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

点击翻译按钮获取中文摘要

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.