Raphidiophrys contractilis Kinoshita, Suzaki, Shigenaka & Sugiyama 1995
Creators: name: Ikeda, Risa
name: Kurokawa, Miki
name: Murai, Momoka
name: Saito, Noboru
name: Ando, Motonori
Publisher: name: Zenodo
Year: 2020
Subjects: Biodiversity
Taxonomy
Chromista
Heliozoa
Centrohelida
Raphidiophryidae
Raphidiophrys
Raphidiophrys contractilis

description: Morphological characteristics of R. contractilis
Rapid axopodial contraction in R. contractilis was examined using video microscopy (Fig. 1). R. contractilis has a spherical cell body surrounded by several radiating axopodia (Fig. 1A). These axopodia have an average length of 60 µm and a maximum length exceeding 100 µm. Each axopodium contains granular kinetocysts that participate in food capture (Fig. 1A, arrowheads). Immediately after mechanical stimulation (see Methods), all axopodia retracted into the cell body at a less-than-video rate. The axopodial length was reduced to less than 10% of the initial length immediately after axopodial contraction (Fig. 1B). Simultaneously, the widths of the contracted axopodia appeared to increase compared with the widths before the onset of contraction (Fig. 1B, arrow). The microtubule orientation in the central and peripheral regions of the cells after rapid axopodial contraction was examined using conventional electron microscopy (Fig. 2). The centroplast, a microtubule-organizing center presenting in the centrohelid heliozoa located at the center of each cell (Fig. 2A). A cross-sectional analysis revealed that each axopodium comprised six microtubules in the peripheral region of the cell (Fig. 2B).
Evaluation of fixation procedures
Next, we investigated the dependence of flow rate on the rapid axopodial contraction in R. contractilis using a micro flow-through chamber. This chamber, equipped with a micro-syringe pump, was expected to mitigate the effect of shear stress against adherent cells during the injection of test solutions (Fig. 3). We examined the effect of the flow rate on the rapid axopodial contraction by changing the flow rate from 0.5 to 500 μl/min. Notably, gentle perfusion with culture medium at a flow rate of <50 μl/min did not evoke rapid axopodial contraction. Therefore, in subsequent experiments, the test solutions were injected into the cell chamber at a flow rate of 12.5 μl/min.
Next, the effects of fixatives on the morphological appearances of the axopodia were examined using light microscopy (Fig. 4). The cells were fixed in solutions containing 4% paraformaldehyde or 0.2% glutaraldehyde in phosphate buffer or PHEM. Initially, the cells were fixed with 4% paraformaldehyde in phosphate buffer. Despite the status of this fixative as the most widely used in immunohistochemical applications, we found that 4% paraformaldehyde caused a reduction in the lengths of axopodia compared with the original lengths before fixation (Fig. 4A). A similar result was obtained when the cells were fixed with 4% paraformaldehyde in PHEM (Fig. 4B), and fluorescence images of α-tubulin labeling revealed the breakdown of axopodial microtubules within the contracted axopodia (data not shown). Second, the cells were fixed using 0.2% glutaraldehyde. The axopodial lengths were not maintained when the cells were fixed with 0.2% glutaraldehyde in phosphate buffer (Fig. 4C). Conversely, the axopodial lengths were maintained when the cells were fixed with 0.2% glutaraldehyde in PHEM (Fig. 4D).
Distribution of α-tubulin before and after rapid axopodial contraction
The cellular distribution of α-tubulin before and after rapid axopodial contraction was examined using confocal microscopy. The cells were fixed using 0.2% glutaraldehyde in PHEM. Positive signals corresponding to α-tubulin were detected along the fully extended axopodia in the absence of induced axopodial contraction (Fig. 5A). Notably, the positive signals radiated from the centroplast (Fig. 5B). A detailed observation of the extended axopodia in the equatorial plane of the cell revealed that the positive signals often appeared to be discontinuously distributed along the axopodia (Fig. 6). Following the induction of axopodial contraction, however, positive signals were detected within the completely contracted axopodia in the cell (Fig. 7A). Those signals accumulated in the peripheral region of the cell (Fig. 7B). Moreover, the signals in the cell with contracted axopodia often exhibited a branched appearance in the distal part of axopodia (Fig. 7B, arrowheads).
description: Published as part of Ikeda, Risa, Kurokawa, Miki, Murai, Momoka, Saito, Noboru & Ando, Motonori, 2020, Immunocytochemical Analysis of α-Tubulin Distribution Before and After Rapid Axopodial Contraction in the Centrohelid Raphidocystis contractilis, pp. 1-12 in Acta Protozoologica 59 (1) on pages 3-5, DOI: 10.4467/16890027AP.20.001.12157, http://zenodo.org/record/10994454