Abstract LB-085: Protein phosphatase 1 and the RNA-binding protein CSTL2 function to constrain daughter centriole number

Abstract

Abstract Centrioles are cylindrical microtubule-based structures that are required for the formation of cilia, flagella and the mitotic spindle. A centrosome is comprised of two centrioles (one mother and one daughter) that are organized in an orthogonal orientation. Centrioles are duplicated only once during each cell cycle and this involves the formation of a single daughter centriole next to each mother. Dysregulation of this process yields an abnormal centriole number and this can result in aneuploidy, a hallmark of cancer cells. Therefore, it is critical that centriole duplication (CD) is tightly regulated. The nematode C. elegans is an excellent model system to study the process of CD because the core components of the CD pathway in C. elegans are conserved in humans. The main purpose of this study is to identify novel regulators of CD using C. elegans as a model system. The kinase ZYG-1 is a functional ortholog of human PLK4 and is absolutely essential for CD. Using a zyg-1 suppression assay, we have identified Protein Phosphatase 1 (PP1) as a critical inhibitor of CD. Using super-resolution microscopy, we found that deactivating PP1 results in the formation of multiple daughter centrioles adjacent to a single mother. Western blot analysis indicated that the mechanism by which PP1 inhibits CD is by decreasing ZYG-1 levels. Our study has also identified an RNA-binding protein with homology to human cleavage stimulation factor subunit 2 tau variant (CSTL-2) as a potential PP1 substrate and a novel component of the C. elegans CD pathway. Utilizing a fluorescently tagged CSTL-2 transgenic worm line, we determined that CSTL-2 localizes to the centrosomes during mitosis. An immunoprecipitation assay followed by mass spectrometry has identified CSTL-2 as an interacting partner of PP1- thereby implicating it as a PP1 substrate. To evaluate the effect of CSTL-2 on ZYG-1-mediated CD, cstl-2-null; zyg-1-hypomorphic double mutants were constructed. At the restrictive temperature, the zyg-1-hypomorphic mutant worms show 100% embryonic lethality due to a failure of CD. However, in the cstl-2-null; zyg-1-hypomorphic double mutants, approximately 20% of the embryos survive indicating a role for CSTL-2 as an inhibitor of CD. We performed cytological analyses on cstl-2-null; zyg-1-hypomorphic double mutant embryos and confirmed that CSTL-2, like PP1, also functions as an inhibitor of CD. Therefore, based upon our data, we conclude that PP1 is an important negative regulator of CD which functions to ensure that only one daughter centriole forms adjacent to a mother centriole and we propose that CSTL-2 is a substrate through which PP1 regulates CD. In summary, this study highlights a novel pathway that controls centriole number in C. elegans, which may be dysregulated in cancer. Citation Format: Jyoti Iyer, Neena Peel, Sean O'Rourke, Bruce Bowerman, Kevin O'Connell. Protein phosphatase 1 and the RNA-binding protein CSTL2 function to constrain daughter centriole number. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr LB-085. doi:10.1158/1538-7445.AM2015-LB-085