Microtubule organisation in epithelial cells

我们的大多数器官都是由上皮细胞片组成的，上皮细胞片的功能是隔室（如血管、分泌腺）或身体内外（皮肤、消化系统和肺部）之间的屏障。这些上皮层的形成取决于细胞的协调极化，因此它们的顶端表面都是向外的，而它们的基面都是向内的。因此，这种顶基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逐渐丧失，这与肿瘤的恶性有关。上皮片的一个关键功能是在上皮间运输营养物质和其他成分。这取决于微管的顶基排列的形成，微管作为马达在细胞内运输成分的轨迹。微管是极细丝，一端是不断生长和收缩的动态端（正端），另一端是更稳定的负端。在分裂细胞中，微管由微管组织中心（MTOCs）组织，称为中心体，其模板和稳定新的MT负端。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）生长而来。在上皮细胞中，ncMTOCs和MT负端位于顶端皮层，正端向细胞基部延伸。虽然中心体的结构和功能已经被很好地理解，但对ncMTOCs知之甚少。我们研究的目的是了解ncMTOCs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式生物，因为它们允许我们在正常的生理环境中研究上皮细胞，并且因为这个系统中强大的遗传学使得使用最近开发的CRISP/Cas9技术修改基因和向感兴趣的蛋白质添加荧光标签变得容易。我们和其他人最近的研究已经确定了苍蝇和哺乳动物ncMTOCs的两个关键成分：人类的巨肌动蛋白微管交联剂Shot （ACF7）和微管负端结合蛋白Patronin （CAMSAP）。这些蛋白相互作用并定位于上皮细胞的顶端。Shot似乎将ncMTOCs招募到细胞皮层，而Patronin则保护MT负端不被解聚。Shot和Patronin不仅对上皮细胞的MT组织很重要，而且对神经元也很重要，其中非中心体MT也起关键作用。因此，Shot或Patronin的减少会影响果蝇和哺乳动物的神经元生长和轴突规范。人们提出了几种假说来解释Shot和Patronin是如何定位和调控的。为了测试这些，我们将在每个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Shot与皮质肌动蛋白的相互作用是否受顶端蛋白激酶的调节，以及Shot如何被外侧极性激酶Par-1排除在上皮细胞的外侧皮质之外。我们还将从上皮细胞中分离ncMTOCs，并通过质谱分析其成分。鉴定与Shot和Patronin相互作用的新蛋白将揭示ncMTOCs的组成，并为其功能和调控提供见解。这也可能为研究细胞分化和影响微管组织的神经疾病（如阿尔茨海默病和其他痴呆症）提供新的标记物。

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.