Raphidiophrys contractilis, Kinoshita, Suzaki, Shigenaka & Sugiyama, 1995, Kinoshita, Suzaki, Shigenaka & Sugiyama, 1995

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, Acta Protozoologica 59 (1), pp. 1-12 : 3-5

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).

Figures

Fig. 1. (A, B) Rapid axopodial contraction induced by mechanical stimulation in R. contractilis. Images (A) before and (B) after rapid axopodial contraction. Arrowheads indicate kinetocysts. Note the synchronized retraction of all axopodia and the apparent increases in the widths of contracted axopodia relative to the features observed before the onset of axopodial contraction (an arrow). Scale bar: 20 µm (A, B).

Fig. 2. (A, B) Fine structures associated with axopodial microtubules. (A) Centroplast in the center of the cell. (B) Cross-section of an axopo- dium in the peripheral region of the cell. Note that bundles of microtubules radiate from the centroplast (arrowheads) and that the axopodium comprises six microtubules. Scale bars: 500 nm (A), 100 nm (B).

Fig. 3. Schematic illustration of the experimental setup. The fixative is injected into the cell suspension via a micro flow-through chamber with a syringe pump. Next, the cells are fixed by injecting the fixative at a rate below the threshold required for inducing rapid contraction. The cells are then observed under a microscope.

Fig. 4. (A–D) Evaluation of the maintenance of the original lengths of axopodia in R. contractilis using different combinations of fixative and buffer. Cells were fixed with (A) 4% paraformaldehyde in phosphate buffer, (B) 4% paraformaldehyde in PHEM, (C) 0.2% glutaralde- hyde in phosphate buffer, or (D) 0.2% glutaraldehyde in PHEM. Note that only fixation with 0.2% glutaraldehyde in PHEM maintained the axopodial length. Scale bar: 20 µm.

Fig. 5. (A, B) The distribution of α-tubulin in the cell before rapid axopodial contraction. (A) Low-magnification images of a cell without the induction of rapid axopodial contraction.A projection image of the whole cell was constructed from 192 optical sections obtained at 0.5 µm intervals (left); the corresponding light micrograph is also shown (right). (B) High-magnification images of the same cell shown in (A). A projection image of the equatorial plane of the cell body constructed from 5 optical sections taken at 0.5 µm intervals, and the correspond- ing light micrograph. The asterisk indicates a centroplast. Note that positive signals were detected along the fully extended axopodia. Scale bars: 20 μm (A), 10 μm (B).

Fig. 6. Confocal analysis of α-tubulin-immunolabeled extended axopodia. Fine reconstructed projection image from 161 optical sections taken at a 0.05 µm (top) and the corresponding light micrograph (bottom). Note the regions of relatively low fluorescence (arrowheads) did not correspond to the location of kinetocysts (arrows). Scale bar: 10 μm.

Fig. 7. (A, B) The distribution of α-tubulin in the cell after rapid axopodial contraction. (A) Low-magnification images of the cell after the induction of rapid axopodial contraction. A projection image of the whole cell constructed from 87 optical sections taken at a 0.5 µm interval (left) and the corresponding light micrograph (right). (B) High-magnification images of the same cell shown in (A). A projection image of the equatorial plane of the cell body constructed from 5 optical sections taken at a 0.5 µm interval (left), and the corresponding light micrograph (right). Note that the positive signals in the distal parts of the axopodia have a branched appearance (arrowheads). Scale bars: 20 μm (A), 10 μm (B).