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

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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 to understand 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.