Immunocytochemical Analysis Of Alpha-Tubulin Distribution Before And After Rapid Axopodial Contraction In The Centrohelid Raphidocystis Contractilis

The centrohelid Raphidocystis contractilis is a heliozoan that has many radiating axopodia, each containing a bundle of microtubules. Although the rapid contraction of the axopodia at nearly a video rate (30 frames/s) is induced by mechanical stimuli, the mechanism underlying this phenomenon in R. contractilis has not yet been elucidated. In the present study, we described for the first time an adequate immunocytochemical fixation procedure for R. contractilis and the cellular distribution of alpha-tubulin before and after rapid axopodial contraction. We developed a flow-through chamber equipped with a micro-syringe pump that allowed the test solution to be injected at a flow rate below the threshold required to induce rapid axopodial contraction. Next, we used this injection method for evaluating the effects of different combinations of two fixatives (paraformaldehyde or glutaraldehyde) and two buffers (phosphate buffer or PHEM) on the morphological structure of the axopodia. A low concentration of glutaraldehyde in PHEM was identified as an adequate fixative for immunocytochemistry. The distribution of alpha-tubulin before and after rapid axopodial contraction was examined using immunocytochemistry and confocal laser scanning fluorescence microscopy. Positive signals were initially detected along the extended axopodia from the tips to the bases and were distributed in a non-uniform manner within the axopodia. Conversely, after the induction of a rapid axopodial contraction, these positive signals accumulated in the peripheral region of the cell. These results indicated that axopodial microtubules disassemble into fragments and/or tubulin subunits during rapid axopodial contraction. Therefore, we hypothesize that the mechanism of extremely rapid axopodial contraction accompanied by cytoskeletal microtubule degradation in R. contractilis involves microtubule-severing at multiple sites.