Isolation of pure pellicles containing intact basal bodies of Tetrahymena pyriformis.
A new procedure for mass isolation of pure pellicles containing intact basal bodies of Tetrahymena pyriformis is reported. The success of the procedure depends on the elimination of the sticky mucocyst contents before fractionation of the cells, which is induced by Alcian Blue 8GS. Under appropriate ionic conditions greater than 95% of the cells are able to form a capsule by simultaneous extrusion of all mature mucocysts. About 50% of these cells are able to escape from their capsules, which are now devoid of mature mucocysts. These cells are separated from the empty capsules and encapsulated cells by passage through layers of gauze of 10 microns pore size. The fractionation of mucocyst-free cells in homogenization buffer yields pure pellicles, which are retained when the homogenate is sieved through steel sieves of 5 microns pore size. Electron-microscopic controls show that the isolated pellicles are not contaminated with subcellular particles. Cells homogenized in the presence of low concentrations of Triton X-100 yield pellicles consisting of the known cell-surface-associated cytoskeletal elements, together with basal bodies. The cilia are detached just above the kinetosomal plate. The basal bodies of isolated pellicles are obviously undamaged, since all the known structures of native basal bodies are preserved. Even the granular matrix, a labile structure in the lumen of the basal body that probably contains RNA, is preserved.
D000818:Animals; D002458:Cell Fractionation / Q000379:methods*; D002478:Cells, Cultured; D002502:Centrioles / Q000648:ultrastructure*; D008854:Microscopy, Electron; D013769:Tetrahymena pyriformis / Q000648:ultrastructure*