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

构成我们身体的细胞显示出极大的多样性，然而，许多类型的细胞都有一些共同的特性。这些能力包括沟通(“信号”)以协调功能，以及采用两极分化的形状或行为。因此，了解细胞信号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，信令和极化是耦合的。一个例子是“平面极性”，即一张纸中的细胞相互作用，使它们在组织平面上指向相同的方向。这导致皮肤上的毛发能够指向一个方向，而纤毛排列在气管和肺的同一方向上。我们将研究两个在建立动物组织平面极性方面起重要作用的分子，钙粘素脂肪(Ft)和纤维粘附素(DS)。钙粘附素通过位于细胞外膜并与相邻细胞上的钙粘附素形成纽带来调节细胞之间的结合。研究最多的钙粘附素--上皮钙粘蛋白(E-cad)--结合在上皮片的细胞之间，在将组织结合在一起的过程中起着结构性的作用。Ft和DS之间的这种异嗜性结合介导了细胞之间的信号传递，因为一个细胞上的高Dd会导致更多的Ft分子结合在接触的细胞表面。此外，Ft-DS异源二聚体在细胞-细胞接触处聚集在一起，在这些簇内分子被‘分类’，使得簇主要由一个细胞中的Ft和相邻细胞中的D组成。这种局部排序似乎促进了Ft和D在细胞水平上的极化，使得大多数Ft去往细胞的一端，而大多数D去往另一端。Ft和DS的这两个特性--细胞之间的异亲结合和细胞内相对边缘的分离--导致了细胞片的组织水平的平面极化，因此每个细胞在一边有Ft，在另一边有Ds。我们将研究Ft和DS如何促进组织的平面极化。我们认为，它们在细胞-细胞接触处的稳定聚集取决于Ft和D之间的顺式和反式相互作用的组合，我们将通过系统地将Ft和D的部分与自身和彼此结合进行映射来检验这一点。此外，我们认为局域分选成极化团簇是由于同一取向的Ft-DS杂二聚体之间的稳定相互作用和相反取向的杂二聚体之间的不稳定相互作用的共同作用。我们将寻求确定这种分类相互作用的分子基础。为了了解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，我们将使用前沿的基因工程技术来辅助对它们的精细解剖。我们将在培养的细胞系统中研究Ft和D的性质，在那里它们可以被快速操纵，也将在一个简单的动物模型中--发育中的果蝇翅膀--特别适合基因操作和成像。这项工作将揭示人类健康所需的一组研究较少的非典型钙粘附素的分子性质。Ft-ds活性的中断与先天性疾病有关，如心脏瓣膜缺陷、Van Maldergem和Hennekam综合征，以及与癌症进展有关。对ft和ds如何调节细胞信号和细胞极化的详细了解将揭示细胞功能基本方面的潜在机制，并为理解如何从治疗角度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.