Gen Omura

Axopodial Contraction in the Heliozoon Raphidiophrys contractilis Requires Extracellular Ca2

Abstract Axopodial contraction of the centrohelid heliozoon Raphidiophrys contractilis was induced by mechanical or electrical stimulation. For inducing contraction, extracellular Ca2+ was required. The threshold level of extracellular Ca2+ was between 10−6−10−7 M. The speed of axopodial contraction was faster than 3.0 mm/sec. Re-elongation of axopodia started just after contraction, and its initial velocity was ∼0.30 μm/sec. Electron microscopic observations were carried out using an improved fixative that contained 1 mg/ml ruthenium red and 15 μM Taxol. This fixative prevented artificial retraction of axopodia and resulted in better fixation. A bundle of hexagonally-arranged microtubules was observed in each axopodium, but no other filamentous structures were detected, suggesting that the contractile machinery of axopodia in R. contractilis may be different from that in actinophryid heliozoons in which Ca2+-dependent contractile filaments are employed for contraction.

Ca2+-dependent contractility of isolated and demembranated macronuclei in the hypotrichous ciliate Euplotes aediculatus

The hypotrichous ciliated protozoan Euplotes aediculatus possesses a characteristic C-shaped somatic nucleus (macronucleus) within the cytoplasm, which shows dynamic shape change during the cell cycle. It is shown that isolated macronuclei possess Ca(2+)-dependent contractility. Macronuclei were isolated, stuck fast on the glass surface, and subjected to different concentrations of Ca(2+) in a Ca(2+)-EGTA buffer. The nuclei became expanded at [Ca(2+)]<10(-7)M, and they contracted on subsequent addition of higher concentrations of Ca(2+). Cycles of expansion and contraction of the nucleus could be repeated many times by alternate addition of EGTA and Ca(2+), indicating that the size of isolated nuclei can be regulated by [Ca(2+)] alone. The nuclear contraction was observed in all phases of the cell cycle, but contractility was less evident around replication bands in the S phase. In addition to the hypotrichous ciliate Euplotes, similar Ca(2+)-dependent nuclear contractility was found to exist in other cell types, including protozoans of different taxa (a heliozoon Actinophrys sol and a peniculine ciliate Paramecium bursaria), and also mammalian culture cells (HeLa cells). Our findings suggest a possibility that Ca(2+)-dependent nuclear contractility may be shared among diverse eukaryotic organisms.

Ca2+-dependent cytoplasmic contractility of the heliozoon Actinophrys sol

A “contractile tubules structure (CTS)” has been described in the radiating axopodia and cytoplasm of the heliozoon Actinophrys sol. Permeabilized cell models with fully-extended axopodia were prepared with 100 mM EGTA. When Ca2+ was added to the permeabilized cells, cytoplasmic swellings formed along the length of axopodia in a concentration-dependent manner. Triton X-100 was also used to prepare cell models of heliozoa. In the Triton-extracted cell model, axopodia were detached, but the cell body remained as a mass of cytoplasm which showed repetitive contraction and relaxation in a Ca2+-dependent manner with 10–8 M as its threshold level. A cell homogenate of A. sol was found to yield a precipitate following the addition of Ca2+. Formation of the precipitate occurred within a few minutes after the addition of Ca2+, and was dependent on the concentration of Ca2+. Electron microscopy showed that the CTS-like filamentous structures were present in the cell homogenate, and that the precipitate obtained from the cell homogenate by adding Ca2+ was composed of granular aggregates morphologically similar to the transformed CTS observed after axopodial contraction.