Creating a CRISPR-engineered Polo-like kinase 4 mutant sensitized to small molecule inhibitors in Drosophila
Fryer, Lily
Fryer, Lily
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Abstract
An essential protein that regulates the centrosome is the serine-threonine kinase Polo-like-kinase 4 (Plk4). Plk4 is the master regulator of centrosome biogenesis. It has been proposed that Plk4 has functions beyond centrosome biogenesis, however, the study of these in Drosophila is a challenge as Plk4 mutants lack centrosomes to observe. I aimed to characterize novel roles for Plk4 in centrosome dynamics using small molecule inhibitors to acutely perturb Plk4 function, while centrosomes are still present. However, I found Drosophila Plk4 is not susceptible to Centrinone, a commercially available Plk4 inhibitor. From the cells treated with centrinone (n=73) 98.6% had 2 or more centrosomes; cytokinesis failure, an off-target effect, was also observed in 56.2% of treated cells. Therefore, I created a gatekeeper mutant (plk4as1) which changes the shape of the ATP binding site, sensitizing the kinase to inhibition by PP1 analogs. Unfortunately, I found that without inhibitor present the gatekeeper mutation caused a loss of Plk4 function; this was quantified by the absence of centrosomes in 97.7% of the mutant cells (n=174) compared to 1.2% of wild type cells (n=82). This was further displayed by decreased survivability of the mutants; plk4as1 homozygous pupae eclosed but died shortly after. Through comparison to other analog sensitive kinases by sequence alignment, I identified a secondary mutation which could rescue kinase function (plk4as2). Excitingly, 100% of the plk4as2 cells measured (n=76) possessed 2 centrosomes. When the plk4as2 line was treated with a PP1 analog inhibitor, the cells displayed a reduction in centrosome number, while the wild-type cells maintained 2 centrosomes. Thus, I have successfully created a Plk4 mutant sensitized to small molecule inhibition. This tool will be used to probe Plk4 functions beyond centrosome biogenesis in Drosophila, and its role in asymmetric cell division.
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Date
1/1/2026
Student Status
Senior (Graduating in 2026)
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Poster
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Molecular Genetics
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College of Agriculture and Life Sciences
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Life Science