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Determination of laminin LN domain roles in tissue function, using Pierson syndrome and the lens capsule as a model.

使用皮尔逊综合征和晶状体囊作为模型，确定层粘连蛋白 LN 结构域在组织功能中的作用。

基本信息

批准号：
1945182
负责人：
金额：
--
依托单位：
University of Liverpool
依托单位国家：
英国
项目类别：
Studentship
财政年份：
2017
资助国家：
英国
起止时间：
2017 至无数据
项目状态：
已结题

来源：
https://gtr.ukri.org/projects?ref=studentship-1945182
关键词：
Determination laminin LN domain roles

项目摘要

Basement membranes (BM) are specialised extracellular matrix structures, critical for tissue integrity. Moreover, BMs regulate key tissue behaviour through three distinct mechanisms: 1) By directly modulating signalling cascades, 2) Through sequestration followed controlled release of growth factors and 3) Through presenting context specific biomechanical cues to attached cells. Each of these properties has been shown to be dependent on BM composition, however, how the 3D organisation and macromolecular assembly status of those components determine tissue behaviour is less understood and has not been rigorously investigated. The laminins are a major structural component of all BMs. Each laminin is a heterotrimer capable of interacting with cell surface receptors (integrins, dystroglycan, syndecan) and other matrix proteins including nidogens and perlecan. Additionally, laminins assemble into networks, a feature controlled by sequences within their LN domains. Missense mutations in this domain are pathogenic and when they occur in LAMB2 they lead to Pierson Syndrome.The aim of this studentship is to determine the mechanisms through which LN domain mutations impact tissue function. Our focus will be Pierson syndrome, where patients present with lens abnormalities including abnormal shape and cataract formation. The lens capsule is a specialised BM that is one of the thickest human BMs, it that can be readily isolated and support the in vitro culture of cells. Moreover, the lens capsule contains a limited subset of laminins making it a tractable system for asking the focused questions proposed hereThis studentship will test the hypothesis that pathogenic mutations will influence the BM function in a context specific manner. The student will therefore characterise the effect of laminin mutants on:1) Laminin Signalling. Analysis of activity level of signalling pathways (candidate and hypothesis independent), comparative analysis of location, expression, interaction with mutants, and activity of laminin receptors.2) Growth factor sequestration by LAMB2. Immunogold EM quantification of perlecan/FGF2 abundance in BMs, immunoprecipitation for perlecan/nidogens, and functional analysis through resistance to staurosporine/etoposide induced apoptosis. 3) Structural/biomechanical properties of laminin networks. Laminin network morphology assessed by electron microscopy, biomechanical properties by atomic force microscopy and tensile strength analysis. To do so we will use CRISPR-Cas9 genome editing technology to introduce Pierson syndrome mutations into human lens epithelial cells (FHL124). We will then combine these cells, with isolated bovine lens capsules that have been treated to remove existing LMs to generate 3D physiologic ally relevant systems in which each of these potential BM functions can be assessed.

基底膜（BM）是专门的细胞外基质结构，对组织完整性至关重要。此外，BM通过三种不同的机制调节关键组织行为：1）通过直接调节信号级联，2）通过隔离随后控制释放生长因子，以及3）通过向附着的细胞呈现背景特异性生物力学线索。这些特性中的每一个都已被证明取决于BM组成，然而，这些组分的3D组织和大分子组装状态如何决定组织行为的了解较少，并且尚未进行严格的研究。层粘连蛋白是所有BM的主要结构组分。每个层粘连蛋白是能够与细胞表面受体（整联蛋白、肌营养不良蛋白聚糖、多配体蛋白聚糖）和其他基质蛋白（包括巢蛋白和串珠蛋白聚糖）相互作用的异源三聚体。此外，层粘连蛋白组装成网络，这是由其LN结构域内的序列控制的特征。错义突变在这个领域是致病的，当他们发生在LAMB 2，他们导致皮尔逊综合征。这个学生的目的是确定LN域突变影响组织功能的机制。我们的重点将是皮尔逊综合征，患者表现为透镜异常，包括形状异常和白内障形成。透镜囊是一种特殊的BM，是最厚的人类BM之一，它可以容易地分离并支持细胞的体外培养。此外，透镜囊包含层粘连蛋白的有限子集，使其成为一个易于处理的系统，用于提出本文提出的重点问题。本研究将检验致病突变将以特定方式影响BM功能的假设。因此，学生将讨论层粘连蛋白突变体对以下方面的影响：1）层粘连蛋白信号传导。信号传导途径的活性水平的分析（候选者和假设独立的），位置、表达、与突变体的相互作用以及层粘连蛋白受体的活性的比较分析。2）通过LAMB 2的生长因子螯合。BM中串珠素/FGF 2丰度的免疫金EM定量、串珠素/巢蛋白的免疫沉淀以及通过对星形孢菌素/依托泊苷诱导的细胞凋亡的抗性的功能分析。3)层粘连蛋白网络的结构/生物力学特性。通过电子显微镜评估层粘连蛋白网络形态，通过原子力显微镜和拉伸强度分析评估生物力学特性。为此，我们将使用CRISPR-Cas9基因组编辑技术将皮尔逊综合征突变引入人类透镜上皮细胞（FHL 124）。然后，我们将联合收割机这些细胞，与分离的牛透镜囊，已处理，以消除现有的LM，以产生三维生理相关的系统，其中每个这些潜在的BM功能可以进行评估。