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

丝状真菌是一类成功的具有巨大生态和经济意义的生物体。在那里，通过土壤生长的能力对大多数陆地植物的生长是有利的，而真菌是植物残渣的重要生态分解者。此外，丝状真菌用于蛋白质的工业生产，也是挑战公共卫生和农作物生产的病原体。丝状真菌的基本单位是菌丝，这是一种细丝细胞，通过称为尖端生长的过程在一端扩张。这种生长模式允许组织和底物的入侵，并被认为涉及将新合成的膜、蛋白质和细胞壁前体运送到扩张的细胞端。然而，对植物病原菌Ustilago maydis的早期研究表明，物质进入细胞的吸收几乎同样重要。被吸收到细胞中的物质的性质还不完全清楚，但到目前为止，很明显，被称为早期内小体(EES)的细胞器是接收这种材料的内部载体。这些胚胎沿着细胞骨架的丝状纤维长距离移动。这种运动是由分子马达驱动的，分子马达利用化学能在细胞内运输货物。有趣的是，EES向菌丝尖端移动，也向菌丝尖端远离。这种双向运动是由相反的马达蛋白介导的，这些马达蛋白似乎是随机切换方向的，这是“拔河”事件的结果。来自动物模型系统的大量证据表明，细胞控制机制改变了运动活动，以确保平衡的双向运输和细胞骨架纤维末端的运动集中。这种浓缩似乎是必需的，以保证到达的货物有效地装载到发动机上，并防止货物在细胞骨架“轨道”的末端脱落。在这个项目中，我们将结合复杂的活细胞成像和详细的建模来了解随机运动行为和EE运动中的高阶控制机制之间的相互作用。我们已经开发了一种微观设置，使我们能够可视化活细胞中单个马达和细胞器的运动，使用特定抑制剂的初步药理学研究已经将我们引向玉米病原菌U.maydis中的一些关键调节因子。我们将确定这些假定的调控因子的确切性质，并通过产生在这些候选基因中有缺陷的突变来研究它们在EE运动和运动合作中的作用。此外，我们将着手更好地了解潜在的细胞骨架纤维的组织，并将调查EE运动性对塑造细胞和真菌毒力的重要性。该项目将通过生成一个数学模型(基于活细胞的测量)来实现这一点，该模型结合了整个细胞中的随机运输和确定性调节。该项目的预期结果将是对丝状真菌如何远距离移动细胞器的新见解。这将提供对细长细胞运动过程的最低要求的全面了解，因此也将告知具有类似过程的其他细胞系统，如神经元。此外，该项目将对真菌研究的各个方面产生根本的兴趣，特别是对了解真菌的致病性具有重要意义。

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.