Language：English   Publisher：EDITRICE COMPOSITORI BOLOGNA  

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Language：English   Publisher：EDITRICE COMPOSITORI BOLOGNA  

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Language：English   Publisher：GEORG THIEME VERLAG  

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Language：English   Publishing type：Book review, literature introduction, etc.   Publisher：GEORG THIEME VERLAG  

Recent experimental studies of microtubule sliding in demembranated sea urchin sperm flagella are described. A local iontophoretic application of ATP to a Triton-extracted flagellum elicits a local bending response whose form is in exact conformity with the predictions of the sliding microtubule model. Cinematographic analysis of the microtubule sliding initiated by treating fragments of demembranated flagella with trypsin in the presence of ATP reveals that the speed of sliding is almost constant. This implies that the speed does not depend on the number of dynein arms participating in the generation of sliding force. The distribution of apparent sliding velocities indicates that there is no difference in sliding velocity among the doublets. The sliding velocity depends on MgATP concentration in a manner consistent with Michaelis-Menten kinetics. The sliding velocity of doublets in trypsin-treated axonemes is close to the maximum velocity of relative sliding taking place between adjacent doublets in beating flagella reactivated at the same MgATP concentration.

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Language：English   Publisher：MACMILLAN MAGAZINES LTD  

The movement of eukaryotic cilia and flagella is caused by ATP-driven active sliding between the doublet microtubules1-3, and the force for the sliding is believed to be generated by the dynein arms of the A-tubule interacting with the B-tubule of the adjoining doublet. To understand the mechanochemical basis of this force-generating reaction and to correlate it with the overt motile behaviour of cilia or flagella, it is important to quantify the force exerted by the dynein arms. Attempts have been made to estimate this force from the bending moment generated by the whole organelle4,5, but the estimation was of necessity hypothetical because the mechanism by which sliding is coupled with bending is poorly understood. Microtubule sliding without bending can be induced in vitro if trypsin-2 or elastase 6-treated axonemes are perfused with a solution containing ATP. By attaching glass microneedles to such axonemes, we have now directly determined the sliding force at various ATP concentrations. © 1981 Nature Publishing Group.

DOI： 10.1038/293566a0

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Language：English   Publisher：MACMILLAN MAGAZINES LTD  

DOI： 10.1038/293566a0

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Language：English   Presentation type：Poster presentation  

Dyneins are phylogenetically classified into three subfamilies, ciliary, cytoplasmic and intraflagellar transport (IFT) dyneins. It is widely known that cytoplasmic and IFT dyneins adopt an inactivated conformation called the phi structure. In contrast, such a distinct inactivation state was not evident for ciliary dynein. Here we report that one of the ciliary dynein species has a novel structure that is very similar to the phi structure. Based on the structural analysis, we propose a novel shutdown mechanism common to all three subfamilies of dynein.

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Language：Japanese   Presentation type：Poster presentation  

Microtubules (MT) are involved in essential cellular functions. One known characteristic of them is rapid disassembly to tubulin subunits upon cooling, a process whose molecular basis is not yet understood. We hypothesize that the conformational changes in tubulin accumulate strain of MT that triggers disassembly. To test this hypothesis, we analyzed the X-ray fiber diffraction patterns of native MTs during rapid cooling from 37 to 10℃ (<20s). We found that shape changes of MT on cooling was anisotropic, i.e., the magnitude of shrinkage was different between longitudinal and diameter directions. Detailed time-course analysis showed that the shrinkage and expansion are hysteretic. The present study would help us to understand how MTs become instable at low temperatures.

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Language：Japanese   Presentation type：Oral presentation (general)  

Chattonella is marine algae that causes HAB at Seto Inland Sea in Japan. When collected in a shallow petri dish, they start gradual accumulation in a few minutes and forms specific spotted or branching patterns with nonhomogeneous cell distributions. Since this phenomenon of bioconvection is expected to be tightly correlated with HAB mechanisms, we are executing the image analysis of accumulation behavior of Chattonella using a spatial statistical technique. Examples to show novel quantitative descriptions of bioconvection we found are shown.

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Language：English   Presentation type：Poster presentation  

Chattonella is single-celled marine algae that cause harmful algal blooms (HAB). When collected in a shallow petri dish, they start gradual accumulation to form specific patterns with a non-uniform distribution. This phenomenon of bioconvection is considered to be closely related to the HAB formation mechanism in fields. In order to understand the mechanism of HAB development, we executed the image analysis of collective swimming behavior of Chattonella marina var. ovata (Raphidophyceae) using spatial statistical techniques as well as high-speed-video microscopy. Here, we present how this quantitative analysis, variogram or correlogram, an empirically tool in geostatics, can be useful to describe the pattern of bioconvection.

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Language：English   Presentation type：Oral presentation (general)  

Microtubules are assembled from tubulin dimers that are arranged in a semi-crystal lattice with high regularity. Dynamics of tubulin dimer within microtubules is thus expected to be directly affecting the stability of microtubules, however, it has been difficult to quantitatively describe such properties. By analyzing the time course of pattern changes in X-ray fiber diffractions from aligned microtubules with a high-flux synchrotron beam of BM40XL (SPring-8), we found microtubules showed rapid elongation in the axial tubulin repeat and the concomitant increase of structural flexibility with a time constant of about 0.2 s after applying paclitaxel, microtubule-stabilizer. Contrasting effects were found for laulimalide, another type of microtubules stabilizer.

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Language：Japanese   Presentation type：Oral presentation (invited, special)  

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Language：Japanese   Presentation type：Poster presentation  

微小管内にはチューブリン分子が、結晶のように整然とラセン状格子配置している。これまで抗がん剤として、種々の微小管安定化剤が開発されて来ているが、微小管構造の安定性とチューブリンの分子構造変化とを関連づけて調べる良い方法がなかった。我々はＸ線繊維回折法を用いて、タキソールとラウリマライドの２種の安定化剤の効果の明確な違いを明らかにできたので報告する。両者ともに秒単位で微小管構造の変化を引き起こすことがわかったが、チューブリン分子の縦・横方向の相互作用に関して異なる効果を示すことも明らかとなった。

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Language：Japanese   Presentation type：Poster presentation  

シャトネラ（Chattonella marina var. obata）は、栄養塩類や海水温の条件が整うと急速に増殖・集積する赤潮の原因となるラフィド藻である。小型シャーレ内に培養した高密度のシャトネラを静置すると、数分内に自発的な集積を開始し、時間経過と共に集団で移動する生物対流現象を示すことがわかった。これが実験室内で再現される赤潮現象であると考えられる。この現象は細胞の密度を上げることで促進され、海水中のCa2+濃度を減らすことで抑制されることがわかった。位相差顕微鏡を使ったシャトネラの遊泳行動観察から、細胞密度を上昇させると互いにぶつかり合う頻度が増加し、その機械的な刺激によって短時間の鞭毛打停止反応が起こり、同時に数秒間遊泳停止することがわかった。この鞭毛打停止反応は海水中のCa2+濃度を減らすことで抑制されることもわかった。ここで発見された鞭毛停止反応が、赤潮における細胞集積反応の1つの重要な要因になっていると考えられる。

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Language：Japanese   Presentation type：Poster presentation  

タコノマクラ（Clypeaster japonicus）は、KCl神経刺激や殻部損傷によって緑色の色素を体外へ分泌するため、この色素は生体防御機構と深く関わっていると考えられる。その色素の特性は、Goodwin & Fox（1955）の吸収スペクトルに関する報告しかない。本研究で緑色色素の特性を詳細に調べた結果、親水性の高い高分子量の成分であることがわかり、他種のウニから採取されるナフトキノン系の赤色色素であるechinochromeやspinochrome等とは大きく異なる点、さらに、スカシカシパンにも共通する特性の色素があることがわかった。4腕幼生でもすでに生成機能が備わっており、腕部の機械的な刺激により内胚葉組織付近で分泌される点、その分泌にAchが関わっていることもわかった。

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Language：Japanese   Presentation type：Poster presentation  

魚類の精子は、生殖器内と異なる環境に暴露されことで運動が誘発される。今回、キンギョの精子ではイオンや糖の種類にかかわらず、240 mOsmより高い浸透圧で運動が停止することがわかり、運動停止に関しては、K+濃度よりも、高浸透圧が重要であることが示唆された。また、精子の細胞膜を除去し再活性化を試みたところ、ATPが存在すればCaCl2やcAMPを添加していない溶液にても精子の再活性化がみられ、Ca2+、cAMPともに、精子運動開始の細胞内シグナル伝達において必須でない可能性が示唆された。

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Microtubules are key components of the cytoskeleton in eukaryotic cells. Their dynamic conversions between assembled microtubules and free tubulin dimers in cytoplasm are precisely controlled along with the states of cell activities. One of the most fundamental questions of us is how the microtubule dynamics and its structural properties are related to the chemical states of tubulin dimers during GTP-hydrolysis, with the existence of other MAPs and tubulin-binding chemicals.|rn||rn| X-ray fiber diffraction is one of the most powerful techniques to collect the structural information of native microtubules in physiological solutions without fixation, crystallization or freezing. In the present study, we examined the structural changes of microtubules on a time scale of seconds by combining our original technique for shear-flow alignment of microtubules [Sugiyama et al, 2009; Oiwa et al., 2009; Kamimura et al., 2016] with a high-flux beam line at SPring-8 (BL40XU). Our observations clearly showed that binding with paclitaxel induced the decrease of mean microtubule diameter as well as the increase of axial tubulin repeat within a second, indicating configuration changes of tubulin di

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Language：English   Presentation type：Poster presentation  

Kinesin-1, the founding member of kinesin superfamily proteins, is known to use only a subset of microtubules for transport in living cells. This biased use of microtubules is proposed as the guidance cue for polarized transport in neurons, but the underlying mechanisms are still poorly understood. Here we report that there is a positive feedback in the binding of kinesin-1 to the GDP-microtubule, which spontaneously produces high affinity microtubules from other low-affinity microtubules. This high affinity state requires the binding of kinesin-1 in the nucleotide-free state. Microtubules return to the initial low affinity state by washing out the binding kinesin-1 or by the binding of AMPPNP to kinesin-1. X-ray fiber diffraction, fluorescence speckle microscopy and second harmonic generation microscopy, as well as cryo-EM, collectively demonstrated that the binding of nucleotide-free kinesin to GDP-microtubule changes the conformation of GDP-microtubule to a conformation close to the GMPCPP-microtubule.

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Language：Japanese   Presentation type：Oral presentation (general)  

マダコは体内受精と体外受精の中間的な生殖戦略を取る。交尾は行わず、オスは精莢と呼ばれるカプセル入りの精子をメスに手渡し、精子はメスの体表上で精莢から放出され、その後、貯精嚢へと移動する。メスの貯精嚢内で精子は数週間保存され適宜使われる。単離した精莢から海水中に放出された精子を通常海水中で観察すると、頭部を折り曲げその場で回転する運動と、頭部を振って前進する運動とを繰り返しながら絡み合って束を形成する。マダコ精子は約330 μmの長い鞭毛をもち、通常海水中で鞭毛の屈曲は根元と先端では顕著だが、根元から100-200 μmの中間部分ではあまり見られない。海水中に分子モーターダイニンの阻害剤であるシリオブレビン（文献1）を加えると、シリオブレビンAでは波形変化は見られなかったがシリオブレビンDでは後半部分が極端に大きく屈曲した（日本動物学会第86回新潟大会）。|rn| 2%メチルセルロース（1500 cPs）を加えて粘度を上げた海水中で

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Language：Japanese   Presentation type：Poster presentation  

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The 22nd International Congress of Zoology & the 87th meeting of the Zoological Society of Japan|rn||rn||rn||rn|Reproduction strategy of octopus is just the intermediate between internal and external fertilization. During mating, male octopus deliver pieces of spermatophore to females. Spermatozoa, after being released from the spermatophores placed on the body surface of females, move or swim towards the spermathicae, sperm storage organs in female oviducts. In spite of detailed descriptions so far on such unique behaviors of Octopus mating, there has been few reports investigating the sperm physiology of Octopus vulgaris. Their flagellar length was about 330 μm with a head of about 25 μm long. We found the manner of flagellar bend propagation was different from usual flagellar motions. It seemed to be propagating along the entire length of flagellar shaft in a non-continuous manner, i.e., smooth bends propagated along the proximal region of ca. 100 μm and then diminished at a middle part of the flagellum (100-200 μm from the base) where flagellum look almost straight, then flagellar bends reappeared around 200 μm from the base. We also found there was difference in the wave

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Language：English   Presentation type：Poster presentation  

Sea urchin spermatozoa released from males swim towards eggs in seawater for fertilization in the intertidal zone where sea urchins live. There are many other species living in the deep sea, or some of them accidentally may fall down to the deep sea by gravity after storm. Thus, the fertilization could be occurring under the stress of high or various different pressures. However, there are no report describing how the sperm swimming and flagellar motility is affected under such high pressure conditions.|rn|In order to understand the effects of pressure on motility of sea urchin spermatozoa, we examined sperm motility of sea urchin, Anthocidaris crassispina, under high pressure of seawater in the presence or in the absence of 10 mM CaCl2. Without Ca2+, consistently with previous studies, we observed the circular swimming path of sea urchin sperm on the glass surface under the condition of atmospheric pressure. The path of circular swimming was similar to that of spermatozoa observed in the seawater containing 10 mM CaCl2. We interpreted that the pumping activity of Ca2+ channels or other transporting systems (e.g. Na+/Ca2+ exchanger) can maintain the intracellular Ca2+ concentration

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Language：Japanese   Presentation type：Poster presentation  

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For the efficient chemotaxis and fertilization, it should be a crucial problem for animal spermatozoa to swim in the medium without disturbing the concentration gradient of chemical attractants derived from unfertilized eggs. Precise analysis and description of micro-flows occurring around swimming sperm cells was expected to help us to understand the chemotaxis phenomena more in details, however, there were many technical prob-lems to be solved. In the present study, using an in-situ storage image sensor (ISIS) equipped on a differential interference contract (DIC) microscope with a high-intensity xenon-lamp illumination, we successfully record-ed clear images of swimming sea-urchin sperm cells at 6,000 fps in a medium containing micro-particles with 0.1 μm diameter to directly visualize medium flows around bending flagella. We processed the acquired DIC images and traced precise motions of sperm flagella as well as moving free micro-particles with a 0.3-ms time-resolution.|rn|By comparing with theoretical flows expected from the slender body theory (SBT), we concluded that ob-served flows around micro-swimmers such as beating flagella under microscopes are classified as stokes f

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Language：Japanese   Presentation type：Poster presentation  

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Language：Japanese   Presentation type：Poster presentation  

The axial repeat of tubulin molecules in microtubules is roughly 4 nm and has conventionally been used as one of the most convenient standard scales to estimate molecular or organelle size for electron microscopy. However, in our previous report, the axial repeat varied depending on experimental conditions, e.g., temperature, with microtubule stabilizer and GTP-hydrolysis states. It was also shown that the tubulin axial repeat in porcine brain GTP-microtubules, which are mainly composed of GDP-tubulin, was almost constant with a low coefficient of thermal expansion of 3.7×10-5/degree. Here, we determined the accurate repeat of GDP-tubulin molecules after careful revision of camera length, X-ray wavelength as well as the angles of specimen, beam and detector tilting.

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Language：Japanese   Presentation type：Oral presentation (general)  

X線繊維回折法には、生体試料中の周期構造を直接反映したの回折信号を、< 0.1nmもの高精度でリアルタイム解析できる点で、他手法にはない優れた利点がある。本研究では、一般に重合・脱重合の平衡状態を繰り返すために、動的な構造ゆらぎも大きいと想像される微小管を使った解析結果を紹介する。GTP加水分解や微小管安定化剤添加による構造変化が見られることがわかったが、通常のポリマーには見られない特性、負の熱膨張特性が観察されることがわかった。細胞骨格の示すのユニークな特性について議論したい。

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One of the most fundamental questions on the molecular mechanisms of microtubule assembly-disassembly dynamics is how the structural stability of microtubules is correlated with the variation of molecular conformation of tubulin dimers within microtubules. To address this issue, we applied our technique for the rapid shear-flow alignment of biological filaments in a physiological buffer medium, enabling us to acquire the structural periodicity data from native microtubules by X-ray fiber diffraction under solution conditions. We could classified microtubules into three main groups on the basis of distinct mean axial tubulin repeats and microtubule diameters that varied depending on GTP hydrolysis and the content of paclitaxel. The paclitaxel effects to induce the elongation of the mean axial repeat of tubulin was observed to be completed within 30 s. It was also suggested that this elongation effects were appearing in a cooperative manner from the observation of microtubules with subthreshold paclitaxel content. Another interesting feature we found was the variation in the temperature dependency of axial tubulin repeat in GTP-, GMPCPP- and GTPγS-microtubules with/without paclitaxe

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Language：Japanese   Presentation type：Public lecture, seminar, tutorial, course, or other speech  

Microtubules play a variety of functions in cells. Recent studies using high-resolution cry-electron micrography and X-ray fiber diffraction revealed that multiple conformational states of tubulin give rise to structural polymorphism in microtubule lattice, which may be crucial for diverse physiological functions of microtubules. Multiple conformations of tubulin also underlie dynamic instability of microtubules, where the balance between assembly and disassembly is stochastically switched in a nucleotide-dependent manner. In this session, we introduce the forefront in the field of microtubule, aiming to share new findings and insights about the conformational dynamics of tubulin and microtubule.

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Our major question is how tubulin states are related to the stability of microtubules. To address the question, we used our novel technique for the quick shear-flow alignment of biological filaments, which enabled us to acquire fine X-ray fiber diffraction signals from native microtubules in a few seconds. We found that microtubules could be classified into three major groups with distinct axial periodicities of tubulin, which varied depending on the temperature of solution, the state of GTP-hydrolysis and the contents of microtubule stabilizers. It was also revealed that the GTPγS-tubulin showed the widest variation in the longitudinal tubulin periodicity in situ, suggesting a highly flexible state of GTP-tubulin in the microtubules.

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Language：Japanese   Presentation type：Poster presentation  

Tubulin is one of the major polypeptides that construct the axonemes of cilia/flagella. Thus, the mechanical or chemical properties of tubulin in axonemes would be optimized for the bending motion or against mechanical stresses given by external liquid. In the present study, with an X-ray fiber diffraction technique, the length changes or variations of tubulin dimer periodicity within microtubules (MT) was investigated with a high precision (~ 0.01 nm). We found there are two main states of tubulin periodicity within usual porcine brain MTs (intracellular singlet MTs), i.e., short and long configurations with a length change by about 3% depending on temperature and taxol binding (MT stabilizer). However, the tubulin repeat within sea-urchin sperm flagella showed just the intermediate of the two states of brain MTs. Here we postulate a new hypothesis, the mechanical capacity of tubulin molecules inside axonemes, which would serve a certain allowance of bending compression-decompression inside MTs.

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Language：Japanese   Presentation type：Poster presentation  

The gliding motion of flatworms (Planarian, Platyhelminthes) is primarily generated by the effective strokes of ventral cilia. The maximum motion rate of ca. 3-5 mm/s appears to be one of the fastest rates reported for the ciliary motility. Our assumption is there would be fine optimization or regulation of the motile mechanisms to accomplish such a high gliding performance. In the present study, we used different types of specific motility blockers, blebbistatin/BDM for muscular myosin II and ciliobrevin for axonemal dyneins. Blebbistatin blocked peristaltic motions and depressed the half-cylindrical shapes of flatworm body (making flatworms more planar). BDM also blocked gliding motions. We also fund ciliobrevin completely inhibited the gliding motion in minutes, and induced peristaltic shape changes. From our observations, it was suggested that both muscle contraction and ciliary motion, some coordination between them, would be required for the efficient motion of gliding.

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Language：Japanese   Presentation type：Poster presentation  

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Language：Japanese   Presentation type：Oral presentation (general)  

ウニ精子にダイニン特異的な低分子阻害剤cliobrevin A（HPI4）を投与すると鞭毛打周波数が濃度依存的に低下し、高濃度では根元の非対称性が大きな運動となることを前回大会で報告した。今回、合成アナログのciliobrevin D をウニ精子に投与したときの阻害効果をciliobrevin A と比較した。周波数への効果はciliobrevin A と同様だが、波形への効果として鞭毛全長にわたり運動非対称性がさらに増し、鞭毛が頭部の周りに巻きつくものが多数みられた。ダイニン種の違いによる阻害効果の差という観点で考察を加える。

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Language：Japanese   Presentation type：Oral presentation (general)  

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溶液内でtubulin dimerは、curved/straight の2 状態をとると考えられている。微小管内でのtubulin の動的構造変化は、微小管の安定性に大きく影響すると考えられるが、まだ、その詳細を調べた研究報告はない。X 線微小管繊維回折の解析から、taxol 濃度、温度、GTP 加水分解に依存してtubulin ピッチが変わることがわかった。微小管内でtubulin dimer がshort/long の2 状態をとる可能性が考えられる。

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Language：Japanese   Presentation type：Oral presentation (general)  

細胞質ダイニンの特異的な阻害剤であるciliobrevin の軸糸ダイニンに対する効果を調べた。この物質は低分子で膜透過性を持つので、除|rn|膜していないウニ精子に直接投与した場合に効果を見ることが期待された。予想通り、ciliobrevin は濃度依存的にウニ精子の鞭毛打周波数を下げ、このとき波形の変化も観察された。つまり、内腕、外腕ダイニンの両方に効果があることが示された。

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To clarify the fluid dynamics of swimming microorganisms, a technique to visualize fluid flows under an optical microscope with appropriate spatial and temporal resolution is required. In the present study, we used sea-urchin spermatozoa that have long flagella with planar beat patterns and are suited for precise beat form analysis. Differential inter ference microscopy (DIC) images of the beating sperm flagella and particles included in the medium were recorded with a rate of 6,000 fps. As has been theoretically expected, spermatozoa were observed to swim in a stable medium without perturbation of surrounding fluids. This is the first direct evidence to show how fluid flows around whipping flagella under the condition of low Reynolds number.

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Language：Japanese   Presentation type：Oral presentation (general)  

タンパク分子の構造は，けっして均一で等方的なものではないために，冷却時には非等方的な収縮を示すと予測される。そのような物性はタンパク分子の機能や構造の安定性を理解する上で重要な知見となるが，直接調べることは難しかった。本研究では，流動配向させたウシ脳微小管を15秒で37から17℃まで急速冷却し，同時にＸ線繊維回折法により構造変化を追跡することに成功した。GDPチューブリンからなる微小管は，低温で脱重合する直前まで，長軸方向・直径方向に，それぞれ60，220×10-6/Kの熱膨張係数を示し，非等方的な冷却収縮を示すことがわかった。

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With conventional light microscopy, precision in the measurement of the displacement of a specimen depends on the signal-to-noise ratio when we measure the light intensity of magnified images. This implies that, for the improvement of precision, getting b

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