Molecular mechanisms of kinesin-5s in fungal mitosis

The purpose of this research is to discover how cell replication is controlled and how it might be blocked. We will focus our studies on cell replication in fungi, both because they are excellent and well-established model organisms for studying replication, and because fungal diseases are medically, environmentally and economically important. In particular, we want to investigate the cell replication machinery in a fungus that infects corn and causes the disease corn smut. This crop disease poses a major threat to global food security, particularly because of the emergence of resistance to currently available fungicides. By understanding how the corn smut fungus replicates, we hope to first, provide general insight into the mechanisms of cell replication. Secondly, we hope to uncover unique features of fungus-specific cell replication because this knowledge promises to help in the development of novel fungicides.

In the same way as our bodies have a skeleton that provides us with support and strength, the cells of fungi have a skeleton - called the cytoskeleton - which also provides support and structure. The cytoskeleton is involved in many important aspects of the life of fungi, including cellular transport, architecture and replication. Studying the cytoskeleton is important both so we can understand how healthy cells work, but also so we can specifically target the cytoskeleton of pathogenic organisms with drugs that kill these organisms and prevent disease.

In particular, this project will focus on a part of the cytoskeleton called microtubules. These are long cylindrical structures that act like tracks along which molecular transport motors carry cellular cargo. The motors that we will study are called kinesins and there are many different types. In this project, we will be studying a kinesin type-5, which is important for accurate cell replication. We want to know how fungal kinesin-5s use cellular fuel to move along microtubules during replication and how this activity might be blocked.

The work by the Birkbeck research team will involve studying the three-dimensional structure of the cytoskeleton, because knowing what the cytoskeleton looks like will contribute to our understanding of how it works in the fungus itself. We will use a very powerful microscope - an electron microscope - to take pictures of individual cytoskeleton molecules and then use computational analysis to combine these pictures and calculate their three-dimensional shape. A powerful aspect of the proposed project is that we will also study the function of kinesin-5 in live corn smut fungus, in collaboration with experts in fungal cell biology at the University of Exeter. Unlike many other fungi, which are very small, the cells of the corn smut fungus are relatively large (10um). This means that cell replication of individual cells can be studied in detail using light microscopy, so that our collaborators will be able to visualise the activity of kinesin-5s in the living fungus and examine the effects of blocking its activity on fungal survival.

Initial analysis suggests that the kinesin-5s from fungi are different from kinesin-5s from other organisms, including humans. This means that we might be able to find drugs that can block fungal kinesin-5 and not human kinesin-5, and such drugs could be very promising for development as new fungicides. Studying the structure and function of the fungal kinesin-5s will allow us to investigate this idea.

The goal of this project is to elucidate molecular mechanisms of the microtubule-based mitotic machinery in fungi. Fungi are important model organisms for mitosis research, but are also significant mediators of pathogenesis in a variety of settings. Kinesin-5 proteins - members of the ATP-driven, microtubule-based kinesin nano-motor superfamily - are essential for mitosis in many eukaryotes. They are involved in generation and maintenance of spindle bipolarity but the molecular basis for this activity is not well understood. Drugs that could specifically inhibit kinesin-5s would block fungal mitosis and are therefore interesting candidates for novel fungicides.

The proposed work will investigate the molecular mechanism of ATP-dependent force generation by fungal kinesin-5s in vitro and in vivo. First, the structure of microtubule-bound fungal kinesin-5 from the model organism fission yeast will be studied using sub-nanometre (<10Å) resolution cryo-electron microscopy. These structures will visualise fungal-specific contacts between the kinesin motor and its microtubule track, and reveal nucleotide-dependent conformational changes that drive force generation. This work will provide a mechanistic backdrop to investigations of kinesin-5 from the pathogenic fungus that causes corn smut, Ustilago maydis. This is an economically important, crop-damaging pathogen, but little is known about the molecular basis of kinesin-5 function in mitosis in this organism. We will study the molecular mechanism of force generation of recombinant U. maydis kinesin-5 motor domain using cryo-electron microscopy. This will allow us to identify elements within this motor that are important for function. We will test our mechanistic hypotheses by studying wild-type and mutant U. maydis kinesin-5 mitotic function in vivo using light microscopy. By studying these mitotic motors in vitro and in vivo, we will provide a platform for future investigations of novel fungicides.

Who will benefit from this research?

- Agrochemical industry
- Crop farmers
- UK economy
- The wider public
- Women in science

How will they benefit from this research?

The work described will lead to a greater understanding of essential mechanisms involved in cell division in fungi. Kinesin-5 motors could be potential targets for novel fungicides that block fungal mitosis and our research will aid development of these. Beneficiaries from this aspect of the research would be the agrochemical industry, as greater understanding from academic studies such as ours could lead to more effective generation of improved fungicides and, therefore, improved sales. We would aim to engage these beneficiaries as soon as possible. This work would also ultimately lead to benefits for crop farmers whose output could be increased by control of fungal crop damage. In the longer term, development of improved fungicides will also benefit the consumers of crop foods leading to general improvement of quality of life.

Science and technology will lie at the heart of global economic recovery, and we will liase with UCL Business (UCLB), who work with Birkbeck researchers on technology development and intellectual property matters, and Exeter University's Research and Knowledge Transfer Management Group, to maximise the impact of our discoveries. This will ultimately have benefits for the economic competitiveness of the United Kingdom. It is essential to retain talented young researchers in the UK, and the proposed research programme will provide an attractive research opportunity for excellent young scientists looking for multi-disciplinary areas of discovery. In addition, transferable skills - such as time- and project-management, presentation and collaboration - that can be applied in all employment sectors will be acquired, particularly through transferable skills training within the Institute of Structural and Molecular Biology and at Exeter University.

We will aim to make the discoveries of our research available not only to the academic community, but also to the general public. The PI has a proven track-record of public communication of science, was the 2006 winner of the prestigious DeMontfort medal for science communication (SET for Britain), has attended a BBSRC Media Training Day, presented her research to a general audience as part of Birkbeck Science Week 2012 and discussed her work at a Coffee for Cancer Club meeting at a local retirement community. The appointed PDRA and the PI will undertake to design web-pages for the PI's lab which are accessible to the general public and will seek to participate in other public understanding of science activities, for example by inviting sixth-form students to visit the lab and experience the day-to-day life of scientists. During the project period, the PI will arrange to visit her former girls' school to inspire future scientists, and will continue to be involved in advancing gender equality in science, engineering and technology through involvement with the Athena SWAN programme at Birkbeck College - she is a member of the steering committee that submitted Birkbeck's recent successful application for a bronze Athena SWAN university award.