Three-dimensional construction of the kinetosomal complex in a ciliate Blepharisma, revealed by isolation technique and serial sectioning

Abstract

In a heterotrichous ciliate, Blepharisma japonicum, pairs of basal bodies (kinetosomes) and their fibrous associates containing microtubular sheets and bundles are closely related to cell elongation in response to light stimulation. Up to date, however, three dimensional construction of them has not been clarified because of their own complexity and difficulty of establishment of their isolation technique. By applying the newly devised isolation medium, however, they were found to be isolated fruitfully from the cell bodies. The present isolation medium was composed of 10 mM MgSO4, 10 mM EGTA, 1 mM dithiothreitol, 32 mM sucrose, AH Triton X-100, 20% glycerol, 100 mM HEPES and 20 mM PIPES adjusted to pH 7.2 with KOH. The isolated complexes were mounted onto Formvar-coated grid, fixed briefly with 2% glutai-aldehyde (GA) buffered with 0.1 mM sodium cacodylate at pH 7.2, rinsed with deionized distilled water and finally stained with 1% aqueous uranyl acetate (UA). For thin sectioning, on the other hand, the organisms were fixed for 10 min with a mixture of 2.5% GA and 1% OsO4 in cacodylate buffer (pH 7.2) at room temperature.