Control of initiation and elongation of cilia during ciliary regeneration in Tetrahymena.

Abstract

Tetrahymena thermophila strain B could regenerate approximately 10% of its somatic ciliary mass in concentrations of cycloheximide believed to block all cytoplasmic protein synthesis. A quantitative study of the relative numbers and lengths of cilia regenerated in the presence and absence of cycloheximide under a variety of conditions suggested that specific initiation and elongation protein factors are involved in the control of ciliary morphogenesis in Tetrahymena.The ciliated protozoan Tetrahymena can regenerate its cilia in both nutrient and non-nutrient medium. It has been shown in a number of laboratories that regeneration is completed within about a 2-h period after deciliation by the Rosenbaum and Carlson calcium shock procedure (1,5,8,10,13). It has also been shown by several laboratories that there is an induced synthesis of ciliary proteins associated with this regeneration (1, 5, 13; S. D. Guttman and M. A. Gorovsky, J. Cell Biol. 67:149a, 1975). From the beginning, however, label dilution studies have consistently indicated that only part of the protein actually incorporated into the regenerating cilia is the result of this induced synthesis (5, 7, 13; Guttman and Gorovsky, J. Cell Biol. 67:149a, 1975). This suggests that a significant proportion of the structural protein in regenerated cilia comes from preexisting cytoplasmic pools. It has also been found recently that tubulin induction occurs only after ciliary regeneration is well under way (1, 5), which leads to the same conclusion. The substantial use of stored proteins in ciliary regeneration by Tetrahymena suggests the operation of specific assembly control mechanisms which may be of general interest. into the postulated assembly control mechanisms. Our results suggest that specific initiation and elongation factors may be involved in the control of ciliary assembly in Tetrahymena. MATERIALS AND METHODST. thermophila strain B stock cultures were maintained axenically in a tryptone medium (3) and grown for experiments in an enriched proteose-peptone medium (PPYGFe) containing yeast extract, glucose, and iron-ethylenediaminetetraacetic acid (15). Flask cultures containing 150 ml of growth medium were grown to a density of 3 x 105 cells/ml at 22°C. The cells were then rinsed by gentle centrifugation three times and suspended in 150 ml of the Carlsberg Institute inorganic medium (CBI-IM) (6). All experiments were begun after maintenance of the cells in inorganic medium for 4 h.The cells were deciliated for regeneration experiments by the method of Rosenbaum and Carlson (10). In this method, the cells are concentrated in a small amount of a deciliation medium and briefly exposed to a high concentration of calcium. The cilia, weakened at the base by the calcium shock, are easily removed by drawing the cell suspension through a no. 16 needle fitted to a syringe. Immediately after the deciliation procedure, the cell suspension is diluted tenfold in inorganic medium, rinsed once, and resuspended in 150 ml of inorganic medium. The deciliated cells we...

Kinesin-13 regulates the quantity and quality of tubulin inside cilia

“…Chlamydomonas cells with a knockdown of kinesin-13 fail to regenerate cilia in the presence of the protein translation inhibitor cycloheximide, which led to a hypothesis that kinesin-13 produces cilia-destined tubulin by depolymerizing the cell body microtubules ( Wang et al , 2013 ). In the presence of 20 μM cycloheximide at a concentration that shuts down protein synthesis in Tetrahymena ( Hallberg and Hallberg, 1983 ), both wild-type (as reported; Hadley and Williams, 1981 ) and 13BC-KO cells regenerated cilia, and the drug decreased the elongation rate and the maximal length in both genetic backgrounds to a similar extent; at 120 min postdeciliation, the wild-type and 13BC-KO cilia were shorter by 23 and 21%, respectively ( Figure 4F ). Thus the loss of kinesin-13 does not make ciliary assembly more dependent on the synthesis of new proteins.…”

The Developmental Process of the Growing Motile Ciliary Tip Region

“…Previous investigations into ciliogenesis primarily studied length of the entire cilium during the elongation phase and/or cell motility 4 , 6 , 23 – 25 . In order to consider tip region development in the context of the growing cilium’s length, we determined both the average ciliary tip length and the average length of the entire cilium at different times of regrowth.…”

A Temperature‐Sensitive Mutation Affecting Synthesis of Outer Arm Dyneins in Tetrahymena thermophila

“…This result would be consistent with the hypothesis that the defect is in the microtubules only if the defective microtubules refolded in situ, and if outer arm dyneins were unstable and degraded if not immediately assembled into axonemes. Since Tetrahymena contain a cytoplasmic pool ofaxonemal components [2, 9,10,21,25,29], these results argue strongly against the hypothesis that the oad 1 mutation affects ciliary microtubules.…”