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Live Imaging and Genetic Dissection of Basement Membrane Development and Repair

基底膜发育和修复的实时成像和基因解剖

基本信息

批准号：
BB/L021927/1
负责人：
Brian Stramer
金额：
$ 54.74万
依托单位：
King's College London
依托单位国家：
英国
项目类别：
Research Grant
财政年份：
2014
资助国家：
英国
起止时间：
2014 至无数据
项目状态：
已结题

来源：
https://gtr.ukri.org/projects?ref=BB%2FL021927%2F1
关键词：
Live Imaging Genetic Dissection Basement

项目摘要

The basement membrane, a thin layer of linked extracellular proteins (extracellular matrix), underlies nearly all epithelial cells in the human body. This specialised cellular "tarmac" is necessary for the function of its overlying cells, and an abnormal basement membrane plays a role in a number of pathologies. Despite this clinical relevance, we know little about how the basement membrane is formed. Furthermore, despite its certain damage during any type of tissue injury, we know nothing about how the basement membrane is capable of repair. The basement membrane is composed of a number of different components, such as Collagen Type IV, which are linked by enzymatic reactions to yield a final stable structure. The production and organisation of these components is also thought to require a number of different cell-types. Due to this complexity, a complete understanding of the basement membrane requires its examination within a living organism, which until recently has been experimentally unfeasible.In this proposal we will exploit our ability to live image basement membrane development and repair within a living organism. Fruit flies (Drosophila melanogaster) are becoming a widely utilised model system to understand the basement membrane as this animal has an assortment of extracellular matrix proteins identical to humans. Furthermore, preliminary data from our laboratory has revealed that basement membrane formation during embryogenesis can be imaged live during animal development. In this proposal we will exploit our ability to live image basement membrane development, along with our capacity in flies to knockout virtually any gene of interest, to fully dissect the mechanisms of basement membrane formation and repair.In the first Objective we will characterise the timecourse of basement membrane formation by time-lapse microscopy. We will subsequently use strategies to specifically remove hypothesised basement membrane components and factors required for its formation within the various cells in the embryo thought to be involved in basement membrane development. This analysis will allow us to highlight the molecular mechanisms behind basement membrane formation and the relative requirement of different cells in its production.In the subsequent Objective we will examine the basement membrane repair response. Preliminary data from the laboratory has revealed that the epithelium and underlying basement membrane can be specifically damaged in the fly by laser ablation; this leads to a healing response whereby over the course of a few hours the basement membrane hole is sealed. We will characterise this response and determine the cellular and molecular mechanisms involved in basement membrane repair. Our analysis suggests that basement membrane healing is an active cellular process that requires recruitment of fly macrophages (Drosophila inflammatory cells), and we will directly test the function of macrophages in repairing the damage. Furthermore, preliminary data reveals that the fly macrophages directly respond to damage to the basement membrane (rather than damage to the overlying epithelial cells), which we will directly test. This data suggests that a damaged basement membrane may be playing a significant role in inflammatory responses, which will have wide reaching clinical implications.

基底膜是一层薄薄的细胞外蛋白（细胞外基质），构成人体几乎所有上皮细胞的基础。这种特殊的细胞“停机坪”对于其上覆盖的细胞的功能是必要的，并且异常的基底膜在许多病理中起作用。尽管具有临床意义，但我们对基底膜是如何形成的知之甚少。此外，尽管基底膜在任何类型的组织损伤中都有一定的损伤，但我们对基底膜的修复能力一无所知。基底膜由许多不同的成分组成，如IV型胶原蛋白，它们通过酶促反应联系在一起，形成最终的稳定结构。这些成分的生产和组织也被认为需要许多不同的细胞类型。由于这种复杂性，对基底膜的全面了解需要在活的有机体中进行检查，直到最近，这在实验上是不可行的。在这个建议中，我们将利用我们的能力，在一个活的有机体中，对基底膜的发育和修复进行实时成像。果蝇（Drosophila melanogaster）正成为一种广泛使用的模型系统来了解基底膜，因为这种动物具有与人类相同的细胞外基质蛋白。此外，我们实验室的初步数据显示，胚胎发生过程中的基底膜形成可以在动物发育过程中实时成像。在本提案中，我们将利用我们对基底膜发育的实时成像能力，以及我们在果蝇中敲除几乎任何感兴趣的基因的能力，全面剖析基底膜形成和修复的机制。在第一个目标中，我们将用延时显微镜描述基底膜形成的时间过程。随后，我们将使用特定策略去除假设的基底膜成分和在胚胎中被认为参与基底膜发育的各种细胞中形成基底膜所需的因素。这一分析将使我们能够突出基底膜形成背后的分子机制和不同细胞在其生产中的相对需求。在随后的目的中，我们将检查基底膜修复反应。实验室的初步数据显示，激光消融可特异性损伤苍蝇的上皮和基底膜；这导致了愈合反应，在几个小时的过程中，基底膜孔被密封。我们将描述这种反应，并确定参与基底膜修复的细胞和分子机制。我们的分析表明，基底膜愈合是一个活跃的细胞过程，需要募集苍蝇巨噬细胞（果蝇炎症细胞），我们将直接测试巨噬细胞在修复损伤中的功能。此外，初步数据显示，苍蝇巨噬细胞直接对基底膜的损伤做出反应（而不是对上覆上皮细胞的损伤），我们将直接对其进行测试。这些数据表明，受损的基底膜可能在炎症反应中起着重要作用，这将具有广泛的临床意义。

项目成果

期刊论文数量（10）

专著数量（0）

科研奖励数量（0）

会议论文数量（0）

专利数量（0）

Extracellular matrix assembly stress drives Drosophila central nervous system morphogenesis

细胞外基质组装应激驱动果蝇中枢神经系统形态发生

DOI：
10.1101/2022.04.18.488510
发表时间：
2022
期刊：
影响因子：
0
作者：
Serna-Morales E
通讯作者：
Serna-Morales E

Convergent Insulin and TGF-ß signalling drives cancer cachexia by promoting aberrant fatbody ECM accumulation in a Drosophila tumour model

果蝇肿瘤模型中，胰岛素和 TGF-β 信号趋同通过促进异常脂肪体 ECM 积累来驱动癌症恶病质

DOI：
10.1101/2023.06.10.544444
发表时间：
2023
期刊：
影响因子：
0
作者：
Bakopoulos D
通讯作者：
Bakopoulos D

Tools Allowing Independent Visualization and Genetic Manipulation of Drosophila melanogaster Macrophages and Surrounding Tissues.

DOI：
10.1534/g3.117.300452
发表时间：
2018-03-02
期刊：
G3 (Bethesda, Md.)
影响因子：
0
作者：
Gyoergy A;Roblek M;Ratheesh A;Valoskova K;Belyaeva V;Wachner S;Matsubayashi Y;Sánchez-Sánchez BJ;Stramer B;Siekhaus DE
通讯作者：
Siekhaus DE

Macrosight: A Novel Framework to Analyze the Shape and Movement of Interacting Macrophages Using Matlab(®).