Abstract: The canonical Wnt signaling pathway promotes well-characterized transcriptional programs that direct cell cycle control, proliferation and cell self-renewal, among other processes. We have recently discovered that Wnt signaling also promotes a rich post-translational program by the stabilization and modulation of proteins other than b-catenin. In dividing cells, this occurs preferentially at the G2/M transition of the cell cycle, when gene transcription is switched off, and is now referred to as Wnt-mediated stabilization of proteins (Wnt/STOP). Loss of the Wnt/STOP signaling during mitosis leads to aneuploidy in human somatic cells, and causes severe mitotic defects in embryos. However, the roles and effectors of Wnt/STOP signaling, and how they contribute to abnormal mitosis and chromosome missegregation, remain unexplored. Thus, we will join forces to analyze how Wnt signaling modulates different aspects of mitosis. For this, we will establish cell models based on human somatic and mouse embryonic stem cells (mESCs), which allow the selective inhibition of Wnt signaling in mitosis in the absence of Wnt driven gene expression. Using live cell microscopy we will investigate the dynamic regulation of chromosome alignment, microtubule assembly, spindle orientation, centrosome positioning, chromosome segregation, and symmetry of the division in dependence of the activation of Wnt signaling during mitosis. To understand the impact of Wnt signaling on mitosis at a molecular level, we aim to identify mitosis-specific target proteins that are regulated by Wnt/STOP. As a first step towards this goal, we have analyzed the phospho-proteome of mESCs upon Wnt signaling activation and identified novel targets, many of them implicated in the regulation of mitosis. This approach will be complemented by SILAC-based mass spectrometry analyses to identify novel Wnt/STOP targets in somatic cells. Subsequently, we will characterize the mitotic functions of the newly identified proteins that are regulated by Wnt signaling during cell division. To this end, we will employ phenotypic siRNA screens that will unravel those candidates identified by the systematic approaches that fulfill key roles in different aspects of mitotic regulation, including the regulation of microtubule dynamics and chromosome segregation. For the validated targets we will further explore their molecular functions in mitosis using biochemical and cell biological methods. By integrating these data we will chart how Wnt signaling promotes faithful execution of mitosis, and unravel how its misregulation leads to chromosome instability and unscheduled proliferation, which are hallmarks of various human diseases including cancer.