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Microtubule organisation in epithelial cells

上皮细胞中的微管组织

基本信息

批准号：
BB/R001618/1
负责人：
Daniel St Johnston
金额：
$ 48.66万
依托单位：
University of Cambridge
依托单位国家：
英国
项目类别：
Research Grant
财政年份：
2017
资助国家：
英国
起止时间：
2017 至无数据
项目状态：
已结题

来源：
https://gtr.ukri.org/projects?ref=BB%2FR001618%2F1
关键词：
Microtubule organisation epithelial cells

项目摘要

Most of our organs are composed of sheets of epithelial cells that function as barriers between compartments (e.g. blood vessels; secretory glands) or between the inside and outside of the body (skin, digestive system and lungs). The formation of these epithelial sheets depends on the coordinated polarisation of the cells, so that all have their apical surfaces facing the outside and their basal surfaces on the inside. Loss of this apical-basal polarity therefore disrupts epithelial organisation and disrupts their barrier function. More than 80% of cancers arise from epithelial tissues and one of their hallmarks is a progressive loss of polarity, which correlates with the malignancy of the tumour. A key function of epithelial sheets is to transport nutrients and other components across the epithelium. This depends on the formation of apical-basal arrays of microtubules that act as tracks along which motors transport components across the cell. Microtubules are polar filaments with one dynamic end that constantly grows and shrinks (the plus end) and a more stable minus end. In dividing cells, the microtubules are organised by microtubule organising centres (MTOCs), called centrosomes, which template and stabilise new MT minus ends. The centrosomes are usually inactivated in differentiated cells, however, and the microtubules grow instead from noncentrosomal microtubule organising centres (ncMTOCs). In epithelial cells, the ncMTOCs and MT minus ends localise to the apical cortex, with plus ends extending towards basal part of the cell. Although the structure and function of centrosomes is well understood, very little is known about ncMTOCs. The aim of our research is to understand how ncMTOCs are formed, regulated and localised in epithelial cells. We use fruit flies as our model organism because they allow us to study epithelia in their normal physiological environment, and because the powerful genetics in this system make it easy to modify genes with the recently developed CRISP/Cas9 technology and to add fluorescent tags to proteins of interest. Recent research by us and others has identified two key components of the ncMTOCs in flies and mammals: the giant actin microtubule cross-linker Shot (ACF7 in humans) and the microtubule minus end binding protein Patronin (CAMSAP). These proteins interact with each other and localise apically in epithelial cells. Shot seems to recruit ncMTOCs to the cell cortex, whereas Patronin protects MT minus ends from depolymerisation. Shot and Patronin are important for MT organisation not only in epithelia, but also in neurons, where non-centrosomal MTs also play a key role. Thus, reduction of Shot or Patronin affects neuronal growth and axon specification in both flies and mammals. Several hypotheses have been proposed to explain how Shot and Patronin are localised and regulated. To test these, we will introduce specific mutations into each protein and analyse their effects on protein localisation, the arrangement of the MTs and the organisation of the epithelium, using 3D and 4D immunofluorescence microscopy. For example, we will investigate whether the interaction of Shot with cortical actin is regulated by apical protein kinases, and how Shot is excluded from the lateral cortex of epithelial cells by the lateral polarity kinase, Par-1. We will also isolate ncMTOCs from epithelial cells and analyse their components by mass spectrometry. Identifying novel proteins that interact with Shot and Patronin should reveal the composition of ncMTOCs and provide insights into their function and regulation. This may also provide new markers for studying cell differentiation and neural diseases that affect the microtubule organisation, such as Alzheimer's disease and other dementias.

我们的大多数器官都由上皮细胞组成，这些上皮细胞作为隔室（例如血管;分泌腺）之间或身体内外（皮肤，消化系统和肺）之间的屏障。这些上皮片层的形成取决于细胞的协调极化，因此它们的顶面都面向外部，而它们的基底面在内部。因此，这种顶端-基底极性的丧失破坏了上皮组织并破坏了它们的屏障功能。超过80%的癌症起源于上皮组织，其特征之一是极性的进行性丧失，这与肿瘤的恶性程度相关。上皮片层的一个关键功能是将营养物质和其他成分运输穿过上皮。这取决于微管的顶基排列的形成，微管作为马达在细胞中运输组分的沿着轨道。微管是极性细丝，具有一个不断生长和收缩的动态端（正端）和一个更稳定的负端。在分裂的细胞中，微管由微管组织中心（MTOC）组织，称为中心体，其模板和稳定新的MT负末端。然而，在分化的细胞中，中心体通常是失活的，微管从非中心体微管组织中心（ncMTOCs）生长。在上皮细胞中，ncMTOC和MT负端定位于顶端皮质，正端向细胞的基底部分延伸。虽然中心体的结构和功能已经很清楚，但对ncMTOC知之甚少。我们研究的目的是了解ncMTOC如何在上皮细胞中形成，调节和定位。我们使用果蝇作为我们的模型生物，因为它们允许我们在正常的生理环境中研究上皮细胞，并且因为该系统中强大的遗传学使得使用最近开发的CRISP/Cas9技术修饰基因变得容易，并将荧光标签添加到感兴趣的蛋白质中。我们和其他人最近的研究已经确定了苍蝇和哺乳动物中ncMTOC的两个关键组成部分：巨大的肌动蛋白微管交联剂Shot（人类中的ACF 7）和微管负末端结合蛋白Patronin（CAMSAP）。这些蛋白质相互作用并定位于上皮细胞的顶端。射击似乎招募ncMTOC的细胞皮质，而Patronin保护MT负端解聚。Shot和Patronin不仅在上皮细胞中对MT组织很重要，而且在神经元中也很重要，其中非中心体MT也起着关键作用。因此，Shot或Patronin的减少影响果蝇和哺乳动物的神经元生长和轴突特化。已经提出了几个假设来解释如何拍摄和Patronin的本地化和监管。为了测试这些，我们将在每种蛋白质中引入特定的突变，并使用3D和4D免疫荧光显微镜分析它们对蛋白质定位，MT排列和上皮组织的影响。例如，我们将研究是否与皮质肌动蛋白的相互作用的镜头是由顶端蛋白激酶，以及如何被排除在上皮细胞的侧皮质的侧极性激酶，Par-1。我们还将从上皮细胞中分离ncMTOC，并通过质谱分析其组分。鉴定与Shot和Patronin相互作用的新蛋白质应该揭示ncMTOC的组成，并提供对其功能和调控的见解。这也可能为研究细胞分化和影响微管组织的神经疾病（例如阿尔茨海默病和其他痴呆症）提供新的标记物。