Centrosomes are microtubule-based organelles that are required for assembly of the mitotic spindle. Spindles function to accurately partition chromosomes during cell division to generate two genetically identical daughter cells. Aberrant expression of many centrosome-localized proteins has been linked to diseases such as cancer and primary microcephaly. Therefore, it is critical to identify new centrosome- associated proteins. Biochemical isolation of centrosomes from C. elegans embryos has never been reported. By isolating and lysing the nematode embryos, we performed ultracentrifugation over a sucrose step gradient to enrich for <i>C. elegans</i> centrosomes. Importantly, the isolated centrosomes behaved like microtubule organizing centers and nucleated asters <i>in vitro</i>. Following this, mass spectrometry was performed to identify the proteins that were present in the centrosomal fraction. In addition to identifying known core centrosomal proteins, additional proteins that localize to the centrosomes were also identified by our mass spectrometry analysis. One such protein is RACK-1 (receptor for activated C kinase 1). RACK-1 is a highly abundant ribosomal protein which has been shown to act as a scaffold protein to assemble diverse signaling complexes. Using CRISPR/Cas9 gene editing, we were able to generate a worm strain containing GFP-tagged RACK-1. Supporting previous studies, confocal live imaging demonstrated that GFP-tagged RACK-1 localizes to the centrosomes in C. elegans embryos. Since RACK- 1 localizes to the centrosomes, we investigated whether RACK-1 plays a role in regulating the process of centrosome duplication. Immunoprecipitation followed by mass spectrometry analysis identified RACK-1 as an interacting partner of the master centrosome duplication kinase ZYG-1. In addition to detecting a physical interaction between RACK-1 and ZYG-1 proteins, we also identified a genetic interaction between the two. Specifically, rack-1 RNAi enhanced the embryonic lethality of zyg-1 mutants, thereby suggesting that the depletion of RACK-1 worsens the centrosome duplication defects of zyg-1 mutants, yielding dead embryos. These data collectively indicate that RACK-1 positively regulates centrosome duplication. Thus, our data have identified RACK-1 as a novel protein that is required for centrosome duplication. Future studies will involve performing experiments to dissect the molecular mechanism by which RACK-1 regulates centrosome duplication.

RACK-1 is a centrosomal-localized protein that is required for proper centrosome duplication

Macromolecular Chemistry: The Second Century

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ACS Spring 2020 National Meeting & Expo

The official archive and repository for all ACS Meetings & Expositions abstracts and select posters or presentations.