Regulation of β‐tubulin function and expression in Drosophila spermatogenesis

Abstract

In this study we examined two aspects of β-tubulin function in Drosophila spermatogenesis: 1) β-tubulin structural requirements for assembly of different categories of microtubules and 2) regulatory requirements for production of the correct tubulin protein level. In normal Drosophila spermatogenesis, the testis-specific β2-tubulin isoform supports multiple microtubule functions. Our previous work showed that another Drosophila isoform, β3, cannot support spermatogenesis, whereas a carboxyl-truncated form of β2, β2ΔC, can at least to some extent provide all of β2′s normal functions, save one: β2ΔC cannot support organization of axonemal microtubules into the supramolecular architecture of the axoneme. Here, to test whether β2 carboxyl sequences can rescue the functional failure of the β3 isoform in spermatogenesis, we constructed a gene encoding a chimeric protein, β3β2C, in which β3 sequences in the carboxyl region are replaced with those of β2. Unlike either β3 or β2ΔC, β3β2C can provide partial function for both assembly of axonemal microtubules and their organization into the supramolecular architecture of the axoneme. In particular, the β2 carboxyl sequences mediate morphogenesis of the axoneme doublet tubule complex, including accessory microtubule assembly and attachment of spokes and linkers. However, our data also reveal aspects of β2-specific function that require structural features other than the primary sequence of the isotype-defining variable regions, the C terminus and the internal variable region. Tests of fecundity in males that co-express Δ2 and the chimeric Δ3Δ2C protein showed that in Drosophila there are differential requirements for sperm motility in the male and in the female reproductive tract. Since some aspects of microtubule function in spermatogenesis are sensitive to the tubulin pool size, we examined the mechanisms for control of tubulin protein levels in the male germ cells. We found that both Δ2-tubulin mRNA accumulation and protein synthesis are dependent on gene dose, and that the level of expression is regulated by 3′ noncoding sequences in the Δ2 gene. Our data show that the regulatory mechanisms that control tubulin pool levels in the Drosophila male germ line differ from those observed in cultured animal somatic cells. Finally, expression of transgenic constructs is consistent with early cessation of × chromosome expression in Drosophila spermatogenesis.