Dissecting the molecular organisation of Fat-Dachsous cadherin complexes to understand mechanisms of coordinated cell polarisation

组成我们身体的细胞表现出很大的多样性，然而许多细胞类型有一些共同的特性。这些包括沟通（“信号”）协调功能的能力，以及采取极化形状或行为的能力。因此，理解细胞信号传导和细胞极化是细胞生物学的基本目标。值得注意的是，在某些情况下，信号和极化是耦合的。一个例子是“平面极性”，即薄片上的细胞相互作用，因此它们在组织平面上都指向同一个方向。这导致皮肤上的毛发能够指向一个方向，而气管和肺部的纤毛能够朝着同一个方向跳动。我们将研究在动物组织中对平面极性的建立起重要作用的两个分子，钙粘蛋白Fat （Ft）和Dachsous （Ds）。钙粘蛋白通过位于外膜上并与邻近细胞上的钙粘蛋白形成键来调节细胞间的结合。研究最多的钙粘蛋白是上皮钙粘蛋白（Epithelial cadherin, E-cad），它结合在上皮薄片的细胞之间，并在将组织结合在一起方面起着结构作用。Ft和Ds钙粘蛋白也在细胞间结合，一个细胞上的Ft与邻近细胞上的Ds结合。Ft和Ds之间的这种“异亲性”结合介导了细胞间的信号传导，因为一个细胞上的高Ds会导致更多的Ft分子结合在接触细胞的表面。此外，Ft-Ds异二聚体在细胞-细胞接触处聚集在一起，并且在这些簇中分子被“排序”，这样簇主要由一个细胞中的Ft和相邻细胞中的Ds组成。这种局部分选似乎促进了Ft和Ds在细胞水平上的极化，使得大多数Ft到细胞的一端，而大多数Ds到另一端。Ft和Ds的这两个特性——细胞间的亲异性结合和细胞内相对边缘的分离——导致细胞片的组织水平平面极化，这样每个细胞的一边是Ft，另一边是Ds。我们将研究Ft和Ds如何促进组织的平面极化。我们提出它们在细胞-细胞接触处的稳定聚集取决于Ft和Ds之间顺式和反式相互作用的组合，我们将通过系统地绘制Ft和Ds与自身和彼此结合相关的部分来验证这一点。此外，我们提出，局部分类进入极化簇是由于相同取向的Ft-Ds异二聚体之间的稳定相互作用和相反取向的异二聚体之间的不稳定相互作用的结合。我们将试图确定这种排序相互作用的分子基础。为了理解Ft和Ds的行为，我们将利用显微镜技术的进步，以高分辨率对单个分子进行成像。此外，由于Ft和Ds是非常大的分子，我们将使用先进的基因工程技术来协助它们的精细解剖。我们将在培养细胞系统中研究Ft和Ds的特性，在培养细胞系统中它们可以被快速操作，也可以在一个简单的动物模型中研究——发育中的果蝇翅膀——这特别适合基因操作和成像。这项工作将阐明对人类健康所必需的一组“非典型”钙粘蛋白的分子特性。Ft-Ds活性的破坏与心脏瓣膜缺陷、Van Maldergem和Hennekam综合征等先天性疾病有关，也与癌症进展有关。Ft和Ds如何介导细胞信号传导和细胞极化的详细知识将揭示细胞功能基本方面的潜在机制，并为理解如何治疗性地调节它们的活动开辟道路。

The cells that compose our bodies show great diversity, nevertheless some properties are shared by many cell types. These include the ability to communicate ('signal') to coordinate functions, and to adopt polarised shapes or behaviours. Thus, understanding cell signalling and cell polarisation are fundamental goals of cell biology. Notably, in some contexts, signalling and polarisation are coupled. An example is 'planar polarity', where cells in a sheet interact together so they all point in the same direction in the tissue plane. This results in hairs on the skin being able to point in one direction, and cilia lining the trachea and lungs to beat in the same direction.We will study two molecules that play important roles in establishing planar polarity in animal tissues, the cadherins Fat (Ft) and Dachsous (Ds). Cadherins mediate binding between cells, by sitting in the outer membrane and forming bonds with cadherins sitting on neighbouring cells. The best studied cadherin - Epithelial cadherin (E-cad) - binds between cells in epithelial sheets, and plays a structural role in holding tissues together.Ft and Ds cadherins also bind between cells, with Ft on one cell binding to Ds on the neighbouring cell. This 'heterophilic' binding between Ft and Ds mediates signalling between cells, as high Ds on one cell results in more Ft molecules being bound on the surface of a contacting cell. Furthermore, Ft-Ds heterodimers cluster together at cell-cell contacts, and within these clusters the molecules are 'sorted', such that clusters consist predominantly of Ft in one cell and Ds in the neighbouring cell. This local sorting appears to promote cell-level polarisation of Ft and Ds, such that most Ft goes to one cell end and most Ds goes to the other. These two properties of Ft and Ds - heterophilic binding between cells and segregation to opposite edges within cells - result in tissue-level planar polarisation of cell sheets, such that each cell has Ft at one edge and Ds at the other.We will investigate how Ft and Ds promote planar polarisation of tissues. We propose that their stable clustering at cell-cell contacts depends on a combination of cis- and trans-interactions between Ft and Ds, and we will test this by systematically mapping parts of Ft and Ds involved in binding to themselves and each other. Furthermore, we propose that local sorting into polarised clusters is due to a combination of stabilising interactions between Ft-Ds heterodimers of the same orientation, and destabilising interactions between heterodimers of the opposite orientation. We will seek to identify the molecular basis of such sorting interactions.To understand the behaviour of Ft and Ds, we will exploit advances in microscopy that allow imaging of individual molecules at high resolution. Moreover, as Ft and Ds are very large molecules, we will use leading-edge genetic engineering techniques to assist their fine-scale dissection. We will study the properties of Ft and Ds in a cultured cell system where they can be rapidly manipulated, and also in a simple animal model - the developing fruit fly wing - which is particularly amenable to genetic manipulation and imaging.This work will shed light on the molecular properties of a poorly studied group of 'atypical' cadherins which are required for human health. Disruption of Ft-Ds activity is associated with congenital diseases such as cardiac valve defects and Van Maldergem and Hennekam syndromes, as well as being implicated in cancer progression. Detailed knowledge of how Ft and Ds mediate cell signalling and cell polarisation will reveal mechanisms underlying fundamental aspects of cell function, and open the way to understanding how to therapeutically modulate their activities.