Dissecting the coupling of cell polarity and the stem cell cycle by chemical genetics

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It has become clear that the correct function of the stem cells in the tissues of our body are intimately linked to the risk of those tissues to develop malignancies. In some tissues stem cells have been identified as the cell-of-origin of cancer. It is therefore important to reveal the mechanisms that protect stem cells from being driven into aberrant proliferation to be able to prevent, control or minimize the impact if they malfunction. Stem cells function to maintain tissues and can do this through asymmetric division. The outcome of such a division is a self-renewed stem cell and a daughter cell that will differentiate. Not all stem cells divide in this way, but in many that do, a set of evolutionarily conserved molecules operates, such as the PAR complex, allowing them to divide asymmetrically and to transmit fate information to the daughter cell that will differentiate. One hypothesis aiming at explaining the role of stem cells in the context of cancer states that failed asymmetric stem cell divisions results in mis-specified cells, that can't be kept in check as they do not respond to the control mechanisms normally in place. This proposal aims at addressing the molecular mechanisms that control asymmetric stem cell division to prevent this from happening. The role of the PAR complex and the establishment of cell polarity is the subject of intense research, which is focussed however on cells that are largely none dividing such as epithelial cells, neurons or migratory cells. The difficulty to single out stem cells without ambiguity and to experimentally manipulate them in many mammalian models has hindered understanding the precise role of polarity in many relevant stem cells. Moreover, stem cells have the ability to continuously proliferate. Studying polarity in the context of cell division by standard genetic analysis such as the use of mutants or knockdown of the messenger RNA is unable to resolve the dynamics of the process as it does not provide temporal control. This proposal aims at addressing the link between proliferation and polarity.Drosophila neural stem cells, called neuroblasts, come from a genetically tractable organism, have a very short cell cycle, can be readily identified, are amenable to live cell imaging and their asymmetry is under the control of conserved proteins such as those of the PAR complex. We have developed chemical genetics in the fly that allow specific and acute inhibition of the activity of kinases. We will use this approach to specifically address how the cell cycle machinery regulates cell polarity in a model system stem cell. The upstream signals that drive stem cell polarity in a cell cycle dependent manner are understudied. Our approaches allow for the first time to temporally dissect the role of signalling by critical kinases on stem cell polarity, asymmetry and cell fate determination. Once the PAR complex achieves a polarized localization in neuroblasts in mitosis, it drives the asymmetric localization of molecules such as the NOTCH signalling regulator NUMB, that are exclusively segregated to daughter cells, where they instruct differentiation. Therefore, the asymmetric localization of fate determinants is also under cell cycle control. This is true for neuroblast, but also for other mammalian stem cells such those of the murine mammary gland, radial glia and haematopoietic stem cells. The molecular mechanism that patterns stem cells allowing the asymmetric localization of molecules such as NUMB are highly unclear. We hypothesise that the actomyosin network provides this patterning information. Our preliminary results further support the idea that posttranslational modification of fate determinants is required for their asymmetric retention at the actomyosin cortex. We aim at revealing the molecules that link phosphorylated determinants to the stem cell cortex in response to cell cycle dependent cues.