Shedding light on differential mRNA localisation and RNP dynamics in vitro and in vivo

为了使细胞发挥其复杂的功能，必须在正确的时间将各种组件运送到正确的位置。马达蛋白是这项任务的核心。这些分子机器停靠在细胞组件上，并通过沿着细胞中的轨道网络将它们运送到目的地。分子发动机的关键“货物”之一是信使RNA（mRNA），这些分子是生产蛋白质的模板。将mRNA定位于细胞内的特定位点是控制其蛋白质产物运作的有效方法。因此，这个过程被用于许多需要区室化细胞活动的过程，包括学习和记忆，胚胎发育和细胞运动。尽管它广泛存在，但我们对mRNAs如何通过马达蛋白传递到细胞内的离散位点的了解很少。解决这个问题的一个容易处理的系统是果蝇的发育卵（卵母细胞）。在这里，将mRNA运输到不同位置指定了动物未来的身体轴：头到尾和从前到后。值得注意的是，将mRNA传递到卵母细胞中的每个位点都涉及相同的马达，动力蛋白，它走向极化微管轨道的“负端”。几十年的遗传学研究已经确定了将动力蛋白相关mRNA引导到卵母细胞中离散位点所需的蛋白质，但它们如何做到这一点尚不清楚。我们最近已经成功地重建核心动力蛋白为基础的mRNA运输机制的细胞外使用纯化的组件，这是一个重大的进步，因为它允许运输过程中被详细解剖，包括可视化的行为的mRNA和蛋白质的单分子。我们将建立在这个系统，以了解卵母细胞内的差异mRNA定位的机制基础。我们将测试的假设，根据我们最近未发表的结果，即集群的RNA和电机分子的颗粒是一个mRNA的目的地在卵母细胞的关键决定因素。这将使用人工构建的RNA-蛋白质组装体或已知对卵母细胞中特定mRNA的定位重要的蛋白质来实现。我们还将测试的微管细胞骨架的结构对运输和不同的RNP物种的锚定的影响，通过在玻璃表面上构建定义的微管模式。在一种补充方法中，我们将破坏一个关键的mRNA运输蛋白的“低复杂性”片段，这些片段在其他情况下与控制颗粒组装有关，并监测卵母细胞内外的影响。这些实验将通过我们小组最近开发的果蝇高效基因组编辑技术来促进。总的来说，这项工作将提供独特的见解，了解mRNA如何在同一个细胞中进行差异化排序，以及RNA和蛋白质组装成颗粒如何影响它们的功能。通过揭示可用于调节基于动力蛋白的运输的策略，我们的研究结果还将为了解汽车如何运输其他货物（包括膜结合囊泡和病毒）的努力提供信息。

In order for cells to perform their elaborate functions, various components must be delivered to the right place at the right time. Motor proteins are central to this task. These are molecular machines that dock onto cellular components and transport them to their destination by walking along a network of tracks in the cell. One of the key 'cargoes' for molecular motors is messenger RNA (mRNA), molecules that are the templates for the production of proteins. Localising an mRNA to a specific site within the cell is an effective way of controlling where its protein product operates. This process is therefore used in many processes that require compartmentalised cell activities, including learning and memory, embryonic development and cell movement. Despite its widespread occurrence we have a poor understanding of how mRNAs are delivered to discrete sites within cells by motor proteins. A tractable system for addressing this issue is the developing egg (the oocyte) of the fruit fly, Drosophila. Here, trafficking of mRNAs to different locations specifies the future body axes of the animal: head-to-tail and front-to-back. Remarkably, delivery of mRNAs to each site in the oocyte involves the same motor, dynein, which walks towards the 'minus-end' of polarised microtubule tracks. Genetic research over several decades has identified proteins that are required to direct dynein-associated mRNAs to discrete sites in the oocyte but how they do this is not clear. We have recently succeeded in reconstituting the core dynein-based mRNA transport machinery outside the cell using purified components; this is a significant advance as it allows the trafficking process to be dissected in detail, including the visualisation of the behaviour of single molecules of mRNA and protein. We will build on this system to understand the mechanistic basis of differential mRNA localisation within the oocyte. We will test the hypothesis, based on our recent unpublished results, that clustering of RNA and motor molecules in granules is a key determinant of an mRNA's destination in the oocyte. This will be achieved using RNA-protein assemblies that are built artificially or with proteins that are known to be important for localisation of specific mRNAs in the oocyte. We will also test the influence of the architecture of the microtubule cytoskeleton on transport and anchorage of different RNP species by constructing defined patterns of microtubules on a glass surface. In a complementary approach we will disrupt a key mRNA trafficking protein's 'low complexity' segments, which have been implicated in controlling granule assembly in other contexts, and monitor the effects inside and outside the oocyte. These experiments will be facilitated by efficient genome editing techniques for the fly that were recently developed in our group. Collectively, this work will provide unique insights into how mRNAs are sorted differentially in the same cell and how the assembly of RNAs and proteins into granules affects their function. By revealing strategies that can be used to regulate dynein-based transport, our findings will also inform efforts to understand how the motor traffics other cargoes, including membrane-bound vesicles and viruses.