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Microtubule Associated Proteins with roles in mitosis: A Systems approach

Reference	BB/K017837/1
Principal Investigator / Supervisor	Professor James Wakefield
Co-Investigators / Co-Supervisors
Institution	University of Exeter
Department	Biosciences
Funding type	Research
Value (£)	351,642
Status	Completed
Type	Research Grant
Start date	19/08/2013
End date	31/01/2017
Duration	41 months

Abstract

Cell division is a fundamental biological process, driven by the formation of a microtubule (MT)-based mitotic spindle that ensures faithful chromosome segregation. The nucleation, length and dynamics of MTs are determined by MT associated proteins (MAPs). Continuing previous proteomics- and bioinformatics-based work in our lab, and working with the model organism, Drosophila melanogaster, we have developed a logistic regression-based model that predicts the likelihood of a Drosophila MAP having a function during mitosis. This has identified 63 Drosophila proteins that currently have not been reported to be involved in mitotic MT organisation, but that have a 90% confidence interval of being novel mitotic MAPs. The aim of this study is to confirm which of our 63 candidate MAPs function during cell division, and what their cellular roles are. We will begin by using RNA interference in S2 tissue culture cells to characterise the consequences on cell division of removing each of the 63 putative mitotic MAPs. For each protein that shows a mitotic phenotype, we will generate transgenic flies to express GFP-fusions of in the early embryo, and use these embryos both to analyse the sub-cellular localisation of the transgenes throughout the cell cycle, and to identify proteins that interact with the putative mitotic MAPs, using co-precipitation and mass spectrometry. This will allow us to categorise the true mitotic MAPs into putative functional groups, alongside existing mitotic MAPs. We will then focus on a small number of the novel mitotic MAPs, undertaking an in-depth functional analysis and placing them within known molecular pathways. Through this study, we expect to identify new regulators of MTs, providing mechanistic insight into mitotic spindle formation in animal cells, and the causes of aneuploidy associated with diseases such as cancer.

Summary

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.

Impact Summary

The main immediate beneficiaries of these studies will be the scientific communities whose research is related to cell division, cytoskeletal organization, chromosome segregation and cell cycle regulation, in all model biological systems. In addition, the fusion of distinct disciplines will help to highlight to the general community the potential benefits of systems-led and multi-disciplinary research that BBSRC are championing. Importantly, however, as study will develop our understanding of microtubule organization during cell division it may ultimately allow the identification of potential new drug targets for the development of anti-cancer treatments. Mitotic regulators, and microtubule associated proteins (MAPs), have provided the basis for a number of small molecule inhibitors, and many of these have originated in work undertaken in model organisms, such as Drosophila. Examples are the mitotic kinases Polo and Aurora, and the kinesin Eg5. As such, the potential beneficiaries of this research are the pharmaceutical industry, health services and the wider public, in terms of health. More widely, the PI has a strong track record in impact activities. As Director of the Life Science Interface DTC at the University of Oxford, they took Physical and Mathematical science graduates wishing to undertake research at the interface between the Life, Physical and Medical sciences and supported their development into multi-disciplinary, collaborative and highly-sought-after researchers. They also have a strong interest in the nature of scientific methodology, and the perception of scientific research both within, and outside of, mainstream science. The PI is committed to communicating the impact of their research through standard scientific activities (high impact journals, conference attendance, and presentations at scientific seminars) in addition to more general fora, such as the lab website, Twitter and YouTube. Biosciences, and Exeter University in general, has beenvery successful in promoting science to the wider public, and we will, with support from our Press Office, ensure that results will be disseminated from our work to maximise publicity opportunities. The impact of the nature of the multi-disciplinary methodology pioneered in the lab will be delivered by establishing a science forum within the University. With support from Research & Knowledge Transfer, and assistance from ESRC-funded Exeter University eGENIS Centre, we will run events that will bring together scientists at the start of their career in a multidisciplinary setting. The objective of the sessions will be to: (i) encourage the next generation of scientists to adopt truly multi-disciplinary approaches; (ii) provide a forum for researchers to meet and explore common interests; (iii) provide a platform for keynote speakers to demonstrate the value of holistic approaches to science. Futhermore, the microscopy images obtained from this BBSRC proposal will be of direct use in a project aimed at exploring the value of, and perceived conflicts between, quantitative and qualitative methodologies in biology. This project, in collaboration with Schumacher College, Dartington, and currently at the planning stage, is designed to test the validity of non-expert inference on cell biological datasets and is based on visualization and interpretation of MTs and DNA from control versus RNAi/mutant cells.

Committee	Research Committee D (Molecules, cells and industrial biotechnology)
Research Topics	X – not assigned to a current Research Topic
Research Priority	X – Research Priority information not available
Research Initiative	X - not in an Initiative
Funding Scheme	X – not Funded via a specific Funding Scheme

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