Uncovering the Molecular Mechanisms of Asymmetric Cell Divisions in Mammalian Adult Epithelia (360G-Wellcome-210077_Z_17_Z)

Loss of asymmetric cell divisions (ACDs) regulation in the normal self-renewing stem cells is entwined with the growth and progression of poorly differentiated cancers. Yet, the molecular mechanisms controlling the execution of symmetric versus asymmetric divisions in adult epithelia are still unfolding. We recently demonstrated that KIF5/kinesin-1 is essential for mammary epithelial cell divisions and cytoarchitecture by governing the trafficking of the spindle orientation and apical polarity components, respectively. Whether KIF5/kinesin-1 couples spindle orientation and polarity machineries during mitosis to promote ACDs; and whether other mechanisms are involved remain open questions. Here, we will address these questions in mammary 3D organoids. We will use CRISPR/Cas9 gene editing to generate cells expressing AID-tagged endogenous KIF5/kinesin-1 to allow inducible and rapid degradation of the microtubule motor specifically during mitosis, for a precise evaluation of the role it plays in ACDs. We generated cells expressing GFP-tagged LGN –a key player in the spindle orientation machinery–to purify and analyse the LGN-containing complex by LC-MS/MS mass spectrometry and identify novel factors that participate to ACDs. These studies will elucidate the molecular mechanisms of ACDs in the mammary epithelia and provide rational for subsequent detailed investigations in vivo of their precise roles in development and homeostasis.