Shinji Kamimura

Comparative structural analysis of eukaryotic flagella and cilia from Chlamydomonas, Tetrahymena, and sea urchins.

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.

Lowered Sperm Motility in Subordinate Social Status of Mice

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.

Influence of social dominance and female odor on the sperm activity of male mice

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.

Asymmetric mandibles of water-scavenger larvae improve feeding effectiveness on right-handed snails.

Asymmetric morphology and function are commonly observed in bilateral animals (Palmer 1996). Recent studies also revealed how the asymmetric morphology of animals is developed (Wood 1997). However, the adaptive significance of directional asymmetries (i.e., the advantage to being asymmetric in a particular direction) is rarely known. Well-known cases are the interspecies interaction between crustaceans and snails. In the case of molluscivorous reef crabs Calappa, they use asymmetric claws to manipulate right-handed snails: a robust left claw to peel the shells and the other smaller one to cut the flesh into pieces (Shoup 1968; Ng and Tan 1985; Smith and Palmer 1994; Seed and Hughes 1995). In another case, hermit crabs develop asymmetric bodies at least in part because of the right-handed shells into which they grow (Harvey 1998). A practical restriction of study in this field is that we cannot easily make antiasymmetric or symmetric animals on which to execute an ideal experiment. Therefore, it is usually difficult to obtain direct evidence indicating how any particular asymmetric structure has any ecological significance or how the morphological chirality is related to the interspecies interaction with other animals. However, some of the practical problems could be solved, although not completely, by comparing the performance of a pred-

Direct measurement of nanometric displacement under an optical microscope.

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 displacementof the pinhole. Using a 5-microm 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.

Ciliated Cells Differentiated from Mouse Embryonic Stem Cells

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 β‐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.

Increase in intracellular pH induces phosphorylation of axonemal proteins for activation of flagellar motility in starfish sperm

SUMMARY 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.

Diameter Oscillation of Axonemes in Sea-Urchin Sperm Flagella

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).

New Open Aquarium System to Breed Larvae of Water Beetles (Coleoptera: Dytiscidae)

Abstract 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.

Imaging of the fluorescence spectrum of a single fluorescent molecule by prism-based spectroscopy

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.