David G. Pennock

Tubulin glutamylation regulates ciliary motility by altering inner dynein arm activity.

How microtubule-associated motor proteins are regulated is not well understood. A potential mechanism for spatial regulation of motor proteins is provided by posttranslational modifications of tubulin subunits that form patterns on microtubules. Glutamylation is a conserved tubulin modification [1] that is enriched in axonemes. The enzymes responsible for this posttranslational modification, glutamic acid ligases (E-ligases), belong to a family of proteins with a tubulin tyrosine ligase (TTL) homology domain (TTL-like or TTLL proteins) [2]. We show that in cilia of Tetrahymena, TTLL6 E-ligases generate glutamylation mainly on the B-tubule of outer doublet microtubules, the site of force production by ciliary dynein. Deletion of two TTLL6 paralogs caused severe deficiency in ciliary motility associated with abnormal waveform and reduced beat frequency. In isolated axonemes with a normal dynein arm composition, TTLL6 deficiency did not affect the rate of ATP-induced doublet microtubule sliding. Unexpectedly, the same TTLL6 deficiency increased the velocity of microtubule sliding in axonemes that also lack outer dynein arms, in which forces are generated by inner dynein arms. We conclude that tubulin glutamylation on the B-tubule inhibits the net force imposed on sliding doublet microtubules by inner dynein arms.

Dynein-2 Affects the Regulation of Ciliary Length but Is Not Required for Ciliogenesis in Tetrahymena thermophila

Eukaryotic cilia and flagella are assembled and maintained by the bidirectional intraflagellar transport (IFT). Studies in alga, nematode, and mouse have shown that the heavy chain (Dyh2) and the light intermediate chain (D2LIC) of the cytoplasmic dynein-2 complex are essential for retrograde intraflagellar transport. In these organisms, disruption of either dynein-2 component results in short cilia/flagella with bulbous tips in which excess IFT particles have accumulated. In Tetrahymena, the expression of the DYH2 and D2LIC genes increases during reciliation, consistent with their roles in IFT. However, the targeted elimination of either DYH2 or D2LIC gene resulted in only a mild phenotype. Both knockout cell lines assembled motile cilia, but the cilia were of more variable lengths and less numerous than wild-type controls. Electron microscopy revealed normally shaped cilia with no swelling and no obvious accumulations of material in the distal ciliary tip. These results demonstrate that dynein-2 contributes to the regulation of ciliary length but is not required for ciliogenesis in Tetrahymena.

Targeted gene knockout of inner arm 1 in Tetrahymena thermophila

Cilia and flagella contain at least eight different types of dynein arms. It is not entirely clear how the different types of arms are organized along the axoneme. In addition, the role each different type of dynein plays in ciliary or flagellar motility is not known. To initiate studies of dynein organization and function in cilia, we have introduced a mutation into one dynein heavy chain gene (DYH6) in Tetrahymena themophila by targeted gene knockout. We have generated mutant cells that lack wild-type copies of the DYH6 gene. We have shown that the DYH6 gene encodes one heavy chain (HC2) of Tetrahymena 18S dynein and that 18S dynein occupies the I1 position in the ciliary axoneme. We have also shown that Tetrahymena I1 is required for normal motility, normal feeding and normal doubling rate.

Biochemical analysis of a mutant Tetrahymena lacking outer dynein arms.

ABSTRACT. Tetrahymena thermophila mutants homozygous for the oad mutation become nonmotile when grown at the restrictive temperature, and axonemes isolated from nonmotile mutants lack approximately 90% of their outer dynein arms. Electrophoretic analyses of axonemes isolated from nonmotile mutants (oad axonemes) indicate they contain significantly fewer of the 22 S dynein heavy chains that axonemes isolated from wild‐type cells (wild‐type axonemes) contain. The 22 S dynein heavy chains that remain in axonemes isolated from nonmotile, oad mutants are assembled into 22 S dynein particles that exhibit wild‐type levels of ATPase activity. Two‐dimensional gel electrophoresis of oad axonemes show that they are deficient in no proteins other than those proteins thought to be components of 22 S dynein. This report is the first formal proof that outer dynein arms in Tetrahymena cilia are composed of 22 S dynein.

New axonemal dynein heavy chains from Tetrahymena thermophila.

ABSTRACT Two dyneins can be extracted from Tetrahymena ciliary axonemes. The 22S dynein contains three heavy chains (HC), sediments at 22S in a sucrose gradient, and makes up the outer arms. The 14S dynein contains two to six HCs, sediments at 14S, and is thought to contribute to formation of the inner arms. We have identified two large proteins that are extracted from Tetrahymena axonemes with high salt and that sediment together at approximately 18S. The two large proteins cleave when subjected to UV light in the presence of ATP and vanadate, suggesting both proteins are dynein HC. Antibodies against one of the 18S HCs do not recognize 22S dynein HCs. Antibodies to 22S dynein HC do not bind appreciably to 18S dynein photocleavage fragments. Taken together, these results indicate that the large proteins that sediment at 18S are axonemal dynein heavy chains.

SELECTION OF MOTILITY MUTANTS

Publisher Summary Inducing and screening for temperature-sensitive mutations affecting cilia regeneration is designed to identify genes that affect cilia regeneration. The strengths of this approach are that one makes no assumptions about which genes are important; any gene affecting the process can be identified. Also, ts mutations can be isolated, which allows analysis of essential genes. There are two limitations to this approach: (1) there is no easy way to identify and clone the mutated genes and (2) the only genes that affect cilia regeneration are identified, although the screen could be adapted to identify mutations that affect motility in growing cells but that have little effect during cilia regeneration. Advances in transformation of Tetrahymena shows that it is possible to disrupt the expression of any gene that is cloned, either by targeting a mutation to that gene using gene replacement techniques or by using antisense ribosomes. The advantages of targeted gene knockout are that one can determine whether any cloned gene is involved in motility and what role that gene plays.

The Dynein Heavy Chain Gene Family In Tetrahymena Thermophila

ABSTRACT The dynein ATPases are a family of motor enzymes that drive microtubule sliding in cilia and flagella and contribute to microtubule‐based transport inside cells. the multi‐dynein hypothesis makes two predictions: 1) Axonemes contain multiple dynein heavy chain (DHC) isoforms, each encoded by a different gene; 2) Each isoform performs a specific role in ciliary beating. We used PCR‐based techniques to clone thirteen different DHC sequences from Tetrahymena genomic DNA. All thirteen genes appeared to be expressed in growing cells. Comparisons of the deduced amino acid sequences of the thirteen DHCs with other known DHCs suggested that we have cloned three outer arm DHCs. two cytoplasmic DHCs, and eight inner arm DHCs.

Analyses of 22S Dynein Binding to Tetrahymena Axonemes Lacking Outer Dynein Arms

ABSTRACT. Tetrahymena thermophila mutants homozygous for the oad mutation become nonmotile when grown at the restrictive temperature of 39° C. Axonemes isolated from nonmotile oad mutants (oad 39° C axonemes) lack approximately 90% of their outer dynein arms and are deficient in 22S dynein. Here we report that oad 39° C axonemes contain 40% of the 22S dynein heavy chains that wild‐type axonemes contain and that oad axonemes do not undergo ATP‐induced microtubule sliding in vitro. Wild‐type 22S dynein will bind to the outer arm position in oad axonemes and restore ATP‐induced microtubule sliding in those axonemes. Unlike wild‐type 22S dynein, oad 22S dynein does not bind to the outer arm position in oad axonemes. These data indicate that the oad mutation affects some component of the outer arm dynein itself rather than the outer arm dynein binding site. These data also indicate that oad axonemes can be used to assay outer dynein arm function.

The dcc Mutation Affects Ciliary Length in Tetrahymena thermophila

ABSTRACT. We have characterized ciliogenesis in a mutant Tetrahymena thermophila that both fails to regain motility following deciliation and that fails to complete cytokinesis. Scanning electron microscopic (SEM) observations revealed that starved deciliated cells regenerated fewer, shorter cilia at the restrictive temperature than similarly treated cells incubated at the permissive temperature. Transmission electron microscopic evaluation of isolated, regenerated cilia revealed no structural abnormalities. Incorporation of S‐35 methionine was similar during ciliary regeneration at both the restrictive and permissive temperatures, indicating the mutant phenotype was not due to a simple failure in translation or transcription. Mutant cells incubated in growth medium at the restrictive temperature arrested in cytokinesis and assembled a large number of abnormally short cilia. These cells also developed irregular surface projections that were not visible on wild‐type cells. These observations suggest that ciliogenesis can be initiated in growing cells as well as in starved deciliated cells but that elongation is inhibited before cilia reach full length. The mutation was named dcc for defective in ciliogenesis and cytokinesis.

p28 dynein light chains and ciliary motility in Tetrahymena thermophila

Dynein light chains are required for the assembly of axonemal dyneins into cilia and flagella. Most organisms express a single p28 dynein light chain and four to nine one‐headed inner arm dynein heavy chains. In contrast, Tetrahymena encodes three p28 dynein light chain genes (p28A, p28B, and p28C) and 18 one‐headed inner arm dynein heavy chains. In this article it is shown that mutations in p28A and p28B affected both beat frequency and waveform of cilia, while mutations in p28C affected only ciliary beat frequency. A similar set of dynein heavy chains were affected in both p28AKO and p28BKO, but a distinct set of heavy chains was affected in p28CKO. The results suggested that the p28s have non‐redundant functions in Tetrahymena and that p28C was associated with a different set of dynein heavy chains than were p28A and p28B.