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Stochastic Versus Deterministic: Mechanisms of Bi-Directional Endosomes Motility in the Plant Pathogen Ustilago maydis

Reference	BB/J009903/1
Principal Investigator / Supervisor	Professor Gero Steinberg
Co-Investigators / Co-Supervisors	Professor Peter Ashwin
Institution	University of Exeter
Department	Biosciences
Funding type	Research
Value (£)	542,613
Status	Completed
Type	Research Grant
Start date	01/08/2012
End date	31/01/2016
Duration	42 months

Abstract

In the filamentous fungus U. maydis, microtubules support bi-directional motility of early endosomes (EEs). By combining powerful quantitative live cell imaging with mathematical modelling we recently showed that kinesin-3 and dynein cooperate in a way that could be described by stochastic motor behaviour. However, these studies also provided strong indication for additional deterministic control mechanisms at a higher level. A major limitation of these studies was that the modelling approaches mainly focused on spatially restricted regions of the cell (e.g. the apical 10 micrometres). In doing so, major features of the bi-directional transport process were not taken into consideration. This includes the fact that long-range EE motility occurs along a bi-polar microtubule array, which enables kinesin-3 to support bi-directional motility of the organelles (Schuster et al., 2011, Mol Biol Cell, in revision*). In this project we address the role of phosphatases and kinases in deterministic control of EE motility and the motor behaviour. Preliminary pharmacological results have strongly implied PPA2 and caseine kinases in controlling EE motility and motor accumulation at MT plus-ends. We will further investigate such a role by generating numerous mutant proteins. In addition, we will analyse organelle behaviour at the beginning of the bi-polar MT array by co-visualizing MT minus-ends and EEs. These data, together with published and unpublished quantitative results will be merged into a sophisticated ASEP-type model. In an iterative process this mathematical approach will be shaped by ongoing experiments, while informing these experiments, to generate a robust mathematical description of the process of EE motility at the level of the entire fungal cell. Finally, we will determine the importance of EE motility for virulence and morphogenesis by expressing an artificial anchor protein that specifically blocks EE motion.

Summary

Filamentous fungi are a successful group of organisms of enormous ecological and economical importance. There ability to grow through soil is instrumental for growth of most land plants and fungi are ecologically important decomposer of plant debris. In addition, filamentous fungi serve in industrial production of proteins and as pathogens that challenge public health and agricultural crop production. The basic unit of a filamentous fungus is the hypha, a thin filament of cells that expands at one end by a process termed tip growth. This mode of growth allows the invasion of tissue and substrate and is thought to involve delivery of newly synthesised membranes, proteins and cell wall precursors to the expanding cell end. However, early studies in the plant pathogen Ustilago maydis have shown that the uptake of material into the cell is of almost equal importance. The nature of the material that is taken up into the cell is not fully understood, but it is clear by now, that organelles named early endosomes (EEs) are internal carriers that receive this material. These EEs travel over long distances along the filamentous fibres of the cytoskeleton. This motility is driven by molecular motors, which utilize chemical energy to transport their cargo throughout the cell. Interestingly, EEs move both towards and away from the hyphal tip. This bi-directional motility is mediated by opposing motor proteins that appear to stochastically switch direction as the result of "tug of war" events. Much evidence from animal model systems indicates that cellular control mechanisms modify the motor activity to ensure a balanced bi-directional transport and a concentration of motors at the end of the cytoskeletal fibres. This enrichment seems to be required to guarantee an efficient loading of the arriving cargo onto motors and to prevent cargo falling off at the end of the cytoskeletal "track". In this project we will combine sophisticated live cell imaging and detailed modelling tounderstand the interplay between random motor behaviour and higher order control mechanisms in EE motility. We have developed a microscopic setup that allows us to visualise the motility of individual motors and organelles in the living cell, and preliminary pharmacological studies using specific inhibitors already directed us to some key regulators in the corn pathogen U. maydis. We will identify the precise nature of these putative regulators and investigate their role in EE motility and motor cooperation by generating mutants defective in these candidates. In addition, we will set out to better understand the organization of the underlying cytoskeletal fibres and will investigate the importance of EE motility for shaping the cell and for virulence of the fungus. The project will do this by generating a mathematical model (based on measurements from the living cell) that combines both stochastic transport and deterministic regulation in the entire cell. The expected outcome of this project will be novel insights into the way that a filamentous fungus moves organelles over long distances. This will provide a comprehensive understanding of the minimum requirement for a motility process in elongated cells and will therefore also inform other cell systems with a similar process, such as neurons. Furthermore, this project will be of fundamental interest to all aspects of fungal research, and of particular importance in understanding fungal pathogenicity.

Impact Summary

Filamentous fungi have a major impact on human welfare. As symbionts they ensure growth of more than 95% of the land plants and play important roles as decomposer of plant debris. Fungi are used in industrial production of vitamins and recombinant proteins. On the other hand this group of organisms provide serious pathogens, which are of medical importance, and as plant pathogens also have significant impact on the crop yield. This proposal addresses a fundamental process in fungal biology and it does this in a corn pathogen that is considered a potential bioweapon in the United States of America. To date work in this organism has already massively stimulated the research areas (eg. motility of endosomes and the underlying motors were first identified in U. maydis [Wedlich-Söldner et al. 2000, EMBO J 19:1974; Wedlich-Söldner et al. 2002, EMBO J 21:2946-2957]). In addition, our research in U. maydis has attracted agricultural companies, such as Syngenta, BASF and Bayer and we have a long history of ongoing industry projects. Therefore, the outcome of this current project promises to stimulate fundamental research in fungal biology, but will also inform the industrial development of novel fungicides. Beside this, the project has a strong capacity to be of relevance for general cell biological research. U. maydis forms elongated cells that show phenotypic similarities with neurons (Steinberg and Perez-Martin 2008, Trends Cell Biol 18:61). Like in U. maydis, early endosomes are moving in a bi-directional fashion and in both neurons and U. maydis the underlying motors are kinesin-3 and dynein. Therefore, the outcome of this project will have impact on a wider scientific society, including medical-related research on neurodegenerative disease. This work also has profound educational impact. It is based on an existing collaboration with the Dept of Maths, which has proven to be remarkably successful (eg. Ashwin et al. 2010, Phys Rev. E 82:051907; Schuster et al. 2011,EMBO J 30:652). In this proposal we are now aiming to understand and mathematically describe a trafficking process at the whole cell level. This is an exciting and challenging step and, in large parts, places the project at the interface of fungal biology, cell biology and mathematical modelling. As such it bridges between disciplines and has a major impact on the education of Systems Biology students in Exeter. Furthermore, the fact that we raise quantitative data from the living cell and merge this with sophisticated mathematics is innovative and timely and as such promises to raises the profile of the UK research. Finally, the project fosters research on the corn pathogen U. maydis. Maize is an important crop with 144.4 million hectares planted globally of which 9.3% are located in the EU (Phillips McDougall, 2006, Crop Life International Annual Report). Fungi pose a serious challenge to this industry, with fungicide sales globally in 2007 being $106 million for maize alone (in the EU $3,211 million; Cropnosis Report, 2008). While considered a serious threat in the USA, the fungus is currently not a major challenge in the UK. However, changes in climate conditions, such as the predicted increase of temperature by 1.8 in the nearer future will change agricultural demands and pathogen demography. Considering this project is timely and will impact on strategies to protect crop from smut fungus infections. In summary, the outcome of this project will have profound positive impact on (1) basic fungal research on hyphal growth, (2) building links to agricultural and medical-related industry to help developing pathogen management strategies, (3) bridging between different disciplines (mathematics, plant pathology, cell biology) to provide a holistic Systems Biology approach (4) training of a PDRA in various techniques; (5) educating undergraduate and postgraduate students; (6) strategies to secure food security under changing climate condition.

Committee	Research Committee B (Plants, microbes, food & sustainability)
Research Topics	Crop Science, Microbiology, Plant Science, Systems Biology
Research Priority	Systems Approach to Biological research
Research Initiative	X - not in an Initiative
Funding Scheme	X – not Funded via a specific Funding Scheme

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