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，我认为它们对伤口修复、血管生长和肿瘤扩散等过程非常重要。通过这些研究，将获得对这些过程的更深入的了解，这反过来可能导致确定新的治疗方法，如慢性或缓慢愈合的皮肤溃疡和癌症。身体的不同组织由特定细胞类型和细胞外蛋白质和糖的混合物组成，称为细胞外基质(ECM)。一些细胞类型存在于细胞外基质中并参与细胞外基质的产生，而另一些类型的细胞则覆盖在细胞外(上皮)和内(内皮)表面。在这些细胞膜的正下方，以及周围的神经和肌肉，有一个被称为基底膜(BM)的有组织的ECM区域。骨髓基质为细胞提供了支撑点，因此对于抗应力和结构完整性很重要。此外，骨髓基质支持多种细胞类型在不同时间的不同行为要求，包括充当皮肤细胞迁移到闭合伤口的道路。所有BMS的主要成分是层粘连蛋白家族的蛋白质。层粘连蛋白组装成相互关联的十字形分子，形成一个网络。这种网络的形成被证明涉及层粘连蛋白交叉短臂末端的一个小区域，被称为LN结构域。这种相互作用的重要性在许多遗传性疾病中得到了例证，在这些疾病中，层粘连蛋白结构域的特定缺陷会影响层粘连蛋白网络和BM组织，导致皮肤起泡、眼睛缺陷、肾功能衰竭或肌肉营养不良。然而，尽管有了这些知识，层粘连蛋白网络形成的方式，网络组织如何在不同的细胞过程中变化，以及是什么驱动了这些变化，我们还没有完全了解。这个项目将集中在LANS上，它们已经被证明在细胞附着和迁移中发挥作用，我的初步数据表明，它们很可能是通过调节BM的形成而起作用的。与层粘连蛋白一样，LANT(层粘连蛋白N末端)也含有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，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扮演的角色不同于正常组织功能。为了确定LANTS在BM形成中的作用以及它们对细胞行为和组织功能的影响，这个项目将追求三个目标。在目标1中，我将使用溶液中的蛋白质来直接评估LANTS与层粘连蛋白和其他BM蛋白相互作用的能力，并确定它们对网络形成的影响。在目标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Lant蛋白水平变化对细胞行为的影响。具体地说，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，它们在不同底物上附着的强度以及它们分裂的速度。在目标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。这些研究获得的数据将极大地扩大对LANT的了解，关于层粘连网络的形成，关于BM组织，并最终关于伤口修复、血管生长和肿瘤进展。从长远来看，这可能导致确定新的治疗策略或新的药物靶点。

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.