Revisiting the role of beta-tubulin in Drosophila development: beta-tubulin60D is not an essential gene, and its novel Pin1 allele has a tissue-specific dominant-negative impact

Abstract

Diversity in cytoskeleton organization and function may be achieved through alternative tubulin isotypes and by a variety of post-translational modifications. The Drosophila genome contains five different {beta}-tubulin paralogs, which may play an isotype tissue-specific function in vivo. One of these genes, the beta-tubulin60D gene, which is expressed in a tissue-specific manner, was found to be essential for fly viability and fertility. To further understand the role of the beta-tubulin60D gene, we generated new beta-tubulin60D null alleles (beta-tubulin60DM) using the CRISPR/Cas9 system and found that the homozygous flies were viable and fertile. Moreover, using a combination of genetic complementation tests, rescue experiments, and cell biology analyses, we identified Pin1, an unknown dominant mutant with bristle developmental defects, as a dominant-negative allele of beta-tubulin60D. We also found a missense mutation in the Pin1 mutant that results in an amino acid replacement from the highly conserved glutamate at position 75 to lysine (E75K). Analyzing the {beta}-tubulin structure suggests that this E75K alteration destabilizes the alpha-helix structure and may also alter the GTP-Mg2+ complex binding capabilities. Our results revisited the credence that beta-tubulin60D is required for fly viability and revealed for the first time in Drosophila, a novel dominant-negative function of missense beta-tubulin60D mutation in bristle morphogenesis. Author summaryDiversity in cell microtubule cytoskeleton organization and function may be achieved through alternative tubulin isotypes and by a variety of post-translational modifications. The expression pattern of different tubulin isotypes (both and {beta} subunits) can vary according to cell type and stage of development, which contribute significantly to cell-specific MT organization and function. In this study, we revisited the role of one of the beta-tubulin isotopes in Drosophila, namely, beta-tubulin60D. This is the first study where a well molecularly defined protein null allele of {beta}Tub60D was generated and characterized. This well-characterized {beta}Tub60D allele demonstrated unambiguity that {beta}Tub60D is not an essential gene, as was described before. Moreover, we identified Pin1, an unknown dominant mutant with bristle developmental defects, as a dominant-negative allele of beta-tubulin60D. We also found a missense mutation in the Pin1 mutant that results in an amino acid (E75K). Analyzing the {beta}-tubulin structure suggests that this E75K alteration destabilizes the alpha-helix structure and may also alter GTP-Mg2+ complex binding capabilities. Thus, our results also revealed for the first time in Drosophila, a novel dominant-negative function of a missense beta-tubulin60D mutation, which has a tissue-specific function.