The ultrastructure of contractile tubules in the heliozoon Actinophrys sol and their possible involvement in rapid axopodial contraction.

We employed an improved fixation procedure for electron microscopy using ruthenium red, and found a bundle of contractile tubules inside the axopodia of the heliozoon Actinophrys sol. Upon food uptake, the tubules shorten and transform into a mass of small granules when rapid axopodial contraction occurs, suggesting that these structures are involved in the process of axopodial contraction. The relationship between transformation of the contractile tubules and accompanying disassembly of the axonemal microtubules was studied by examining the ultrastructure of the contractile tubules after disassembly of the microtubules was artificially induced by cold or colchicine treatment. Granulation of the contractile tubules was induced by cold but not by colchicine treatment. During recovery from cold treatment, granular forms of the contractile tubules became re-elongated and their initial tubular appearance was restored. These results suggest that the contractile tubules in heliozoon axopodia play a role in repetitive cytoplasmic contraction.

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.

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.

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.

Studies on the mechanism of cell elongation in Blepharisma japonicum: 4. Three dimensional construction of the kinetosomal complex and its functional role on cell elongation.

The kinetosomal complex of a heterotrich ciliate Blepharisma japonicum was investigated at ultrastructural level, with special reference to its cell elongation in response to light stimulation. In serial sections, vacuole-associated microtubules were found to be originated from both anterior fiber sheet and left surface of the anterior kinetosome. These microtubules form a bundle and extend toward the anterior end of the organism. The postciliary microtubular sheet is attached to the proximal half of the posterior kinetosome. Two types of transverse microtubules are present: the anterior 8-9 microtubules arising at the base of the anterior kinetosome and the posterior 2-3 microtubules arising between the paired kinetosomes. Based on these results, a three dimensional model of the kinetosomal complex was proposed and the mechanism of cell elongation in Blepharisma was discussed.

Cell models of Blepharisma: Ca(2+)-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.

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.

Studies on the mechanism of cell elongation in Blepharisma japonicum. II. Changes of the membrane potential measured by an electrode sensitive to tetraphenyl phosphonium.

An electrode sensitive to tetraphenyl phosphonium (TPP+) was applied to monitor the changes in the membrane potential of the ciliate Blepharisma japonicum during the process of cell elongation. Uptake of TPP+ by Blepharisma cells was observed when they were incubated in a medium containing 2 x 10(-6)M TPP+. Subsequent light stimulation (3,000 lux) led to an efflux of accumulated TPP+ from the cells, indicating that depolarization of the plasma membrane occurred with the concomitant cell elongation. TPP+ efflux was always observed whenever cell elongation was induced in response to various stimuli: valinomycin (10(-6)M), K+ (30 mM), Co2+ (5 mM), and Ca2+ (5 mM). Furthermore, elongation was also evoked by electrical stimulation. These observations indicate that membrane depolarization is involved in the process of cell elongation in Blepharisma.

Electron microscopy of the continuous membrane system in a large heliozoon Echinosphaerium.

Fine structure of the membrane system was examined in a large heliozoon, Echinosphaerium akamae, especially by means of negative staining and thin sectioning under the electron microscope. In addition to solitary membranous organelles such as mitochondrion, Golgi apparatus and nuclear envelope, there was found a continuous membrane system within the axopodium and the peripheral region of the cell body. By applying the isolation medium containing 4 mM glycerol, 2 mM MgSO(4), 1 mM EGTA, 0.05% Triton X-100 and 5 mM MES (pH 6.8), the membranous structures could be easily isolated without giving much distortion. However, pretreatment with 0.5 mM NaN(3) and 50 mM EDTA was necessarily required for isolating them fruitfully. The membrane system isolated from the cell showed tubular and balloon-like appearances both of which were connected with each other. Finally, the system was discussed about its function.

Studies on the mechanism of cell elongation in Blepharisma japonicum I. A physiological mechanism how light stimulation evokes cell elongation.

In a heterotrichous ciliate, Blepharisma japonicum, longitudinal elongation of the cell body is induced by light stimulation (1000 to 3000 lux). This light-induced response was inhibited under the existence of cyclic mononucleotide phosphodiesterase (PDE) antagonists such as papaverine (10(-4)M), theophylline (10(-3) M), dibutyril-cAMP (10(-4)M), 3-isobutyl-1-methyl-xanthine (10(-4) M) and dibutyril-cGMP (10(-4) M). Microinjection of cyclic mononucleotides, especially cGMP, inhibited cell elongation. These observations suggest that the cell elongation was mediated by intracellular cyclic mononucleotide. K(+) specific ionophore valinomycin (10(-8)-10(-7) M) enhanced light-induced cell elongation. This effect of valinomycin became more remarkable when valinomycin coexisted with PDE antagonists, while it was diminished under high K+ conditions. Moreover, the K(+) channel blockers tetraethylammonium (TEA) and CsCl inhibited cell elongation. These observations suggest that the cell elongation is also mediated by K(+) hyperpolarization, and that this electrical change is probably elicited after the intracellular concentration of cyclic mononucleotide decreased.

Genetic analysis of developmental mechanisms in hydra. XVIII. Mechanism for elimination of the interstitial cell lineage in the mutant strain Sf-1.

The interstitial cell lineage in mutant strain sf-1 of hydra is temperature sensitive and is lost rapidly from tissue when the animal is cultured at a restrictive temperature of 23 degrees C or higher. The mechanism responsible for this cell elimination process was investigated. Sf-1 polyps were treated at a restrictive temperature of 27 degrees C for varying lengths of time, their tissues were macerated, and the resultant dissociated cells were examined for evidence of phagocytosis after Feulgen staining. It was found that large phagocytic vacuoles were present in the cytoplasm of some epithelial cells. These vacuoles contained partially degraded cells, whose nuclei had highly-condensed and intensely Feulgen-positive chromatin granules. This indicated that, as in colchicine-treated (Campbell, 1976) or starved (Bosch and David, 1984) wild-type hydra, the epithelial cells in strain sf-1 engulfed and disintegrated other cells in the phagocytic vacuoles. The incidence of phagocytosis was higher in sf-1 tissue maintained at elevated temperature than in sf-1 tissue maintained at normal temperature. However, the observed incidence was relatively low (maximally 0.14 phagocytosed cells per epithelial cell) and appeared to be too low to account for the very rapid interstitial cell loss occurring in this strain. We concluded that elimination of the interstitial cell lineage at a restrictive temperature in strain sf-1 takes place in part by phagocytosis and in part by other yet-unidentified mechanisms (cf., Marcum et al., 1980).

A model of contractile tubules showing how they contract in the heliozoan Echinosphaerium.

In the large heliozoan Echinosphaerium, contractile tubules (formerly called X-bodies) are located between the axopodial membrane and the axonemal microtubules. When axopodial contraction occurs, the tubules have been thought to be transformed from a tubular to a granular form, as seen in ultra-thin sections. Our detailed morphological observations of the contractile tubules, however, have revealed that this so-called granulation of the contractile tubules is mediated by self-twisting and supercoiling during contraction. We also examined the localization of calcium during axopodial contraction using a potassium pyroantimonate assay. Ca-Sb deposits were detected on contractile tubules only during the twisting and coiling of this organelle. Our results indicate that axopodial contraction is enforced by the twisting and coiling of contractile tubules, which action probably is mediated by Ca2+ ions.

Effects of KCl-EGTA solution on the isolated Mytilus smooth muscle: a method for denervation.

The ABRM of M. edulis was immersed in KCl-EGTA solution (540 mM KCl + 5 mM EGTA) for 30 min. Then the muscle was returned to normal ASW, effects of the KCl-EGTA solution being examined on the ultrastructure of the neuromuscular junctions and also on the mechanical responses of the muscle to several kinds of stimuli. By the KCl-EGTA treatment of the muscle, synaptic vesicles in the nerve terminals at the junctions were found to be markedly deformed in shape and materially reduced in number. In most of the muscles tested, the contractile response to ACh and catch-relaxing responses to serotonin and dopamine were not depressed by the present treatment, though in some other muscles those responses were depressed a little. Contractile and catch-relaxing responses to repetitive electrical pulses of stimulation were markedly depressed or almost blocked by the treatment. All of the catch-relaxing responses to hexylamine (10(-3)M), phenylethylamine (10(-4)M), Na+-free ASW and 8-bromo-cyclic GMP (10(-3)M) were also markedly depressed or almost completely blocked. These results indicate that the function of intramuscular nerve fibres in the ABRM is markedly impaired by treating the muscle with KCl-EGTA solution while that of muscle fibres is little impaired. The treatment seems to be a useful method for denervation in the isolated ABRM.

Resorption of microtubules and nucleus during the encystation process in Echinosphaerium nucleofilum.

The cyst formation process was investigated in a large heliozoan Echinosphaerium nucleofilum, with particular reference to the resorption of axonemal microtubules and nuclei into the cytoplasm. As the encystation proceeded, the axonemal microtubules were found to disassemble from their distal ends by being surrounded or invaded with many tubular vesicles, followed by detaching at their proximal ends from the nuclear envelope and by disassembling completely at the final stage. On the other hand, several nuclei were found to be enclosed by large vacuoles before they were destroyed or digested and finally absorbed into the cytoplasm. At this stage, one or two large multi-layered bodies were formed inside the nucleus. The meanings of the resorption of microtubules and nuclei were considered and discussed.

Food capture and ingestion in the large heliozoan, Echinosphaerium nucleofilum.

Using its microtubule-containing axopodia, a heliozoan Echinosphaerium nucleofilum feeds on various kinds of protozoans and small metazoans. The present study revealed that food capture and ingestion were carried out in 2 different ways or by a combination of them. The first one was by the rapid contraction of axopodia, by which the food organism was conveyed directly toward the body surface. After such a contraction, many of the microtubules which had been present inside the axopodia degraded and were replaced by C-shaped microtubules. Bundles of tubular bodies were also detected alongside the axonemal microtubules, especially following the use of glutaraldehyde fixative containing ruthenium red. The second method was by means of axopodial flow, by which a food organism attached to an axopodium was conveyed to the body surface along the axopodial surface without accompanying axopodial degradation or contraction. Subsequently the food organism was surrounded by several small pseudopodia to form a food vacuole; many filamentous structures (5-10 nm in diameter) were observed inside the pseudopodia. During the ingestion process many cytoplasmic extensions, including rosary-like filaments, were observed to protrude from the contracted axopodia and the cell body. Mottled dense granules were observed to be discharged from the axopodial surface just when the prey was captured.

Microtubules in protozoan cells. III. Ultrastructural changes during disintegration and reformation of heliozoan microtubules.

In the heliozoan, Echinosphaerium nucleofilum strain MA, cold temperature (2 degrees C) induced axopodial retraction to about 36% of the initial length after 3 h. By electron microscopy, it was found that such axopodial shortening is accompanied by degradation of axonemal microtubules (25 nm in diameter), followed by the appearance of macrotubles (37 nm in diameter) and filamentous structures (14 nm in diameter) of a tubular and twisted appearance. All of these structures (microtubules, macrotubules and filamentous structures) were found to be depolymerized completely by applying 10 mM colchicine for 1-2 h, and to be replaced by regions with low electron density. Axopodial re-extension was induced rapidly by returning the cold-treated organisms to room temperature (20 degrees C). At a very early stage of axopodial re-extension, the filamentous structures were often observed to be continuous with the macrotubules. At a late stage of axopodial re-extension, the reforming axoneme was composed only of normal microtubules, while the macrotubules and filamentous structures had disappeared. On the basis of these results, the processes of disintegration and reformation of microtubules are discussed.