Determining the role and mechanism of spatially restricted basement membrane remodelling in regulating mesenchymal-epithelial interactions in skin and

角膜利姆布斯和上皮毛囊由于其在组织稳态中的重要作用和作为胚胎后组织重塑的有力模型的价值，长期以来一直是人们强烈关注的区域。越来越多的关注将这两个区域作为支持上皮干细胞和间充质干细胞群体的位点。最近的研究，包括该监督团队的研究，揭示了干细胞及其微环境的许多决定因素。重要的是，我们已经共同确定了这些壁龛中的上皮-基质串扰对于干细胞的维持和功能是必不可少的。此外，使用连续块面扫描电子显微镜（SBF SEM），我们已经显示了角膜缘上皮和基质干细胞之间的直接物理接触。这些发现是令人惊讶的，因为中心法则是上皮和基质区室之间的基底膜充当这种相互作用的屏障。在这个学生奖学金，我们将结合联合收割机的主管1与主管2和3的干细胞专业知识，以确定如何基底膜的组成和组织局部破坏，以促进这些上皮基质的相互作用。每个基底膜包括两个蛋白质家族的互连结构网络; IV型胶原蛋白和层粘连蛋白。存在至少16种不同的层粘连蛋白家族成员，其聚合能力和对细胞表面受体的相对亲和力不同。免疫荧光显微镜显示富集特定的层粘连蛋白在高干细胞密度的区域，层粘连蛋白表达谱在干细胞活化过程中发生变化，并且不同的层粘连蛋白可以维持培养物中细胞的干细胞性或驱动谱系特异性分化。虽然这些发现强烈牵连层粘连蛋白在定义干细胞龛，有没有足够的分辨率来定义的上皮间充质相互作用的网站内发生的龛的研究。该研究将使用免疫金透射电子显微镜，SBF SEM和超分辨率光学显微镜的组合来精细绘制接触点周围的层粘连蛋白和层粘连蛋白结合蛋白分布（目的1）。第二部分的学生将解决的机制，通过该机制，基底膜局部破坏内尼斯。在这里，我们有两个假设。首先，根据肿瘤如何侵入的模型，基质降解酶的富集或局部活化以受控的方式发生在基底膜破裂的部位。该过程将是能量昂贵的，具有相对缓慢的动力学。因此，我们提出了第二个假设，即局部基质破坏是通过层粘连蛋白网络破坏蛋白的作用发生的。这个想法，这还没有提出在这种情况下，确实有平行的发现层粘连蛋白网络暂时中断免疫细胞外渗。与这一概念相一致，Supervisor 1已经鉴定出一种层粘连蛋白相关蛋白LaNt 31，其标志性特征表明它可以发挥这种作用。具体而言，这种蛋白质携带适当的结构基序以与层粘连蛋白相互作用，影响体外层粘连蛋白组织（4），在整个角膜缘区域显示差异分布，并在毛囊中富集，并且在干细胞活化期间上调（5，6）。使用我们建立的体外模型，人类外植体培养物和一套活体成像和基于抗体的工具，我们将直接检验这两个假设。这将涉及观察方法的组合，以研究LaNt 31和结构同源物在静态和动态条件下的定位和分布（目的2），以及确定层粘连蛋白网络破坏蛋白在上皮-间充质相互作用中表达增加/减少的影响的操纵策略（目的3）。在一起，这个项目

The corneal limbus and epithelial hair follicles have long been a region of intense interest due to their essential roles in tissue homeostasis and their value as powerful models of post-embryonic tissue remodelling. Increasingly attention has focused these two regions as the sites supporting epithelial and mesenchymal stem cell populations. Recent studies, including those of this supervisory team, have revealed many determinants of the stem cells and their microenvironment. Importantly, collectively we have established that epithelial-stromal cross-talk in these niches is essential for the maintenance and the function stem cell. Moreover, using serial block-face scanning electron microscopy (SBF SEM) we have shown direct physical contact between limbal epithelial and stromal stem cells. These findings were surprising as the central dogma was that basement membranes between the epithelial and stromal compartments act as a barrier to such an interaction. In this studentship, we will combine the basement membrane expertise of supervisor 1 with the stem cell expertise of supervisors 2 and 3 to determine how basement membrane composition and organisation is locally disrupted to facilitate these epithelial-stromal interactions. Every basement membrane comprises an interconnect structural networks of two families of proteins; type IV collagens and laminins. There are at least 16 different laminin family members which differ in their ability to polymerise and their relative affinity for cell surface receptors. Immunofluorescence microscopy has revealed enrichment of specific laminins in areas with high stem cell density, that laminin expression profiles change during stem cell activation, and that different laminins can maintain the stemness of cells in culture or drive lineage-specific differentiation. Although these findings strongly implicate laminins in defining the stem-cell niche, there are no studies of sufficient resolution to define the sites of epithelial-mesenchymal interactions occur within the niche. This studentship will use a combination of immunogold transmission electron microscopy, SBF SEM and superresolution light microscopy to fine-map the laminin and laminin-binding protein distribution around contact points (aim 1). The second part of the studentship will address the mechanism through which basement membranes are locally disrupted within the nice. Here, we have two hypotheses. First, following a model of how tumours invade, enrichment or localised activation of matrix degradative enzymes occurs in a controlled manner at sites of basement membrane breach. This process would be energy expensive, with comparatively slow dynamics. We, therefore, propose a second hypothesis where localised matrix disruption occurs through the action of laminin-network disrupting proteins. This idea, which has not been proposed in this context, does have parallels to the discovery of temporary disruption of laminin networks during immune cell extravasation. Consistent with this concept, supervisor 1 has identified a laminin-related protein, LaNt 31, with hallmark features that suggest it could play this role. Specifically, this protein carries appropriate structural motifs to interact with laminins, influences laminin organisation in vitro (4), displays differential distribution throughout the limbal region and is enriched in the hair follicle, and is upregulated during times of stem cell activation (5, 6).Using our established in vitro models, human explant culture and a suite of live imaging and antibody-based tools, we will directly test these two hypotheses. This will involve a combination of observational approaches to study the localisation, distribution of LaNt 31 and structural homologues in static and dynamic conditions (aim 2), and manipulative strategies to determine the effect of increased/decreased expression of laminin-network disrupting proteins in epithelial-mesenchymal interactions (aim 3). Together, this proj