Re-organisation of microtubule minus-ends during apico-basal epithelial polarisation and differentiation

Development of elongated epithelial cells such as those of the gut and kidney involves a dramatic rearrangement of tubular structures called microtubules into apico-basal bundles that run from the apex to the base of the cells. Microtubule rearrangement is likely to be dependent on a change in the distribution of molecules such ninein that anchor one end of the microtubules and organises them into specific patterns like the apico-basal bundles and this is likely to be vital for the normal function of these cells. Defects in epithelial elongation and microtubule bundle formation lead to loss of function, abnormal cell migration and cancer. How the microtubules become organised into bundles in elongating epithelial cells is not fully understood. Most of what we do know has come from investigations of columnar shaped flat sheets of cells in culture that do not have the natural tissue architecture. Importantly, we have now established a novel live three dimensional gut organoid culture system that mimics normal gut development producing mini gut-like structures with elongated epithelial cells. This system provides a powerful new way to study microtubule reorganisation during epithelial elongation and tissue development using live microscope imaging and studies that inhibit the function of potential key molecules. We have shown that ninein travels along microtubules and relocates to apical peripheral attachments where the ends of the microtubule bundle become anchored. We have also shown that elongating epithelial cells first form a radial pattern of microtubules that run from the cell centre and out to the cell periphery and that microtubule associated molecules such as CLIP-170 are important for mediating contact between the microtubules and the cell periphery. This contact is likely to be important for the relocation of ninein and therefore also for microtubule reorganisation. Ninein and CLIP170 are therefore likely to play critical roles in microtubule reorganisations and thus also in epithelial development. Studies by others have shown that ninein is important for the development of new blood vessels, neurons, skin and for stem cell determination. All in all, this points to ninein having a major role in cell and tissue development. The aims of this project are to determine the mechanisms responsible for ninein relocation and its role in microtubule reorganisation and epithelial development. Findings from this project are likely to have far-reaching implications for our understanding of cell development, stem cell fate and diseased states.

Differentiation and formation of epithelial layers with apico-basal polarity is a fundamental process in development and this is dependent on microtubule reorganisation. Polarisation and generation of apico-basal microtubules is critical for the normal function of epithelia and defects in polarisation lead to loss of function, epithelial cell invasion and cancer. However, the molecular mechanisms underlying microtubule reorganisation remain to be determined. We have shown that polarisation of columnar epithelial cells not only involves a dramatic reorganisation of the microtubules but also of centrosomal components such as the minus-end anchoring protein ninein. Ninein relocates to apical adherens junctions, where the minus-ends of apico-basal microtubules subsequently become anchored, and this is likely to be central and critical to microtubule reorganisation and epithelial differentiation. Furthermore, efficient redeployment of ninein is likely to be facilitated by plus-end tracking proteins such as CLIP-170 that mediate microtubule junctional interaction. Most of our knowledge to date has been based on investigations of polarised flat epithelial cell layers that do not develop the in vivo tissue architecture. However, novel organoid cultures now provide a powerful new way to study microtubule reorganisation during epithelial polarisation and differentiation using live-imaging and functional inhibition studies. We have recently established such an in vitro system based on gut organoid-generating stem cells that develop cysts and crypt-like structures mimicking the in vivo gut epithelial architecture and morphogenesis process. The aims of this project are to exploit in vitro organoids and adhesive micropatterns to determine the molecular mechanisms responsible for ninein relocation and maintenance at the junctional sites and its role in microtubule reorganisation and epithelial differentiation

This project will exploit the novel in vitro gut organoid culture system that mimics the in vivo gut morphogenesis process. The potential exploitations of this in vitro organoid system are immense not only for future research involving academic institutions but also for medical, drug and bio-technology companies. It will fundamentally change our ability to investigate developmental and differentiation processes live in a system that closely replicates the in vivo tissue architecture and morphogenesis. The project will generate a platform of tools for imaging and delivery of molecules, which together with the organoid model would have great potential for future drug testing, nano-technology research and tissue and organ replacement therapy. The organoids could potentially also benefit the food industry and health care companies by being used to analyse and test for example the impact of natural products such as fish oils and the effect of microorganisms on the gut.
The project will also help to develop The 3R's (Replacement, Reduction and Refinement) further with refinements to the organoid culture system leading to a reduction in the number of mice needed for future gut research. The aim is to refine the culture system so that the organoid-generating stem cells will continually propagate and largely replace in vivo gut research.