Investigating the Mechanical Loading of Desmosomes

桥粒是细胞间粘附复合物，其连接相邻细胞的中间丝细胞骨架，并且对于哺乳动物组织的机械完整性是必不可少的。桥粒蛋白质的突变导致严重的人类病理学，包括上皮起泡和心肌功能障碍，因此目前的模型假定桥粒在细胞之间传递机械力中起核心作用。然而，这些大分子结构的承载性质的直接证据仍然缺乏，因此在上一个资助期，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，以测量桥粒斑蛋白（一种将连接桥粒斑连接到中间丝细胞骨架的专性桥粒蛋白）所经历的皮牛顿级力。我们的实验表明，我们的桥粒斑蛋白张力传感器在细胞中是功能性的，并且定量活细胞FRET分析显示，桥粒斑蛋白在稳态条件下不经历显著的张力。然而，我们的实验表明，桥粒斑蛋白成为机械负荷响应外部机械应力。桥粒斑蛋白的应力诱导负荷是力特异性的、瞬时的，并且对施加的组织变形的大小和方向敏感。我们的数据表明，桥粒履行一个独特的机械功能比以前分析的细胞粘附复合物，似乎作为分子应力吸收上皮组织。在这里，我们建议使用我们新开发的技术来阐明通过桥粒斑蛋白，并通过扩展，桥粒力转导的分子机制。我们将测试先前描述的翻译后桥粒斑蛋白修饰如何影响桥粒负载，以及不同的角蛋白变体和斑嗜蛋白亚型在何种程度上调节桥粒力传播。最后，我们将使用我们最近开发的张力传感器多路复用的方法，同时研究桥粒和粘附连接的机械负荷。总而言之，预期的结果应该有助于解释不同的细胞间连接如何允许上皮细胞的建设，这是动态的和物理上的强大，两个看似矛盾的属性，但对哺乳动物的生活是必不可少的。

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.