Henry D. Hoyle

Conserved axoneme symmetry altered by a component β-tubulin

Abstract Ninefold microtubule symmetry of the eukaryotic basal body and motile axoneme has been long established [1–3]. In Drosophila , these organelles contain distinct but similar β -tubulin isoforms [4–10]: basal bodies contain only β 1-tubulin, and only β 2-tubulin is used for assembly of sperm axonemes. A single α -tubulin functions throughout spermatogenesis [11,12]. Thus, differences in organelle assembly reside in β -tubulin. We tested the ability of β 1 to function in axonemes and found that β 1 alone could not generate axonemes. Small sequence differences between the two isoforms therefore mediate large differences in assembly capacity, even though these two related organelles have a common evolutionarily ancient architecture. In males with equal β 1 and β 2, β 1 was co-incorporated at equimolar ratio into functional sperm axonemes. When β 1 exceeded β 2, however, axonemes with 10 doublets were produced, an alteration unprecedented in natural phylogeny. Addition of the tenth doublet occurred by a novel mechanism, bypassing the basal body. It has been assumed that the instructions for axoneme morphogenesis reside primarily in the basal body, which normally serves as the axonemal template. Our data reveal that β -tubulin requirements for basal bodies and axonemes are distinct, and that key information for axoneme architecture resides in the axonemal β -tubulin.

Axoneme-dependent tubulin modifications in singlet microtubules of the Drosophila sperm tail

Drosophila melanogaster sperm tubulins are posttranslationally glutamylated and glycylated. We show here that axonemes are the substrate for these tubulin C-terminal modifications. Axoneme architecture is required, but full length, motile axonemes are not necessary. Tubulin glutamylation occurs during or shortly after assembly into the axoneme; only glutamylated tubulins are glycylated. Tubulins in other testis microtubules are not modified. Only a small subset of total Drosophila sperm axoneme tubulins have these modifications. Biochemical fractionation of Drosophila sperm showed that central pair and accessory microtubules have the majority of poly-modified tubulins, whereas doublet microtubules have only small amounts of mono- and oligo-modified tubulins. Glutamylation patterns for different beta-tubulins experimentally assembled into axonemes were consistent with utilization of modification sites corresponding to those identified in other organisms, but surrounding sequence context was also important. We compared tubulin modifications in the 9 + 9 + 2 insect sperm tail axonemes of Drosophila with the canonical 9 + 2 axonemes of sperm of the sea urchin Lytichinus pictus and the 9 + 0 motile sperm axonemes of the eel Anguilla japonica. In contrast to Drosophila sperm, L. pictus sperm have equivalent levels of modified tubulins in both doublet and central pair microtubule fractions, whereas the doublets of A. japonica sperm exhibit little glutamylation but extensive glycylation. Tubulin C-terminal modifications are a prevalent feature of motile axonemes, but there is no conserved pattern for placement or amount of these

Axoneme β-Tubulin Sequence Determines Attachment of Outer Dynein Arms

Axonemes of motile eukaryotic cilia and flagella have a conserved structure of nine doublet microtubules surrounding a central pair of microtubules. Outer and inner dynein arms on the doublets mediate axoneme motility [1]. Outer dynein arms (ODAs) attach to the doublets at specific interfaces [2-5]. However, the molecular contacts of ODA-associated proteins with tubulins of the doublet microtubules are not known. We report here that attachment of ODAs requires glycine 56 in the beta-tubulin internal variable region (IVR). We show that in Drosophila spermatogenesis, a single amino acid change at this position results in sperm axonemes markedly deficient in ODAs. Moreover, we found that axonemal beta-tubulins throughout the phylogeny have invariant glycine 56 and a strongly conserved IVR, whereas nonaxonemal beta-tubulins vary widely in IVR sequences. Our data reveal a deeply conserved physical requirement for assembly of the macromolecular architecture of the motile axoneme. Amino acid 56 projects into the microtubule lumen [6]. Imaging studies of axonemes indicate that several proteins may interact with the doublet-microtubule lumen [3, 4, 7, 8]. This region of beta-tubulin may determine the conformation necessary for correct attachment of ODAs, or there may be sequence-specific interaction between beta-tubulin and a protein involved in ODA attachment or stabilization.