Regulation of β‐tubulin function and expression in Drosophila spermatogenesis

Abstract

In this study we examined two aspects of beta-tubulin function in Drosophila spermatogenesis: 1) beta-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 beta 2-tubulin isoform supports multiple microtubule functions. Our previous work showed that another Drosophila isoform, beta 3, cannot support spermatogenesis, whereas a carboxyl-truncated form of beta 2, beta 2 delta C, can at least to some extent provide all of beta 2's normal functions, save one: beta 2 delta C cannot support organization of axonemal microtubules into the supramolecular architecture of the axoneme. Here, to test whether beta 2 carboxyl sequences can rescue the functional failure of the beta 3 isoform in spermatogenesis, we constructed a gene encoding a chimeric protein, beta 3 beta 2C, in which beta 3 sequences in the carboxyl region are replaced with those of beta 2. Unlike either beta 3 or beta 2 delta C, beta 3 beta 2C can provide partial function for both assembly of axonemal microtubules and their organization into the supramolecular architecture of the axoneme. In particular, the beta 2 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 beta 2-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 coexpress beta 2 and the chimeric beta 3 beta 2C 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 beta 2-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 beta 2 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 X chromosome expression in Drosophila spermatogenesis.

Segregation distortion induced by wild-type RanGAP in Drosophila

“…To overexpress wild-type RanGAP in the male germ line, we used the ␤2-tubulin promoter, which specifically drives high levels of expression in the postmitotic male germ line (24,25). The ORF of wild-type RanGAP flanked with EcoRI sites was cloned into the EcoRI site of the testis vector, which contains the ␤2-tubulin promoter inserted into the pCasPeR4 transformation vector.…”

“…To investigate whether even wild-type RanGAP is capable of causing segregation distortion if it could somehow be made to accumulate inside nuclei, we examined the effect on chromosome transmission of overexpressing wild-type RanGAP in the male germ line. For this purpose, we drove expression of wild-type RanGAP from the ␤2-tubulin promoter, which is expressed specifically in the male germ line (24,25). Expression of RanGAP in the testes of transgenic males was increased about 20-fold compared with controls ( Fig.…”

Auxilin is required for formation of Golgi-derived clathrin-coated vesicles during Drosophila spermatogenesis

“…As the b2-tubulin promoter is active at the primary spermatocyte stage (Kemphues et al, 1982), rescue of aux male sterility by b2tub-dAux FL -mRFP would suggest that the requirement for Aux function during spermatogenesis is autonomous. To ensure that the b2tub expression cassette (Huh et al, 2004) recapitulates the specificity of the b2-tubulin promoter (Hoyle et al, 1995), we made a b2tub-luciferase construct and showed that its activity was restricted to males and to the testes, and was absent in bag of marbles mutants, in which the differentiation of male germ cells is blocked (McKearin and Ohlstein, 1995;McKearin and Spradling, 1990) (see Fig. S1 in the supplementary material).…”

Auxilin is required for formation of Golgi-derived clathrin-coated vesicles during Drosophila spermatogenesis

“…As the β2-tubulin promoter is active at the primary spermatocyte stage (Kemphues et al, 1982), rescue of aux male sterility by β2tub-dAux FLmRFP would suggest that the requirement for Aux function during spermatogenesis is autonomous. To ensure that the β2tub expression cassette (Huh et al, 2004) recapitulates the specificity of the β2-tubulin promoter (Hoyle et al, 1995), we made a β2tub-luciferase construct and showed that its activity was restricted to males and to the testes, and was absent in bag of marbles mutants, in which the differentiation of male germ cells is blocked (McKearin and Ohlstein, 1995;McKearin and Spradling, 1990) (see Fig. S1 in the supplementary material).…”