Microtubule organisation in epithelial cells

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Most of our organs are composed of sheets of epithelial cells that function as barriers between compartments (e.g. blood vessels; secretory glands) or between the inside and outside of the body (skin, digestive system and lungs). The formation of these epithelial sheets depends on the coordinated polarisation of the cells, so that all have their apical surfaces facing the outside and their basal surfaces on the inside. Loss of this apical-basal polarity therefore disrupts epithelial organisation and disrupts their barrier function. More than 80% of cancers arise from epithelial tissues and one of their hallmarks is a progressive loss of polarity, which correlates with the malignancy of the tumour. A key function of epithelial sheets is to transport nutrients and other components across the epithelium. This depends on the formation of apical-basal arrays of microtubules that act as tracks along which motors transport components across the cell. Microtubules are polar filaments with one dynamic end that constantly grows and shrinks (the plus end) and a more stable minus end. In dividing cells, the microtubules are organised by microtubule organising centres (MTOCs), called centrosomes, which template and stabilise new MT minus ends. The centrosomes are usually inactivated in differentiated cells, however, and the microtubules grow instead from noncentrosomal microtubule organising centres (ncMTOCs). In epithelial cells, the ncMTOCs and MT minus ends localise to the apical cortex, with plus ends extending towards basal part of the cell. Although the structure and function of centrosomes is well understood, very little is known about ncMTOCs. The aim of our research is to understand how ncMTOCs are formed, regulated and localised in epithelial cells. We use fruit flies as our model organism because they allow us to study epithelia in their normal physiological environment, and because the powerful genetics in this system make it easy to modify genes with the recently developed CRISP/Cas9 technology and to add fluorescent tags to proteins of interest. Recent research by us and others has identified two key components of the ncMTOCs in flies and mammals: the giant actin microtubule cross-linker Shot (ACF7 in humans) and the microtubule minus end binding protein Patronin (CAMSAP). These proteins interact with each other and localise apically in epithelial cells. Shot seems to recruit ncMTOCs to the cell cortex, whereas Patronin protects MT minus ends from depolymerisation. Shot and Patronin are important for MT organisation not only in epithelia, but also in neurons, where non-centrosomal MTs also play a key role. Thus, reduction of Shot or Patronin affects neuronal growth and axon specification in both flies and mammals. Several hypotheses have been proposed to explain how Shot and Patronin are localised and regulated. To test these, we will introduce specific mutations into each protein and analyse their effects on protein localisation, the arrangement of the MTs and the organisation of the epithelium, using 3D and 4D immunofluorescence microscopy. For example, we will investigate whether the interaction of Shot with cortical actin is regulated by apical protein kinases, and how Shot is excluded from the lateral cortex of epithelial cells by the lateral polarity kinase, Par-1. We will also isolate ncMTOCs from epithelial cells and analyse their components by mass spectrometry. Identifying novel proteins that interact with Shot and Patronin should reveal the composition of ncMTOCs and provide insights into their function and regulation. This may also provide new markers for studying cell differentiation and neural diseases that affect the microtubule organisation, such as Alzheimer's disease and other dementias.