Microtubule Associated Proteins with roles in mitosis: A Systems approach

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How do complex animals and plants ensure that, when their cells divide, they do so properly? And why does disruption of this process lead to disease?These are fundamental questions in biology. We know that cells use protein fibres, called microtubules, and get them to change shape and size, into a complex, self-regulating structure termed the mitotic spindle. This spindle ensures the right number of chromosomes end up at opposite sides of the cell; in this way a single cell ends up producing two identical new cells. The organization of microtubules is controlled by other proteins called microtubule associated proteins, or MAPs. If these MAPs fail to organise the spindle properly, the process of cell division goes wrong, and can lead to many different genetic diseases, including cancer. We are investigating how cell division happens in the fruit fly. Not only do human and fruit fly cells use similar proteins to build the mitotic spindle, and behave similarly through mitosis, but one can also take a fruit fly, disrupt a single protein and investigate the consequences to the whole animal, in a way impossible to do with tissue culture cells. Following on from previous work in our lab, we have developed a bioinformatics model that predicts the likelihood of over 1000 Drosophila proteins being a MAP with a function during mitosis. This model has identified 63 Drosophila proteins that currently have not been reported to be involved in mitotic microtubule organisation, but that have a 90% confidence interval of being novel mitotic MAPs. We will reduce the levels of each of these 63 proteins in the cell, using a technique called RNA interference, and assess the effect on microtubule organisation during mitosis. For those proteins where we see an effect, we will make fruit flies that express fluorescent-tagged versions of the mitotic MAPs. We will take embryos from these flies and use microscopy to analyse where in the cell the proteins go, throughout the cell cycle. We will also use these tagged proteins to isolate other proteins in the cell that interact with them. We will compare the properties of these proteins with what is known of existing mitotic MAPs, allowing us to categorise the novel mitotic MAPs into functional groups. We will then focus on a small number of these novel mitotic MAPs, to move towards a full understanding of their roles in organising microtubules. This will include imaging and analysing microtubule organisation and dynamics in flies that have reduced levels of the MAPs, and investigating the functional relationships between the novel MAPs and their known interacting proteins.We believe this study is a very important one that will provide a greater understanding of microtubule organization during mitosis. Indeed, a proof-of-principle investigation already carried out shows that reducing levels of some of these proteins does affect microtubule organsation, suggesting that the in depth study proposed in this application will significantly contribute to our understanding of cell division and the problems in mitotic microtubule organisation that lead to diseases such as cancer.