Reactivation of Glycerinated Cilia from Tetrahymena Pyriformis

The ease with which Tetrahymena can be grown in relatively large quantity makes it an exceptionally useful source of cilia for biochemical study. Several methods of isolating the cilia have been described (3, 11). Although excellent for many purposes, these procedures all suffer from the limitation that the isolated cilia seem incapable of being reactivated by ATP. This paper describes a method for isolating cilia from Tetrahymena by treatment with 60 per cent glycerol. The preparations of isolated cilia are highly pure and the majority of cilia in them become motile when treated with ATP. The method is based on that used by Hoffmann-Berling (9) and by Brokaw (1) to reactivate sperm tails and protozoan flagella, but certain modifications were necessary in this case. MATERIALS AND METHODS One-liter cultures of Tetrahyrnena pyriformis, strain W, were grown in medium containing 1 per cent peptone, 0.1 per cent yeast extract, and phosphate buffer pH 6.5. The cells were harvested by centrifugation, washed at room temperature with a solution containing 0.18 per cent NaC1 and 0.2 M sucrose, and resuspended in a sufficient volume of fresh wash solution to give a total volume of 20 ml. A typical culture yielded about 10 ml packed volume of cells after washing. The concentrated cell suspension was cooled to 0 °, and then mixed with 100 ml of solution containing 70 per cent (v/v) glycerol, 50 mM KC1, 2.5 mM MgSO4, and 20 mM Tris-thioglycolate buffer, pH 8.3 at 0 °. As soon as it was thoroughly mixed, the suspension of cells in glycerol was cooled to-20 °, and maintained at this temperature. Vigorous agitation of the suspension on a Vortex mixer for 1 minute caused the majority of the cilia to become detached, but left the cell bodies intact. The bodies were removed by centrifugation at 12,000 g for 10 minutes. The supernatant, which consisted of a suspension of pure cilia, was removed and stored at-20 °. This stock cilia suspension was used in re-activation experiments simply by diluting it with the appropriate solution containing ATP. The cilia could be recovered from the suspension by centrifuging them at 80,000 g for 3 hours, but it was found impractical to maintain the temperature below-10 ° during this centrifugation and there was some loss of potential for motility. To obtain pure preparations of isolated cilia with this procedure, it is essential that there be no lysis of …