The regulation of dynein mechanochemistry in vivo

细胞含有一种名为微管的细丝系统，它由一种名为微管蛋白的蛋白质聚合物组成。微管作为细胞内的轨道，将小结构从一个区域输送到另一个区域。这种传递系统依赖于蛋白质在微管上“行走”，充当微小的马达。这些马达可以连接到细胞中的各种不同的结构上，就像铁路发动机可以拉客车或货运车厢一样。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动到细胞中心。这种运输在神经细胞中尤其重要，从周围到中心的距离确实很长；即使有了这种活跃的运输系统，从大脚趾的神经细胞尖端一直运送到脊髓的细胞体的物质也需要2.5天的路程。出于这个原因，可能只会轻微影响动力蛋白功能的突变会导致神经退行性疾病。然而，值得注意的是，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，这项拨款将调查运动单位以最高效率工作所需的条件。动力蛋白本身是非常复杂的，由几种不同的蛋白质组成。还需要其他蛋白质将动力蛋白连接到它的货物上，并组装一个功能正常的运动单位。总而言之，该装置包括驱动动力蛋白马达的发动机，将动力蛋白连接到微管轨道和货物上的装置，以及改变齿轮以使动力蛋白能够以一定范围的速度移动的装置。这笔拨款将检验这样一个假设，即与运动单位内的动力蛋白相互作用的特定蛋白质对货物附着或齿轮传动都是重要的。验证这一假设需要一个实验系统，在这个实验系统中，动力蛋白的活动可以被跟踪到微小的细节。出于这个原因，我们将利用动力蛋白移动一组特殊的货物的事实，称为内小体。这些是由脂肪和蛋白质组成的小袋子，用来将重要物质从细胞外输送到细胞中心。出于这个原因，它们必须使用动力蛋白以一种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。黄金就是这样一种材料。我们的实验室配备了以惊人的高帧速率(每秒10,000帧以上)成像的摄像头。这使我们能够追踪使用动力蛋白的金标内体运动的细节，以至于可以识别发动机的单个行程或步骤。人们认为，这些台阶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，通过观察内体加速或减速时它们是如何变化的。此外，通过操纵被认为影响马达货物结合或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.