Regulation of long-distance dynein motility in the model fungus Ustilago maydis

细长的细胞，如脊椎和大脑中的神经元，必须克服很长的距离，才能在外围和细胞体之间进行交流，这对大脑发育和学习过程至关重要。长途运输是由所谓的分子马达实现的。在化学能量的推动下，它们沿着细胞骨架的纤维“行走”，细胞骨架就像铁路系统一样，连接着细胞的各个部分。在哺乳动物的大脑中，微管细胞骨架提供了这个系统的主要部分，单个微管充当运动蛋白Kinesin和Dynein的“轨迹”。这两个分子马达以相反的方向携带膜运输容器(例如囊泡)，从而允许蛋白质、膜和信号的双向交换。由于这一基本过程对细胞功能和生存至关重要，因此许多神经元疾病与这种运输机制的突变有关也就不足为奇了。马达动力蛋白将货物从外周突触移动到细胞体。要想长跑，动力蛋白需要其他辅助因素的支持。其中包括动力蛋白复合体和可能的动力蛋白调节因子，如Lis1和Nudel。这些蛋白质还被发现与退行性神经元疾病有关，例如无脑畸形和肌萎缩侧索硬化症。尽管在这些因素方面做了很多工作，但一些基本问题，如它们在膜贩运中的行动方式，仍然没有得到解决。例如，我们关于dynactin作用的许多知识仅限于无细胞检测，而且这些结果是否可以转化为活细胞存在一些疑问。理想的情况是，人们希望看到在膜性货物运输过程中动力蛋白及其调节器。然而，由于技术限制，这一点尚未实现。丝状真菌与人类神经元有一些惊人的相似之处。例如，它们的细胞高度伸长，在一个细胞极上扩张。向这个生长顶端的运输是通过沿微管的长距离运输来调节的，与神经元类似，这个过程涉及动力蛋白、动蛋白-1和动蛋白-3。我们所知道的关于真菌长途运输的大部分知识是我们使用模式真菌Ustilago maydis发现的。这个模型系统与人类细胞有显著的相似之处，但它结合了强大的技术优势，包括公布的基因组序列，众多的遗传工具，以及许多细胞学工具，如不同颜色的荧光蛋白。最近，我们成功地可视化了膜运输中的单个动力蛋白马达。这一技术进步为解决动力蛋白和辅助因子在逆行贩运中的作用开辟了新的途径。我们将利用这些技术优势来解决以下问题：(1)动力蛋白辅助因子Lis1、Dynactin和Nudel如何控制动力蛋白活性并支持单个动力蛋白马达的远程运动？(2)已知的人类肿瘤抑制因子动力蛋白轻链Lc7/Roadblock/Km93起什么作用？(3)延长细胞中动力蛋白是如何动态的？(4)是什么因素将动力蛋白带到微管末端的加载区，并锚定或调节那里的活动？该项目将为真菌逆行膜运输的机制提供新的见解。因此，它将对真菌研究的各个方面产生根本的兴趣，但它的特殊性将刺激对真菌致病性的研究。因此，我们的工作将有利于英国的制药和农业生物技术行业。然而，更具潜在意义的是，动力蛋白运输机制对于神经元中长距离的轴突运输是必不可少的。因此，拟议的研究还有望更好地了解动力蛋白和辅助因子在哺乳动物细胞运动神经元障碍中的作用。

Elongated cells such as neurons in the spine and the brain have to overcome long distances in order to communicate between the periphery and the cell body, which is essential for brain development and learning processes. Long-distance transport is achieved by so-called molecular motors. Fueled by chemical energy they 'walk' their cargo along the fibers of the cytoskeleton, which, much like a railway system, connect all parts of the cell. In the mammalian brain the microtubule cytoskeleton provides the major part of this system, and individual microtubules serve as 'tracks' for the motor proteins kinesin and dynein. Both molecular motors take membranous transport containers (e.g. vesicles) in opposite direction, thereby allowing bi-directional exchange of proteins, membranes and signals. As this fundamental process is essential for cell function and survival, it is not surprising that many neuronal diseases are related to mutations in this transport machinery. The motor dynein moves cargo from the peripheral synapse towards the cell body. In order run over long-distances, dynein needs support of other accessory factors. Among these is the dynactin complex and putative regulators of dynein, such as Lis1 and NudEl. These proteins have also been found to be involved in degenerative neuronal disorders, such as Lissencephaly and Amyotrophic Lateral Sclerosis. Despite much work done on these factors some basic questions, such as their mode of action in membrane trafficking, are still unresolved. Much of our knowledge about the role of e.g. dynactin is restricted to cell-free assays, and some doubt exists whether or not these results can be translated into the living cell. Ideally, one would like to visualize dynein and its regulators during transport of membranous cargo. However, due to technical limitations this was not yet achieved. Filamentous fungi share some striking similarities with human neurons. Their cells, for instance, highly elongated and expand at one cell pole. Transport towards this growing apex is mediated by long-range transport along microtubules, and similar to neurons this process involves dynein, kinesin-1 and kinesin-3. Most of what we know about long-distance transport in fungi was discovered by us using the model fungus Ustilago maydis. This model system shows remarkable similarities to human cells, but it combines powerful technical advantages, including a published genome sequence, numerous genetic tools and many cytological tools such as fluorescent proteins in different colors are established. Very recently we succeeded in visualizing individual dynein motors in membrane trafficking. This technical advancement opens new avenues for addressing the role of dynein and accessory factors in retrograde trafficking. We will make use of these technical advantages in order to address the following questions: (1) How do the dynein accessory factors Lis1, dynactin and NudEl control dynein activity and support long-range motility of individual dynein motors? (2) What is the role of the dynein light chain LC7/roadblock/Km93, which is known to be a tumor suppressor in humans? (3) How dynamic is dynein in elongated cells? (4) What are the factors that take dynein to the loading area at microtubule ends, and that anchor or regulate activities there? The project will provide novel insight into the mechanism of retrograde membrane transport in fungi. It will therefore be of fundamental interest to all aspects of fungal research, but will particularity stimulate research on fungal pathogenicity. Therefore, our work will be of benefit to the UK pharmaceutical and agricultural biotechnology industries. Of even greater potential significance, however, is that the dynein transport machinery is essential for long-distance axonal transport in neurons. Therefore, the proposed studies promise also to provide a better understanding of the role of dynein and accessory factors in motor neuron disorders in mammalian cells.