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

Reference	BB/V001353/1
Principal Investigator / Supervisor	Dr Jens Januschke
Co-Investigators / Co-Supervisors	Dr Philip Murray
Institution	University of Dundee
Department	School of Life Sciences
Funding type	Research
Value (£)	427,881
Status	Current
Type	Research Grant
Start date	01/12/2021
End date	30/11/2024
Duration	36 months

Abstract

Cell polarity is important for many aspects of the physiological roles of organs, tissues and cells. The PAR polarity complex has been linked to the ability of stem cells to self-renew while generating daughter cells that differentiate during an asymmetric division and perturbations of this process are suspected to contribute to the development of malignancies. This proposal aims to elucidate how proliferative cell, like stem cells, couple polarity and cell fate determination to the cell cycle, which is not understood. We use the highly proliferative asymmetrically dividing Drosophila neuroblasts as a model system and have recently established chemical genetics, the engineering of kinase alleles allowing specific and acute inhibition of the activity of the protein product in Drosophila. Using new alleles of CDK1 that allow its acute inhibition, we already observed phenotypes on the localization of PAR3 in neuroblasts. In this proposal we will shed light on the molecular mechanism of how CDK signalling precisely regulates PAR polarity and cell fate. Despite being one of the best examples of an asymmetric dividing cell, the molecular nature establishing opposing membrane domains, a prerequisite for asymmetry in this system, is unknown. Given the co-evolution of the PAR polarity system and actomyosin regulation, we hypothesise that asymmetries in the actomyosin cortex are established during the cell cycle providing an underlying patterning mechanism that establishes membrane domains. Such asymmetries might be read by fate determinants in response to direct phosphorylation by aPKC facilitating their asymmetric localization. We anticipate that our project will reveal how signals from the cell cycle machinery, the activity of the PAR complex and the actomyosin network are integrated to ensure robust cell fate specification and the fidelity of asymmetric stem cell division.

Summary

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.

Impact Summary

This research proposal aims at addressing the molecular mechanism that couple the cell cycle to the ability of stem cells to produce specialized cells. In our bodies this activity of stem cells is crucial to replace damaged and worn out differentiated cells in the adult to maintain tissue integrity and physiology. Failure to do so has been linked to the risk to develop tumours in mammalian systems, humans and flies. It is therefore of biomedical relevance to reveal the molecular logic that controls normal asymmetric stem cell division by keeping proliferation in check and preventing that stem cells derail from their normal developmental programmes. Based on research designed to reveal fundamental biological principles conducted in an invertebrate model system, our work is not anticipated to have an immediate impact improving quality of life of patients. Nonetheless, Drosophila provides a powerful system in which to test hypothesis and reveal basic principles of the function of evolutionarily conserved molecules and their regulation that have disease relevance in humans. Therefore, this project helps to reduce to use of higher animals for research, which may benefit patients in the longer term. For instance, the cell cycle machinery is an important target of cancer therapy. Interfering with CDK signalling pharmaceutically, may help to block or attenuate proliferation of malignant epithelial cells in patients. Given that stem cells have been identified as cells-of-origin in several cancers, it may be important to understand how interfering with the cell cycle machinery affects their function to prevent unwanted effects on stem cells in otherwise unaffected tissues of patients. Our project has the potential to provide a basic conceptual framework that might help to inform the design of better cancer therapies. This project also aims at addressing the consequences that phosphorylation by a key effector of the evolutionarily conserved PAR complex, atypical protein kinase C (aPKC) has on its substrates. aPKC functions in many different cellular contexts and a large range of substrates have been identified, but precise molecular consequences of phosphorylation by aPKC are unclear. aPKC is found to be deregulated in many human diseases ranging from hypertension to neurodegenerative diseases and cancer. Our work using an invertebrate model system might help to further our knowledge on the basic principles of how this important kinase functions to help shed light on its role in diseases.

Committee	Research Committee C (Genes, development and STEM approaches to biology)
Research Topics	Neuroscience and Behaviour, Stem Cells
Research Priority	X – Research Priority information not available
Research Initiative	X - not in an Initiative
Funding Scheme	X – not Funded via a specific Funding Scheme

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