Diamond (dind) is an essential Drosophila gene identified by five mutant alleles induced by EMS. Larvae homozygous for these mutations die at the third instar stage due to extensive defects in chromosome structure and mitosis. Cytological analysis of DAPI stained larval brains revealed that in dind mutants dividing cells are arrested in metaphase and exhibit a severe pleiotropic phenotype. 50% of metaphases are aneuploid or polyploid, 2% are endoreduplicated, 65% exhibit irregularly condensed chromosome and 33% contain broken chromosomes. In addition, in dind brains immunostained for tubulin, we consistently observed diploid cells with defective spindles and anaphase-like figures that contain chromosomes with unseparated sister chromatids. The latter defect does not appear to be due to kinetochore defects, as the localization of several kinetochore components is regular in brain cells of dind mutants. Nevertheless, loss of Dind severely compromises chromosome biorentation and segregation. In dind metaphase figures the Spindle Assembly Checkpoint (SAC) component Zw10 accumulates at the kinetochores and does not ‘‘stream’’ along the spindle microtubules, suggesting a failure to satisfy the SAC. Immunostaining of larval brains for the centrosomal and centriolar markers DSpd-2 and Asterless (Asl) revealed that dind mutants exhibit fragmented centrioles surrounded by pericentriolar material (PCM). These centriole-containing centrosomal fragments give rise to aberrant numbers of microtubule organizing centers (MTOCs) that lead to the assembly of disorganized spindles. Analysis of male germline cells revealed that dind mutant spermatogonia and spermatocytes exhibit most of the phenotypic traits observed in somatic cells, pointing to a pleiotropic role of dind in both mitotic and meiotic divisions. The lethality and the complex mitotic and meiotic phenotypes elicited by dind mutations are completely rescued by the expression of GFP-tagged dind transgenes. The analysis of GFP-Dind and Dind-GFP expressing brains stained with anti-GFP antibodies revealed that Dind has a diffuse localization in both interphase and mitotic cells, with no detectable signal accumulation in any intracellular structure. The same pattern was observed in wild type larval brains immunostained with an anti-Dind antibody. However, Western blotting on larval cell extracts obtained by separation of cytoplasmic, nucleoplasmic and chromatin fractions showed that Dind is present in the fraction deriving from the pelleted nuclear material, suggesting that at least part of the Dind protein is associated with the chromatin throughout most of the cell cycle. We thus investigated possible interactions between Dind and chromosomal proteins whose loss leads to defects similar to those observed in dind mutants such as Separase (Sse) and Topoisomerase 2 (Top2). Both Sse and Top2 levels resulted significantly reduced in brain extracts from dind mutants. Strikingly, also the amount of Dind was lowered in extracts from both Sse and Top2 mutants, pointing to a mutual dependence between Dind and these two chromosomal proteins. To the best of our knowledge, dind is the first Drosophila gene so far identified specified by mutations that cause the variety of phenotypic traits described here: defects in chromosome condensation, CABs, metaphase arrest, centriole fragmentation, polyploidy, aneuploidy and endoreduplication. The defects in chromosome morphology and integrity might be explained by assuming that loss of Dind alters the chromatin structure, preventing proper recruitment and stability of Sse and Top2, whose loss would in turn destabilize Dind. On the other hand, the metaphase arrest phenotype, the formation of aneuploid and polyploid cells and centriole fragmentation are more difficult to reconcile with a single chromatin-related function of dind. We thus speculate that the unique pleiotropic phenotype of dind mutants is due to the “moonlighting” functions of the Dind protein, which might be able to perform different roles in different contexts. Further experiments will help to verify this hypothesis and elucidate the Dind functions at the molecular level.
The role of the diamond gene in the control of Drosophila genome stability / Graziadio, Lucia. - (2018 Feb 15).
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