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 β-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-tubulin60D M) 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 Pin 1, an unknown dominant mutant with bristle developmental defects, as a dominant-negative allele of beta-tubulin60D. We also found a missense mutation in the Pin 1 mutant that results in an amino acid replacement from the highly conserved glutamate at position 75 to lysine (E75K). Analyzing the β-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.