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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 β-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 modifications. We conclude their functions are likely species-specific. Cell Motil. Cytoskeleton 2008. © 2008 Wiley-Liss, Inc. [ABSTRACT FROM AUTHOR]

Focuses on a testis-specific ...-tubulin (...2) Drosophila melanogaster which is required for spremato-genesis. Identification of a sequence motif in carboxyle termini of axonemal ...2 in diverse taxa; Tests of differences of orthologous ..2 from different species.

Using Drosophila spermatogenesis as a model, we show that function of the beta-tubulin C-terminal tail (CTT) is not independent of the body of the molecule. For optimal microtubule function, the beta-tubulin CTT and body must match. beta2 is the only beta-tubulin used in meiosis and spermatid differentiation. beta1-tubulin is used in basal bodies, but beta1 cannot replace beta2. However, when beta1 is co-expressed with beta2, both beta-tubulins are equally incorporated into all microtubules, and males exhibit near wild type fertility. In contrast, co-expression of beta2beta1C and beta1beta2C, two reciprocal chimeric molecules with bodies and tails swapped, results in defects in meiosis, cytoskeletal microtubules, and axonemes; males produce few functional sperm and few or no progeny. In these experiments, all the same beta-tubulin parts are present, but unlike the co-assembled native beta-tubulins, the "trans" configuration of the co-assembled chimeras is poorly functional. Our data thus reveal essential intra-molecular interactions between the CTT and other parts of the beta-tubulin molecule, even though the CTT is a flexible surface feature of tubulin heterodimers and microtubules. In addition, we show that Drosophila sperm tail length depends on the total tubulin pool available for axoneme assembly and spermatid elongation. D. melanogaster and other Drosophila species have extraordinarily long sperm tails, the length of which is remarkably constant in wild type flies. We show that in males of experimental genotypes that express wild type tubulins but have half the amount of the normal tubulin pool size, sperm tails are substantially shorter than wild type., (Copyright 2008 Wiley-Liss, Inc.)

The relationship between the primary structure of the beta-tubulin C-terminal tail (CTT) and axoneme structure and function is explored using the spermatogenesis-specific beta2-tubulin of Drosophila. We previously showed that all beta-tubulins used for motile 9 + 2 axonemes contain a conserved sequence motif in the proximal part of the CTT, the beta-tubulin axoneme motif. The differential ability of tubulin isoforms and abilities of beta2-tubulin C-terminal truncations to form axonemes led us to hypothesize that the axoneme motif is essential for axoneme formation and the distal half of the CTT was less important. The studies we report here indicate that it is not that simple. Unexpectedly, some changes in the core sequence of the axoneme motif did not disrupt formation of motile axonemes. And, while deletion of the distal CTT did not disrupt the ability to produce functional sperm [Popodi et al., Cell Motil Cytoskeleton 2005;62:48-64], changing the amino acid sequence in this region can. Thus both regions are important. The deep conservation of the axoneme motif in all eukaryotic groups implies that the presence of the sequence motif confers a functional advantage. The central pair is the axoneme structure most sensitive to perturbations in tubulin molecules; we hypothesize central pair assembly is facilitated by the presence of this motif. Our data reveal that beta2-tubulin has robust properties for axoneme assembly, and that axonemal specializations are embedded in both the CTT and the body of the beta2 molecule.

Marine embryos and larvae reflect distinct life histories and body plans from their adults, and are relatively simple in morphology and genetic regulation. Evolution of development to produce highly modified larval forms can be rapid among closely related species. We have studied the mechanisms by which the non-feeding direct-developing larva of the sea urchin Heliocidaris erythrogramma evolved from an indirect-developing feeding larva, the pluteus. H. erythrogramma diverged within 4 million years from its sister species, H. tuberculata, which has a typical pluteus larva. Radical evolution of H. erythrogramma early development allows it to reach metamorphosis in three to four days versus the several weeks required for the pluteus. We have used embryology, cross species hybrids, and manipulation of gene expression in embryos to dissect developmental changes and the genic controls that underlie these changes. Evolution of a new larval form resulted largely from several heterochronies in which conserved regulatory pathways are shifted in timing, producing new temporal relationships to other developmental events. Other changes in gene regulation also have contributed to rapid evolution of larval features, including the origin of unexpected and novel tissue identities that transcend changes within homologous features.

We have used Drosophila testis-specific beta2-tubulin to determine sequence requirements for different microtubules. The beta2-tubulin C-terminal tail has unique sperm-specific functions [Dev Biol 158:267-286 (2003)] and is also important for forming stable heterodimers with alpha-tubulin, a general function common to all microtubules [Mol Biol Cell 12(7):2185-2194 (2001)]. beta-tubulins utilized in motile 9 + 2 axonemes contain a C-terminal sequence "axoneme motif" [Science 275 (1997) 70-73]. C-terminal truncated beta2-tubulin cannot form the sperm tail axoneme. Here we show that a partially truncated beta2-tubulin (beta2Delta7) containing only the proximal portion of the C-terminal tail, including the axoneme motif, can support production of functional motile sperm. We conclude that these proximal eight amino acids specify the binding site for protein(s) essential to support assembly of the motile axoneme. Males that express beta2Delta7, although they are fertile, produce fewer sperm than wild type males. Beta2Delta7 causes a slightly increased error rate in spermatogenesis attributable to loss of stabilizing properties intrinsic to the full-length C-terminal tail. Therefore, beta2Delta7 males would be at a selective disadvantage and it is likely that the full-length C-terminus would be essential in the wild and in evolution., ((c) 2005 Wiley-Liss, Inc.)

Through evolutionary history, some features of the phenotype show little variation. Stabilizing selection could produce this result, but the possibility also exists that a feature is conserved because it is developmentally constrained--only one or a few developmental mechanisms can produce that feature. We present experimental data documenting developmental constraint in the assembly of the motile sperm tail axoneme. The 9+2 microtubule architecture of the eukaryotic axoneme has been deeply conserved. We argue that the quality of motility supported by axonemes with this morphology explains their long conservation, rather than a developmental necessity for the 9+2 architecture. However, our functional tests in Drosophila spermatogenesis reveal considerable constraint in the coevolution of testis-specific beta-tubulin and the sperm tail axoneme. The evolution of testis beta-tubulins used in insect sperm tail axonemes is highly punctuated, indicating some pressure acting on their evolution. We provide a mechanistic explanation for their punctuated evolution by testing structure-function relationships between testis beta-tubulin and the motile axoneme in D. melanogaster. We discovered that a highly conserved sequence feature of beta-tubulins used in motile axonemes is needed to specify central pair formation. Second, our data suggest that cooperativity in the function of internal beta-tubulin amino acids is needed to support the long axonemes characteristic of Drosophila sperm tails. Thus, central pair formation constrains the evolution of the axoneme motif, and intramolecular cooperativity makes the evolution of the internal residues path dependent, which slows their evolution. Our results explain why a highly specialized beta-tubulin is needed to construct the Drosophila sperm tail axoneme. We conclude that these constraints have fixed testis-specific beta-tubulin identity in Drosophila.