A genetic screen for mechanisms that counter extra centrosomes

2020-04-20T22:15:12Z (GMT) by Erin Jezuit Don Fox

Metazoan cells carefully regulate centrosome number. With the exception of multiciliated cells, the presence of more than two centrosomes per cell can be detrimental, causing a host of disease phenotypes including cellular aneuploidy and aberrant cell motility. Understanding the cellular mechanisms that counter extra centrosomes and associated phenotypes can reveal fundamental centrosome regulation and potential therapies of centrosome-related diseases. We previously found a tissue, the Drosophila rectal papillar cells of the hindgut, that are extremely tolerant to extra centrosomes and multipolar divisions. When the centrosome duplication regulator Polo-like kinase 4 (Plk4) is overexpressed in this tissue, ~100% of cells contain extra centrosomes, and ~75% of these cells in this developing organ undergo tripolar division. Despite this aberrant centrosome production, no cell death occurs and the tissue develops and functions normally. In order to better understand the mechanism(s) by which these cells tolerate extra centrosomes and the extreme aneuploidy generated by tripolar cell division, we are conducting a forward genetic screen for mutants that are lethal in papillar tissue only in the presence of extra centrosomes. To do so, we have generated 1300 recessive mutations on the X chromosome and are generating mosaic homozygous clones in papillar tissue to screen for lethality specifically in a Plk4-overexpression background. This screen takes advantage of the ability of wild type rectal papillar tissue to excrete excess salt. Animals with compromised papillae, including large patches of mosaic mutant cells, die rapidly on a high-salt diet. Using this simple feeding assay, we have already screened 807 EMS-generated lines and identified seven mutants that reproducibly yield phenotypes only when centrosomes are amplified. To further analyze mutants, we will evaluate wild type and mutant clones during mitosis using centrosome, chromatin and microtubule markers. Results from this screen are beginning to reveal new, physiologically relevant responses to centrosome amplification.