Microtubule organisation in epithelial cells

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.

Most differentiated cells inactivate their centrosomes and organise microtubules from noncentrosomal microtubule organising centres (ncMTOCs). This is particularly important in epithelial cells, where apical ncMTOCs nucleate an apical-basal microtubule array that underlies the polarised functions of the cell. We and others have identified the actin-microtubule cross-linker, Shot, the microtubule minus end capping protein, Patronin, and the microtubule severing complex, Katanin 60/80 as conserved components of these ncMTOCs. We now propose to investigate how these ncMTOCs are assembled and localised at the apical cortex of Drosophila epithelial cells.
1) Apical recruitment of Shot requires its actin-binding domain (ABD), but the ABD on its own localises all round the cortex. We will use transgenesis and CRISPR/Cas9-mediated homologous recombination to examine if the activity of the ABD is spatially regulated by intramolecular inhibition by the Shot C-terminus or by activation by Src/FAK phosphorylation.
2) Shot is excluded from the lateral cortex by the lateral polarity kinase, Par-1, and contains 3 putative Par-1 phosphorylation sites. We will investigate whether Par-1 regulates Shot localisation directly or through Src phosphorylation.
3) Although Patronin binds to Shot, there must be a parallel localisation pathway, as Patronin localises apically in shot mutants. We will map the apical-targeting domain(s) of Patronin and screen for proteins that interact with this domain.
4) We will take several approaches to purify ncMTOCs and identify novel components by mass-spectrometry. Candidate proteins will be verified by examining their localisation with GFP-tagged constructs and by creating mutants to test if they are required for ncMTOC function. Our results will provide insights into the composition and function of ncMTOCs and will reveal how they localise apically in epithelial cells, which is a key step in the establishment of apical-basal polarity.

The expected beneficiaries of the research detailed in this proposal include i) Other researchers, (ii) The medical and pharmaceutical industries; iii) Businesses recruiting graduate-level staff; iv) School students and the general public.

Other researchers
We will make all of our reagents freely available and will disseminate our results through conference talks, seminars, preprints and peer-reviewed publications.

The medical and pharmaceutical industries:
The proposed research will identify novel components of noncentrosomal microtubule organizing centres. These are likely to play a role in forming the noncentrosomal microtubule cytoskeleton in neurons and could therefore be valuable markers in research on diseases that disrupt the cytoskeleton, such as congenital forms of frontal-temporal dementia and Alzheimer's disease. Since differentiated cells often inactivate their centrosomes and organize their microtubules from such ncMTOCs, these components may also prove to be very useful markers for following cell differentiation in the development of protocols for producing replacement organs for regenerative medicine. In the long-term (>10 years) our results could impact the medical and pharmaceutical industries, helping to improve health and quality of life.

Businesses recruiting graduate-level staff:
The research proposal involves training that will ultimately prepare our staff for highly
skilled employment in the private and public sectors. Former members of the lab
have progressed to successful careers in the biotechnology industry, in consulting
and in publishing, as well as in the medical, charitable and public sectors. The skills
obtained in our lab are likely to produce individuals who will have a major
impact on both the economy and the well-being of society.

School students and the general public:
Our group is heavily involved in science communication and outreach activities, both
in schools and to the general public. Current activities for students include mobile lab visits to primary schools, discovery challenge projects for 6th form students at a local technical college, workshops in the University's widening participation programme, and internships for students considering science degrees. Our main goal is to encourage students from less advantaged backgrounds to consider careers in science. We also organize many activities for the general public, such as an Institute open day as part of the Science Festival and demonstrations at other events and festivals.

We will disseminate the results of our research to the widest possible audience.