Microtubule Associated Proteins with roles in mitosis: A Systems approach

复杂的动物和植物如何确保它们的细胞分裂正常进行？为什么这个过程的中断会导致疾病？这些都是生物学的基本问题。我们知道，细胞使用蛋白质纤维，称为微管，并让它们改变形状和大小，成为一个复杂的，自我调节的结构，称为有丝分裂纺锤体。这种纺锤体确保正确数量的染色体在细胞的两侧结束;这样，一个细胞最终产生两个相同的新细胞。微管的组织是由称为微管相关蛋白（MAP）的其他蛋白质控制的。如果这些MAP不能正确组织纺锤体，细胞分裂过程就会出错，并可能导致许多不同的遗传疾病，包括癌症。我们正在研究果蝇的细胞分裂是如何发生的。不仅人类和果蝇细胞使用相似的蛋白质来构建有丝分裂纺锤体，并且在有丝分裂过程中表现相似，而且人们还可以采取果蝇，破坏单个蛋白质并研究整个动物的后果，这在组织培养细胞中是不可能做到的。在我们实验室以前的工作之后，我们开发了一个生物信息学模型，预测了超过1000种果蝇蛋白质在有丝分裂期间具有功能的MAP的可能性。这个模型已经确定了63果蝇蛋白，目前还没有报道参与有丝分裂微管组织，但有90%的置信区间是新的有丝分裂MAP。我们将使用一种称为RNA干扰的技术降低细胞中这63种蛋白质中每一种的水平，并评估有丝分裂期间对微管组织的影响。对于那些我们看到效果的蛋白质，我们将使果蝇表达有丝分裂MAP的荧光标记版本。我们将从这些果蝇身上取出胚胎，用显微镜分析蛋白质在整个细胞周期中的去向。我们还将使用这些标记的蛋白质来分离细胞中与它们相互作用的其他蛋白质。我们将比较这些蛋白质的性质与已知的现有的有丝分裂MAP，使我们能够将新的有丝分裂MAP分类为功能组。然后，我们将集中在少数这些新的有丝分裂的地图，走向一个全面了解他们的作用，组织微管。这将包括成像和分析微管组织和动态的苍蝇，有减少的地图水平，并调查新的地图和他们已知的相互作用proteines. We之间的功能关系，我们相信这项研究是一个非常重要的，将提供一个更好的理解微管组织在有丝分裂。事实上，已经进行的原理验证研究表明，降低这些蛋白质中的一些蛋白质的水平确实会影响微管组织，这表明本申请中提出的深入研究将大大有助于我们理解细胞分裂和有丝分裂微管组织中导致癌症等疾病的问题。

How do complex animals and plants ensure that, when their cells divide, they do so properly? And why does disruption of this process lead to disease?These are fundamental questions in biology. We know that cells use protein fibres, called microtubules, and get them to change shape and size, into a complex, self-regulating structure termed the mitotic spindle. This spindle ensures the right number of chromosomes end up at opposite sides of the cell; in this way a single cell ends up producing two identical new cells. The organization of microtubules is controlled by other proteins called microtubule associated proteins, or MAPs. If these MAPs fail to organise the spindle properly, the process of cell division goes wrong, and can lead to many different genetic diseases, including cancer. We are investigating how cell division happens in the fruit fly. Not only do human and fruit fly cells use similar proteins to build the mitotic spindle, and behave similarly through mitosis, but one can also take a fruit fly, disrupt a single protein and investigate the consequences to the whole animal, in a way impossible to do with tissue culture cells. Following on from previous work in our lab, we have developed a bioinformatics model that predicts the likelihood of over 1000 Drosophila proteins being a MAP with a function during mitosis. This model has identified 63 Drosophila proteins that currently have not been reported to be involved in mitotic microtubule organisation, but that have a 90% confidence interval of being novel mitotic MAPs. We will reduce the levels of each of these 63 proteins in the cell, using a technique called RNA interference, and assess the effect on microtubule organisation during mitosis. For those proteins where we see an effect, we will make fruit flies that express fluorescent-tagged versions of the mitotic MAPs. We will take embryos from these flies and use microscopy to analyse where in the cell the proteins go, throughout the cell cycle. We will also use these tagged proteins to isolate other proteins in the cell that interact with them. We will compare the properties of these proteins with what is known of existing mitotic MAPs, allowing us to categorise the novel mitotic MAPs into functional groups. We will then focus on a small number of these novel mitotic MAPs, to move towards a full understanding of their roles in organising microtubules. This will include imaging and analysing microtubule organisation and dynamics in flies that have reduced levels of the MAPs, and investigating the functional relationships between the novel MAPs and their known interacting proteins.We believe this study is a very important one that will provide a greater understanding of microtubule organization during mitosis. Indeed, a proof-of-principle investigation already carried out shows that reducing levels of some of these proteins does affect microtubule organsation, suggesting that the in depth study proposed in this application will significantly contribute to our understanding of cell division and the problems in mitotic microtubule organisation that lead to diseases such as cancer.