Characterisation of LaNt regulation of Basement Membrane organisation in wound repair and angiogenesis.

在这项工作中，我建议研究最近发现的LaNt蛋白家族，我认为它对伤口修复、血管生长和肿瘤扩散等过程很重要。通过这些研究，将对这些过程有更深入的了解，进而可能导致对慢性或缓慢愈合的皮肤溃疡和癌症等疾病的新治疗方法的确定。身体的不同组织是由特定细胞类型和细胞外蛋白质和糖的混合物组成的，称为细胞外基质（ECM）。一些细胞类型存在于ECM中并促进其产生，而另一些细胞类型则以细胞片的形式覆盖在ECM区域的外（上皮）和内（内皮）表面。在这些细胞片的正下方，以及周围的神经和肌肉，有一个有组织的ECM区域称为基底膜（BM）。脑基为细胞提供锚定点，因此对抗应力和结构完整性很重要。此外，脑转移瘤支持多种细胞类型在不同时间的不同行为需求，包括作为皮肤细胞迁移到闭合伤口的道路。所有脑转移瘤的一个主要成分是层粘连蛋白家族。层粘连蛋白聚集成十字形的分子，相互结合形成一个网络。该网络的形成已被证明涉及层粘连蛋白交叉短臂末端的一个小区域，称为LN结构域。这种相互作用的重要性通过许多遗传疾病得到例证，其中LN结构域的特定缺陷影响层粘连蛋白网络和BM组织，导致皮肤起泡、眼睛缺陷、肾衰竭或肌肉萎缩。然而，尽管有这些知识，层粘连蛋白网络形成的方式，网络组织在不同的细胞过程中如何变化以及驱动这些变化的因素尚未完全了解。该项目将重点关注已被证明在细胞附着和迁移中发挥作用的LaNts，我的初步数据表明，这可能是通过调节BM的形成。像层粘连蛋白一样，LaNt（层粘连蛋白n端）也包含一个LN结构域，这表明它们可以直接与层粘连蛋白相互作用，并改变层粘连蛋白网络的组织方式。重要的是，在层粘连蛋白家族中也存在组织特异性差异，这些差异可能意味着LaNts的影响是细胞类型特异性的。这也可能意味着LaNts在血管生长或伤口修复过程中发挥的作用不同于正常组织功能。为了描述LaNts在BM形成中的作用及其对细胞行为和组织功能的影响，该项目将追求3个目标。在Aim 1中，我将使用溶液中的蛋白质直接评估LaNts与层粘胶蛋白和其他BM蛋白相互作用的能力，并确定它们对网络形成的影响。在Aim 2中，我将使用皮肤和角膜上皮细胞以及血管内皮细胞来研究改变LaNt蛋白水平对细胞行为的影响。具体来说，我们将确定细胞沉积的BM是否会随着LaNt水平的增加或减少而变化，我们将评估细胞的运动速度，它们在不同基质上的附着强度和分裂速度。在Aim 3中，我将使用皮肤、眼睛和血管生长的三维模型来研究LaNt在这些更复杂的组织模型中的作用。从这些研究中获得的数据将极大地扩展我们对LaNts、层粘连蛋白网络形成、基底膜组织以及最终对伤口修复、血管生长和肿瘤进展的了解。从长远来看，这可能会导致新的治疗策略或新的药物靶点的确定。

In this work I propose to study the LaNt family of proteins which were recently identified and which I believe are important for processes such as wound repair, blood vessel growth and the spread of tumours. Through these studies, a deeper understanding of these processes will be obtained and this, in turn, may lead to identification of new treatment approaches for conditions such as chronic or slow-healing skin ulcers and cancer.The different tissues of the body are composed of defined combinations of specialised cell types and a mixture of proteins and sugars outside the cells, termed the extracellular matrix (ECM). Some of the cell types reside in and contribute to the production of this ECM, whereas others cover the outer (epithelia) and inner (endothelia) surfaces of regions of ECM as sheets of cells. Directly beneath these cell sheets, as well as surrounding nerves and muscles, there is an organised region of ECM termed basement membrane (BM). BMs provide the anchorage point for cells and are therefore important for stress resistance and structural integrity. In addition, BMs support the different behavioural requirements of a wide range of cell types at different times, including acting as the road upon which the skin cells migrate to close wounds. A major component of all BMs is the laminin family of proteins. Laminins assemble into cross shaped molecules that associate with one another to form a network. Formation of this network has been shown to involve a small region at the very end of the short arms of the laminin cross, which is termed a LN domain. The importance of this interaction is exemplified by a number of genetic diseases where specific defects in LN domains impact the laminin network and BM organisation resulting in skin blistering, eye defects, kidney failure or muscular dystrophy. However, despite this knowledge, the ways in which laminin networks form, how network organisation changes during different cellular processes and what drives those changes is yet to be fully understood.This project will focus on the LaNts which have been demonstrated to play a role in cell attachment and migration and which my preliminary data indicate is likely to be through regulating BM formation. Like the laminins, the LaNt (Laminin N-terminus) also contain a LN domain, this suggests that they can interact directly with laminins and modify the ways in which laminin networks are organised. Importantly, there are also tissue specific differences within the laminin family and these differences are likely to mean that the impact of the LaNts is cell type specific. This may also mean that LaNts play different roles during blood vessel growth or wound repair than during normal tissue function. In order to characterise the roles of LaNts in BM formation and the impact they have on cell behaviour and tissue function, this project will pursue 3 aims.In Aim 1, I will use proteins in solution to directly assess the ability of LaNts to interact with laminins and other BM proteins and to determine their impact on network formation. In Aim 2, I will use skin and corneal epithelial cells and blood vessel endothelial cells to study the impact of changing LaNt protein levels on cell behaviour. Specifically, we will determine if the BM deposited by cells changes in response to increasing or decreasing LaNt levels and we will assess cell movement speeds, how strongly they attach and how rapidly they divide on the different substrates.In Aim 3, I will use three dimensional models of skin, eye and of blood vessel growth to study LaNt roles in these more complex tissue models.Together the data obtained from these studies will dramatically expand what is known about LaNts, about laminin network formation, about BM organisation and ultimately about wound repair, blood vessel growth and tumour progression. In the longer term, this may lead to identification of new treatment strategies or new drug targets.