Abstract P035: The Nucleolar Protein Nucleophosmin Regulates Mitosis Progression in Cardiac Progenitor Cells

Abstract

Besides its notorious function during ribosome biogenesis, emerging evidence suggests a central role for the nucleolus in controlling many other important cellular processes such as stress response, proliferation and mitosis. Here we show that the nucleolar protein Nucleophosmin (NPM) is necessary for the correct progression of mitosis in primary cultures of cardiac progenitor cells (CPCs). Methods: CPCs were cultured on Permanox chamber slides at high confluence and fixed with 4% PFA. CPCs were blocked in cytokinesis with 2μ M cytochalasin B (CytB) for 18 hours. Silencing of NPM (ShNPM) or scramble (ShSc) was performed by specific shRNA expressing lenti viral vectors. Confocal microscopy, co-immunoprecipitation and proximity ligation assay (PLA), FACS analysis and Cyquant assay were employed to study protein localization and interaction, cell death and proliferation respectively. Results: ShNPM infected CPCs showed reduced proliferation with no apparent cell death compared to scramble. Cells in prometa and metaphase significantly increased after NPM knock down. ShNPM-CPC showed 50% decrease in the number of cells that complete mitosis as assessed by counting binucleated cells after CytB treatment. NPM localized in the nucleolus during interphase as assessed by co-staining with nucleostemin (NS), a highly expressed nucleolar protein. NS and NPM decorate the chromosome periphery from prometaphase to anaphase and return to the newly forming nucleolus at the end of telophase. The phosphorylated form of NPM at threonine 199 (pNPM) was found to co-localize with spindle components such us NUMA and α-tubulin specifically at methaphase. NPM was also found to co-precipitate and directly interact with α-tubulin as shown by in-situ protein interaction assay (PLA) in the cytoplasm during interphase and at the centrosome and spindle during mitosis. Conclusion: This is the first study to detail the role of nucleolar proteins during CPCs mitosis. Our findings demonstrate a central role for NPM during metaphase transition, possibly thorough interaction with components of the spindle and in particular with α-tubulin.