Microtubule Associated Proteins with roles in mitosis: A Systems approach

复杂的动植物如何确保当它们的细胞分裂时，它们能正确地进行分裂？为什么这个过程的中断会导致疾病？这些都是生物学中的基本问题。我们知道，细胞使用蛋白质纤维，称为微管，并使它们改变形状和大小，变成一个复杂的自我调节结构，称为有丝分裂纺锤体。这个纺锤体确保了正确数量的染色体最终位于细胞的两边；通过这种方式，单个细胞最终产生两个相同的新细胞。微管的组织由其他称为微管相关蛋白或MAP的蛋白质控制。如果这些图谱不能正确地组织纺锤体，细胞分裂的过程就会出错，并可能导致许多不同的遗传病，包括癌症。我们正在研究果蝇的细胞分裂是如何发生的。人类和果蝇细胞不仅使用相似的蛋白质来构建有丝分裂纺锤体，并在有丝分裂中表现相似，而且人们还可以取一只果蝇，破坏单一蛋白质，并研究其对整个动物的影响，这是组织培养细胞不可能做到的。在我们实验室以前工作的基础上，我们开发了一个生物信息学模型，该模型预测了1000多个果蝇蛋白质作为有丝分裂期间具有功能的图谱的可能性。这个模型已经鉴定了63个果蝇蛋白质，这些蛋白质目前还没有被报道参与有丝分裂微管组织，但有90%的可信区间是新的有丝分裂图谱。我们将使用一种名为RNA干扰的技术来降低细胞中这63种蛋白质的水平，并评估在有丝分裂期间对微管组织的影响。对于那些我们看到影响的蛋白质，我们将制造表达有丝分裂图的荧光标记版本的果蝇。我们将从这些苍蝇身上提取胚胎，并使用显微镜分析整个细胞周期中蛋白质在细胞中的位置。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。我们将把这些蛋白质的性质与已知的现有有丝分裂图谱进行比较，从而使我们能够将新的有丝分裂图谱归类为功能群。然后，我们将专注于这些新的有丝分裂图谱中的一小部分，以全面了解它们在组织微管中的作用。这将包括成像和分析MAP水平降低的果蝇的微管组织和动力学，以及调查新MAP和它们已知的相互作用蛋白之间的功能关系。我们相信这项研究是一项非常重要的研究，将提供对有丝分裂过程中微管组织的更多了解。事实上，已经进行的一项原则验证研究表明，降低其中一些蛋白质的水平确实会影响微管组织，这表明在这一应用中提出的深入研究将大大有助于我们理解细胞分裂和导致癌症等疾病的有丝分裂微管组织中的问题。

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.