Investigating the Mechanical Loading of Desmosomes

研究桥粒的机械负载

• 批准号: 273412230
• 负责人: Professor Dr. Carsten Grashoff
• 金额: --
• 依托单位: Institut für Integrative Zellbiologie und Physiologie (IIZP)
• 依托单位国家: 德国
• 项目类别: Priority Programmes
• 财政年份: 2015
• 资助国家: 德国
• 起止时间: 2014-12-31 至 2022-12-31
• 项目状态: 已结题
• 来源: https://gepris.dfg.de/gepris/projekt/273412230?language=en
• 关键词: Investigating; Mechanical; Loading; Desmosomes

Desmosomes are intercellular adhesion complexes that connect the intermediate filament cytoskeletons of neighboring cells and are essential for the mechanical integrity of mammalian tissues. Mutations in desmosomal proteins cause severe human pathologies including epithelial blistering and heart muscle dysfunction, thus current models assume a central role of desmosomes in transmitting mechanical force between cells. However, direct evidence for the load-bearing nature of these macromolecular structures is still lacking.In the previous funding period, we therefore focused our efforts on the development and analysis of Förster resonance energy transfer (FRET)-based tension sensors to measure the piconewton-scale forces experienced by desmoplakin, an obligate desmosomal protein that links the junctional desmosomal plaque to the intermediate filament cytoskeleton. Our experiments showed that our desmoplakin tension sensors are functional in cells, and quantitative live cell FRET analyses revealed that desmoplakin does not experience significant tension under homeostatic conditions. However, our experiments demonstrate that desmoplakin becomes mechanically loaded in response to external mechanical stresses. The stress-induced loading of desmoplakin is force-specific, transient and sensitive to the magnitude and orientation of applied tissue deformation. Our data indicate that desmosomes fulfill a distinct mechanical function than previously analyzed cell adhesion complexes and seem to act as molecular stress absorbers in epithelial tissues. Here, we propose to use our newly developed technologies to elucidate the molecular mechanisms governing force transduction through desmoplakin, and by extension, the desmosome. We will test how previously described post-translational desmoplakin modifications affect desmosome loading, and to which extend different keratin variants and plakophilins isoforms modulate desmosome force propagation. Finally, we will use our recently developed tension sensor multiplexing approach to investigate the mechanical loading of desmosomes and adherens junctions simultaneously. Altogether, the expected results should help explain how the distinct intercellular junctions allow the construction of epithelia that are both dynamic and physically robust, two seemingly contradictory properties that are nonetheless essential for mammalian life.

桥粒是连接相邻细胞中间丝细胞骨架的细胞间黏附复合体，对哺乳动物组织的机械完整性是必不可少的。桥粒蛋白的突变会导致严重的人类疾病，包括上皮起泡和心肌功能障碍，因此目前的模型承担了桥粒在细胞间传递机械力的中心作用。然而，对于这些大分子结构的承载性质的直接证据仍然缺乏。因此，在之前的资助期间，我们集中精力开发和分析基于Förster共振能量转移(FRET)的张力传感器，以测量桥粒蛋白所经历的皮牛顿尺度的力，桥粒蛋白是一种专有的桥粒蛋白，连接连接桥粒斑块和中间细丝细胞骨架。我们的实验表明，我们的桥粒蛋白张力传感器在细胞中起作用，定量的活细胞FRET分析显示，在稳态条件下，桥粒蛋白没有经历显著的张力。然而，我们的实验表明，桥粒蛋白在外界机械应力的作用下成为机械负荷。应力诱导的桥粒蛋白载荷是力特异性的，是瞬时的，对所施加的组织变形的大小和方向很敏感。我们的数据表明，桥粒履行了与先前分析的细胞黏附复合体不同的机械功能，并且似乎在上皮组织中扮演着分子应力吸收者的角色。在这里，我们建议使用我们最新开发的技术来阐明通过桥粒蛋白以及延伸到桥粒的力传递的分子机制。我们将测试前面描述的翻译后桥粒修饰如何影响桥粒负载，以及不同的角蛋白变体和蛋白异构体对桥粒力量传播的调节作用。最后，我们将使用我们最近开发的张力传感器多路复用方法来同时研究桥粒和粘连连接的机械载荷。总之，预期的结果应该有助于解释不同的细胞间连接如何允许构建既动态又健康的上皮细胞，这两个看似矛盾的特性对哺乳动物的生命来说仍然是必不可少的。