The role of microtubule plus-end capture at cortical sites in the assembly of apico-basal microtubules in polarised epithelial cells.

Microtubules are tubular structures, which are important for many cellular functions including the transport of vesicles and molecules within cells. It is therefore very important that the microtubules are assembled and positioned correctly within cells. The specific pattern that microtubules form within cells vary depending on cell type and function. Most animal cells have a radial array of microtubules anchored at a centrally located structure called the centrosome. In these cells the centrosome's job is to start off the assembly of the microtubules and to keep them tightly bound to it. However, many specialised cells like polarised epithelial cells found in the gut and kidneys arrange their microtubules in a different way. Here most of the microtubules are anchored at the cell apex away from the centrosome and run towards the base forming a so called apico-basal array. Our recent findings suggest that the apico-basal microtubules are assembled at the centrosome, released and moved to apical sites where they are captured and anchored. The aim of this project is to find out whether the microtubules first grow out from the centrosome, make contact with the cell cortex, are released from the centrosome and then move downward by the pulling action of dynein motor proteins located at the cortex. The end result would be that one end (plus-end) of the microtubules is pulled down to the cell base while the other end (minus-end) becomes anchored at the cell apex. We want to find out whether proteins such as EB1 and CLIP-170, which stick to the growing end of microtubules, are important for the capture by dynein or other proteins at the cortex. It is very important to know if for example CLIP-170 is vital for the normal assembly of the microtubule in real life. We will therefore analyse tissue from the inner ear (cochlea) isolated from mice, which do not produce CLIP-170. Finally, we would also like to know whether thin filaments known as actin help the microtubules to get to their destination. We will use special fluorescent (glowing) molecules called GFPs linked to proteins so that the microtubules or their plus-ends glow and we will make movies of their movements over time and look at the localisation of these important proteins using fluorescent dyes and specialised microscopes. Microtubules are clearly vital for the normal function of a cell and we need to establish how they are organised and controlled before we can unravel the causes and consequences of many diseases.

This project will capitalise on our recent very promising findings concerning the assembly and organisation of non-centrosomal Mts in epithelial cells. It will benefit from the use of a combination of inner ear tissue and cell culture model systems and an exciting opportunity to analyse the role of mammalian CLIPs and CLASPs in vivo. Epithelial polarisation involves major reorganisation of the microtubule cytoskeleton from a radial centrosomally focused array to an apico-basal array associated with apical non-centrosomal anchoring sites. Correct assembly and positioning of these non-centrosomal microtubules is vital for many of the highly specialised functions performed by differentiated cells. Evidence from the applicant's lab. suggests that the non-centrosomal microtubules originate from the centrosome and that a microtubule 'release and capture' mechanism is exploited in many epithelial cells as a means of generating these microtubules. This proposal specifically sets out to determine whether microtubule plus-end capture at cortical sites is a vital intermediate step in the assembly of apico-basal arrays in polarised epithelial cells. The aims are to determine whether dynein acts as a protein linking microtubule +TIPs such as EB1 and CLIP-170 to the cell cortex and act as a motor protein pulling released microtubules towards the periphery and cell base. The aim is also to resolve whether microtubule-actin filaments interactions are important for microtubule guidance towards the cortical sites. Furthermore, we aim to identify protein interacting partners for EB1 and CLIP-170 in polarised epithelial cell. This investigation will use GFP- and RNAi-technology, protein biochemistry and high resolution widefield fluorescence, confocal and electron microscopy to pursue these objectives. Findings from this proposal will not only help to identify the molecular mechanisms involved in the assembly of non-centrosomal Mt arrays but also advance our understanding of the role of Mt plus-end cortical interactions in cells generally.