Molecular mechanisms of kinesin-5s in fungal mitosis

Lead Research Organisation: University of Exeter
Department Name: Biosciences


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.

Technical Summary

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.

Planned Impact

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.


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Description IMPORTANT:
We were only participating in this project (I was not the PI, as the data here suggest). We had a <10% share in the award.

See below, summary of the approaches and the outcome (which was, unfortunately, negative);
I should highlight I was only co-PI and we received only minor funds, we were, therefore, depending on the PIs input.
Exploitation Route I cannot tell- maybe someone wants to take the challenge and try the hypothesis in other fungi- however, the clearly negative results are not encouraging. Unfortunately, negative results cannot e published, so we cannot make others aware of them
Sectors Healthcare

Description We were contribution live cell imaging to a project led by Carolyn Moores (Birkbeck University, London); in the course of this collaboration, we did the following: (a) We did 3 GFP-fusions to various truncated versions of kinesin-5 from Ustilago maydis to test for binding to microtubules (data that determined truncations provided by the Birkbeck group); we introduced these constructs into cells that express red-fluorescent tubulin; (b) we introduced 1 full length and 3 truncated kinesin-5 alleles into a kinesin5-temperature sensitive strain to see if the constructs rescue the mitotic defect; (c) we generated a conditional kinesin-5 mutant where the Kin5 gene was expressed under the repressible nitrate reductase promoter; (d) We overexpressed the truncated Kin5 region (see "a") to monitor an effect on mitosis; OUTCOME: (a) no GFP signal was detectable along red spindle microtubules, probably microtubule binding is a cooperative mechanism and truncations do not bind on their own; (protein-folding issues may add to this); (b) surprisingly, the full-length Kinesin-5 was not able to rescue the temperature-sensitive kinesin-5ts mutant ; we recon that this is due to the presence of inactive kinesin5 protein in the temperature-inactivated mutants;. This finding excluded the use of the Kin5ts mutant; (c) We never obtained the conditional mutants; this is most likely due to high overexpression of kinesin-5 under "ON" conditions- we therefore could not follow up any of the suggested approaches; (d) We intended to block to function by overexpression of 3 truncated regions of kinesin-5 (as determined by Birbeck collaboration partner); we obtained the mutants, but could not find any effect of overexpression of the genes on mitosis- this corresponds to the finding that truncated kinesin-5 versions did not localise to mitotic spindles.
First Year Of Impact 2017
Sector Other