The regulation of dynein mechanochemistry in vivo

细胞包含一个被称为微管的细丝系统，它是由一种被称为微管蛋白的蛋白质的聚合物组成的。微管在细胞内充当轨道，用于将小结构从一个区域运输到另一个区域。这种传递系统依赖于蛋白质，蛋白质沿着沿着微管“行走”，充当微小的马达。这些发动机可以连接到细胞中的各种不同结构或“货物”上，就像铁路发动机可以拉动客车或货车一样。这项拨款申请涉及一种名为动力蛋白的马达的功能。动力蛋白将货物从细胞的外围区域移向细胞中心。这种运输在神经细胞中尤其重要，从外周到中心的距离确实很长;即使有这种主动运输系统，这个旅程也需要2.5天，因为材料从大脚趾的神经细胞尖端一直运送到脊髓的细胞体。出于这个原因，可能仅轻度影响动力蛋白功能的突变导致神经退行性疾病。然而，重要的是要注意，动力蛋白对体内所有细胞的功能都很重要。由于完整的动力蛋白活性对细胞功能至关重要，这项资助将研究运动单位以最大效率工作所需的条件。动力蛋白本身非常复杂，由几种不同的蛋白质组成。其他蛋白质需要连接动力蛋白的货物，并组装一个功能性的运动单位。总而言之，该装置包含驱动动力蛋白马达的发动机，将动力蛋白与微管轨道和货物连接的装置，以及变速装置，以便动力蛋白可以以一定的速度移动。这项资助将检验这样一种假设，即与运动单位内的动力蛋白相互作用的特定蛋白质对于货物附着或齿轮传动都很重要。验证这一假设需要一个实验系统，其中动力蛋白的活动可以在微小的细节。出于这个原因，我们将利用动力蛋白移动一组称为内体的特定货物的事实。这些由脂质和蛋白质组成的微小袋子用于将重要物质如营养物质从细胞外运送到细胞中心。由于这个原因，它们必须通过动力蛋白以向内的方式快速运输。使用内体作为模型货物的一个关键优势是它们可以很容易地可视化，因为它们可以装载在显微镜下可以看到的材料。其中一种材料就是黄金。我们的实验室配备了以极高的帧速率（每秒10，000帧及以上）成像的相机。这使我们能够利用动力蛋白来跟踪金标记的内体的运动，从而可以识别马达发动机的各个冲程或步骤。人们认为这些步骤的长度或频率会影响货物移动的速度。我们能够直接测试这一点，通过观察它们如何随着内体加速或减速而变化。此外，通过操纵被认为影响货物结合或马达传动的蛋白质的拷贝数和功能，我们将测试这些蛋白质是否确实影响内体速度，如果是，它们是否通过调节步长或频率来发挥这些作用。

Cells contain a system of filaments, called microtubules, that are made up of polymers of a protein call tubulin. Microtubules act as tracks within the cell for the transport of small structures from one region to another. This delivery system relies on proteins that 'walk' along microtubules, acting as minute motors. These motors can be attached to a variety of different structures in the cell, or 'cargoes', in much the same way that a railway engine can pull passenger coaches or freight wagons. This grant application addresses the function of a motor called dynein. Dynein moves cargoes from the peripheral regions of a cell towards the cell centre. Such transport is especially important in nerve cells, where the distance from the periphery to the centre can be very long indeed; even with this active transport system, this journey will take 2.5 days for material carried all the way from the tip of a nerve cell in your big toe to the cell body in your spinal cord. For this reason, mutations that may only mildly affect dynein function lead to neurodegenerative diseases. It is important to note, however, dynein is important for the function of all cells in the body. Since full dynein activity is so vital for cell function, this grant will investigate what is needed for the motor unit to work at maximum efficiency. Dynein itself is very complicated, being made up of several different proteins. Other proteins are needed to link dynein to its cargoes, and to assemble a functional motor unit. Altogether, the unit contains the engine that drives the dynein motor, the means to link dynein both to the microtubule track and to the cargoes, and the means to change gears so that dynein can move at a range of speeds. The grant will test the hypothesis that specific proteins that interact with dynein within the motor unit are important, either for cargo attachment or gearing. Testing this hypothesis requires an experimental system in which dynein activity can be followed in minute detail. For this reason, we will exploit the fact that dynein moves a particular set of cargoes called endosomes. These are tiny bags made up of lipid and protein that are used to deliver important material such as nutrients from outside the cell to the cell centre. For this reason they must be transported rapidly in an inward fashion, using dynein. One critical advantage of using endosomes as a model cargo is that they can be visualized quite easily, since they can be loaded with materials that can be seen under the microscope. One such material is gold. Our laboratories are equipped with cameras that image at astonishingly high frames rates (10,000 frames per second and over). This allows us to follow gold-labelled endosomes moving using dynein in such detail that individual strokes, or steps, of the motor's engine can be identified. It is thought that the length of these steps, or their frequency, influences how fast the cargo moves. We are in a position to test this directly, by seeing how they change as endosomes speed up or slow down. In addition, by manipulating the copy number and function of the proteins thought to affect cargo binding or gearing of the motor, we will test whether these proteins do influence endosome speed, and if so, whether they exert these effects by modulating step length or frequency.