Toshinobu Suzaki

Ultrastructure and Rapid Axopodial Contraction of a Heliozoa, Raphidiophrys contractilis Sp. Nov.

ABSTRACT. In the present study, we isolated a species of heliozoans from a brackish pond in Shukkeien Garden, Naka‐ku, Hiroshima City, Japan. Electron‐microscopic observations showed that the axonemal microtubules in this heliozoan constituted a complex pattern of hexagons and triangles. By applying SDS‐polyacrylamide gel electrophoresis and subsequent immunoblotting, molecular weights of α and β‐tubulins were determined to be 48 and 45 kDa, respectively. X‐ray microanalysis demonstrated that the numerous scales coating the cell body surface were silicic structures. Size and shape of the cell body and the scales were examined and compared with other known species of heliozoans, which led us to conclude that this is a new species belonging to the genus Raphidiophrys. This heliozoan was also found to carry out rapid axopodial contraction during food uptake at a velocity of about 1 mm/s. With reference to this characteristic contractile behavior, this new species was named Raphidiophrys contractilis.

Cell models of Blepharisma : Ca2+-dependent modification of ciliary movement and cell elongation

Ionic mechanisms were examined with reference to modification of swimming velocity and cell elongation in Triton-extracted cell models of Blepharisma. The extracted cells swam forward at Ca(2+) concentrations below 10(-6) M. The forward swimming velocity of the cell models increased with a decreased Ca(2+) concentration in the surrounding medium. At Ca(2+) concentrations above 10(-6) M, the models swam backward or rotated. The elongation of the models occurred at Ca(2+) concentrations below 10(-7) M. Results suggest that swimming velocity, cell elongation and contraction of intact cells may be regulated by intracellular Ca(2+) concentration.

Structure and function of the cytoskeleton in heliozoa: 3. Rapid microtubule disorganization during axopodial contraction in Echinosphaerium

Summary The heliozoan Echinosphaerium feeds on various kinds of small protozoans by slow retraction or rapid contraction of microtubule-containing axopodia. The axonemal microtubules showed an intense positive birefringence that was clearly visualized when the organism was observed under a highly sensitive polarization microscope. In order to know if the axopodial contraction involves rapid disassembly of the cytoskeletal microtubules, dynamic changes of the axonemal birefringence were analyzed here during the process of rapid axopodial contraction by video microscopy with an image digitizing system. The number of microtubules in an axopodium was estimated from its birefringence by applying dielectric theories for shell-covered ellipsoids in concentrated suspensions. Quantitative analysis showed that the fractional volume occupied by the microtubules increased at the proximal region of the axopodium concomitantly with the axopodial contraction, and it was maintained for at least 10 s after the contraction. Electron microscopic observations showed that two bundles of axonemal microtubules crossed each other in the proximal part of a contracted axopodium. These results indicate that, during rapid axopodial contraction, the distal portion of the axonemal microtubules moves as a bundle into the axopodial base without a distinct disassembly of the microtubules themselves.

Structure and function of the cytoskeleton in heliozoa: 2. Measurement of the force of rapid axopodial contraction in Echinosphaerium

The large heliozoan Echinosphaerium extends a number of needle-like axopodia by which it captures food organisms. Every axopodium contains a bundle of several hundreds of axonemal microtubules as a cytoskeletal element. When the tip of a poly-L-lysine-coated glass micro-needle came into contact with the distal part of an axopodium, a rapid axopodial contraction (2.6 mm/s) occurred with a concomitant bending of the needle toward the cell body. In this report, we measured the force of the axopodial contraction by utilizing the relation between force and bending displacement of the micro-needle, and examined a possibility that the axopodial contraction is ascribed to the axopodial tension (surface tension and/or cytoplasmic elasticity) that is developed as a result of microtubule degradation. The force of the axopodial contraction was estimated in the order of 10(-9) N. Treatment with 10 mM colchicine induced disassembly of the axopodial microtubules and a resulting slow retraction of the axopodia (0.1 μm/s) occurred. The force of the slow retraction was also measured by the same procedure to estimate the axopodial tension, and was in the order of 10(-11) N. It was thus demonstrated that the motive force for axopodial contraction cannot be explained as an axopodial tension generated as a result of disassembly of the microtubules.

Flagellar adenosine triphosphatases from annelid spermatozoa: Electrophoretic identification of dyneins

Abstract Axonemes of sperm flagella were prepared from the annelid, Tylorrhynchus heterochaetus . Dialysis of the axonemes against 1 m m Tris-HCl buffer (pH 8.3)-0.1 m m EDTA-0.1 m m dithiothreitol (Tris-EDTA solution) caused disintegration of typical 9 + 2 microtubules into each doublet, resulting in extraction of one-third of the protein and almost all ATPase activity. Agarose polyacrylamide gel electrophoresis of the extract showed the presence of three kinds of dyneins actively stained for ATPase (designated as bands I, II, and III) and two non-ATPase proteins (bands IV, V). The polypeptide components of each dynein molecule and intact axoneme were analyzed by subsequent sodium dodecyl sulfate-polyacrylamide gel electrophoresis to obtain the following results: (1) In the highmolecular-weight region, the intact axonemes yield two major polypeptides with molecular weights of 365,000 and 345,000 (designated as bands A and B, respectively) and three minor polypeptides, 310,000, 290,00, and 270,00 (C1, C2, C3). (2) All three dyneins contain A-band polypeptide as a common polypeptide component. In addition, band I dynein and band II dynein also contain B and C1 polypeptides, and C3 polypeptide, respectively, as high-molecular-weight components. (3) Band III dynein also contains four polypeptides in the lower molecular-weight region, which migrate similarly with those of 21 S dynein from sea urchin sperm flagella or 18 S dynein from Chlamydomonas .

Effect of Mg2+ on Ca2+-dependent contraction of a Spirostomum cell model

Summary A detergent-extracted cell model of the heterotrichous ciliate Spirostomum ambiguum showed ATP-independent contraction on addition of > 10−7 M Ca2+, which is caused by contraction of the myoneme. To further understand the mechanism of cell contraction, the effects of Mg2+ and other cations were examined. Among various kinds of cations tested, Ba2+, Mn2+, Sr2+ and Cd2+ induced slight contraction, but they required higher concentrations (> 10−3 M). Contraction of the cell model was not induced by Mg2+. However, it shifted the threshold of Ca2+ concentration for inducing contraction to higher levels. The Hill constant, denoted as the number of calcium ions cooperatively bound to a contractile element in the myoneme, was 2–3 at 0–1 mM Mg2+, while it decreased with increasing Mg2+ concentration. The Ca2+-induced contraction was not influenced by changing ionic strength of the reactivation medium. These results suggest that Mg2+ ions interact with the Ca2+-binding sites which induce contraction of the myoneme.

Isolation and properties of the axopodial cytoskeleton of a heliozoan, Echinosphaerium akamae

The axopodia of a large heliozoan, Echinosphaerium akamae, were efficiently liberated from the cell body by treatment with 65% D(2)O solution containing 5 mM MgCl(2),2.1 mM EGTA, 1 mM KCl and 5 mM HEPES (pH 6.9). After D20 treatment, the cell bodies were removed by centrifugation at a low speed for 30 sec and the resulting supernatant was recentrifuged at 10,000 × g for 10 min. The axopodia were obtained as the pellet fraction without any contamination from the cell body. The isolated axopodia maintained their regular arrangement of cytoskeletal microtubules and were shortened by treatment with Ca(2+). When the isolated axopodia was subjected to SDS-polyacrylamide gel electrophoresis, two major protein bands were detected. The molecular weights of the proteins, tentatively identified as the heliozoan tubulins, were estimated to be about 46 and 50 kD, and an antibody against rat brain tubulin reacted with only the 46 kD protein species.

Quantitative analysis of superoxide anion generation in living cells by using chemiluminescence video microscopy

Superoxide anions (O2-) generated by rabbit neutrophils were detected and quantified by a video microscope equipped with a photon-counting camera. One count obtained by this system was equivalent to 59 amol of O2-. Maximum O2- production was observed at 6-8 min after stimulation and was estimated as 1.9 fmol/min per cell on the average.

Ultrastructure and calcium-dependent contraction of the myonemal network in a heterotrich ciliate, Blepharisma japonicum

This laboratory has previously demonstrated that shortening of the cell body of a heterotrich ciliate, Blepharisma japonicum, could be induced as a step-down photophobic response. Here, we examined the structure and contractility of the myonemes in detergent-extracted cell models and in isolated cortical fragments. Ultrastructural observation showed that the myoneme was connected to the basal ends of the posterior kinetosomes and constructed a systematic network as a whole. Shortening of the cell model was induced by > 10(-4) M Ca(2+), while the rounded cell model did not re-elongate even when it was washed in a calcium-free solution either with or without addition of ATP. Fluffy fibrils, which were tentatively identified as aggregated bundles of the myonemes, were isolated with the kinetosomal complex and showed calcium-dependent and ATP-independent contraction. The minimum concentration of Ca(2+) required for inducing contraction was at the level of 10(-6) M. These results suggest that the cell body shortening in Blepharisma is caused by the Ca(2+)-dependent contraction of the myonemal network.

Studies on the mechanism of cell elongation in Blepharisma japonicum: 3. Cytoplasmic calcium ions may correlate to cell elongation in calmodulin-dependent manner

Among many heterotrichous ciliates, Blepharisma japonicum especially demonstrates negative phototaxis in response to light stimulation, which is attributed to be caused by swimming acceleration accompanied by cell elongation and ciliary reversal. When Blepharisma cells were treated with 0.1 mM EGTA, cell elongation gradually decreased in its degree as the adaptation time lapsed. Calmodulin inhibitors such as W-7 (10(-5) M) and chlorpromazine (10(-5) M) inhibited cell elongation. These results suggest that a rise in cytoplasmic free Ca(2+) concentration might cause cell elongation through the Ca-calmodulin system. On the other hand, Ca(2+)-channel blockers (La(3+), Co(2+), Cd(2+), Zn(2+), Mn(2+) and verapamil) did not inhibit cell elongation. Ca2+ localization examined by calcium pyroantimonate cytochemistry suggests that Ca(2+) ions required for cell elongation might be supplied from the vacuoles located in the cortical region of the cell instead of Ca(2+) influx from the surrounding medium.