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

Abstract

We have characterized a novel, temperature-sensitive mutation affecting motility in Tetrahymena thermophila. Mutants grew and divided normally at the restrictive temperature (38 degrees C), but became nonmotile. Scanning electron microscopic analysis indicated that nonmotile mutants contained the normal number of cilia and that the cilia were of normal length. Transmission electron microscopic analysis indicated that axonemes isolated from nonmotile mutants lacked outer dynein arms, so the mutation was named oad 1 (outer arm deficient). Motile mutants shifted to 38 degrees C under conditions that prevent cell growth and division (starvation) remained motile suggesting that once assembled into axonemes at the permissive temperature (28 degrees C) the outer arm dyneins remain functional at 38 degrees C. Starved, deciliated mutants regenerated a full complement of functional cilia at 38 degrees C, indicating that the mechanism that incorporates the outer arm dynein into developing axonemes is not affected by the oad 1 mutation. Starved, nonmotile mutants regained motility when shifted back to 28 degrees C, but not when incubated with cycloheximide. We interpret these results to rule out the hypothesis that the oad 1 mutation affects the site on the microtubules to which the outer arm dyneins bind. Axonemes isolated from mutants grown for one generation at 38 degrees C had a mean of 6.0 outer arm dyneins, and axonemes isolated from mutants grown for two generations at 38 degrees C had a mean of 3.2 outer arm dyneins. Taken together, these results indicate that the oad 1 mutation affects the synthesis of outer arm dyneins in Tetrahymena.

Tubulin Glutamylation Regulates Ciliary Motility by Altering Inner Dynein Arm Activity

“…To test whether the TTLL6-mediated tubulin glutamylation affects the microtubule sliding generated specifically by IDAs, we constructed a strain that lacks TTLL6A and TTLL6F and is homozygous for the temperature-sensitive oad1-1 allele. When oad1-1 mutants are grown at restrictive temperature (38°C), ODAs fail to assemble and cells are nearly paralyzed [15, 16]. We assessed microtubule sliding in axonemes isolated from wildtype, oad1-1 , 6AF-KO and 6AF-KO; oad1-1 triple mutant cells, all grown for 12 hr at 38°C.…”

“…For rescue, vegetatively growing 6AF-KO cells were biolistically transformed with a fragment encoding MTT1-GFP-TTLL6A targeted to BTU1 [2]. A strain lacking TTLL6A and TTLL6F and homozygous the oad1-1 mutation [16] was constructed by standard crosses.…”

22S axonemal dynein is preassembled and functional prior to being transported to and attached on the axonemes

“…Both Schroeder et al [ 19901 and Larsen et al [1991] reported the three-headed and one-headed axonemal dyneins to be 22s and 12S, respectively. Additional evidence that the outer arm of Tetrahyrnena axonemes is composed of 22s dynein has been obtained from a temperature-sensitive mutant whose outer dynein arms are reduced by 90% at the restrictive temperature as a result of a reduced synthesis of 22s dynein [Attwell et al, 1992;Ludmann et al, 19931. While considerable information has been obtained on the function and structure of the axonemal dyneins, the questions of how and where the three-headed 22s or the 19s axonemal dynein is assembled and in what form it is transported to the ciliary or flagellar axonemes have remained unknown. In our laboratory we have been optimizing our previously published protocol for the purification of cytoplasmic dynein [Schroeder et al, 19901 and have determined that the use of leupeptin and pepstatin not only greatly improved the yield but also enabled us to purify two other dyneins.…”

Dephosphorylation of inner arm 1 is associated with ciliary reversals in Tetrahymena thermophila

“…Axonemes were removed from cell bodies, and dyneins were extracted from axonemes as described by Johnson [1986] and Attwell et al [1992]. At this step, the dynein preparation contains all dyneins extracted by high salt treatment and is referred to in this report as crude dyneins.…”