Measurement of Mechanical Tension Across Desmosomes

 描述（由申请人提供）：皮肤被认为是人体最大的器官，其主要功能是充当生物体与环境之间的屏障。由粘附连接、紧密连接和桥粒形成的强细胞-细胞连接对于表皮的完整性及其抵抗机械应力的能力至关重要。桥粒靶向的遗传、自身免疫和感染性疾病在临床上存在于皮肤和心脏这两个器官中，这两个器官的组织受到显著的机械力，这表明桥粒的主要功能是抵抗机械应力。虽然已经显示桥粒和角蛋白的表达对于皮肤的机械完整性是关键的，但是尚不知道桥粒主要作为机械分子还是信号分子。由于施加于桥粒的机械力从未被直接测量，因此也不知道强化桥粒的疗法是否是治疗皮肤起泡和伤口愈合的成功策略。该提议的中心假设是桥粒受到角蛋白细胞骨架施加的张力，并且张力水平在伤口愈合和皮肤疾病中改变。这个项目的主要创新是使用我的实验室开发的一种新技术，通过使用基于桥粒芯糖蛋白-2和-3 FRET的张力传感器直接测量桥粒张力。这种新的方法将提供重要的洞察力桥粒力的大小和调节，IF细胞骨架的能力，以传输和施加机械力，以及桥粒力在皮肤生理和病理的作用。在目标1中，将检查桥粒形成过程中桥粒张力的动力学，并鉴定桥粒张力的调节剂。此外，将在经历循环拉伸的细胞中测量桥粒张力的变化。在目标2中，将在伤口愈合过程中以及在上皮向间充质转化中测量桥粒张力。在目标3中，将使用寻常型天疱疮和单纯大疱性表皮病的体外模型来确定皮肤起泡疾病中桥粒张力是否改变。我们将讨论在理解组织的作用方面的一个重大差距， 在由桥粒或角蛋白IF突变或自身免疫引起的皮肤起泡和创伤性疾病中的优势。也就是说，我们将解决如果桥粒的主要作用是机械或信号。这些基本的机械生物学研究将提供重要的洞察力的机械力的动力学桥粒在正常的稳态和疾病的条件下，并确定新的治疗目标伤口愈合和桥粒相关的皮肤病理学是必不可少的。此外，桥粒在力传递中的作用也可以提供为什么桥粒相关疾病也经常表现为心肌病的机制性见解。最后，我们对IF传递机械力的能力缺乏了解。因此，这些研究将从根本上影响对细胞生物力学的理解。

 DESCRIPTION (provided by applicant): Considered to be the largest organ of the body, the primary function of skin is to act as a barrier between the organism and the environment. Strong cell-cell junctions, formed by adherens junctions, tight junctions, and desmosomes, are critical to the integrity of the epidermis and its ability to resist mechanical stress. Desmosome-targeting genetic, autoimmune, and infectious diseases present clinically in both the skin and heart, two organs with tissues subjected to significant mechanical forces, which suggest that a major function of desmosomes is to resist mechanical stress. While it has been shown that expression of desmosomal and keratin proteins are critical to the mechanical integrity of skin, it is not known if desmosomes act primarily as mechanical or signaling molecules. Because the mechanical force applied to desmosomes has never been directly measured it is also not known if therapies that strengthen desmosomes would be a successful strategy for treatment of skin-blistering and wound healing. The central hypotheses of this proposal is that desmosomes are subject to tensile forces applied by the keratin cytoskeleton, and that the level of tension is altered in wound healing and skin diseases. The major innovation in this project is the use of a new technique developed by my lab to directly measure desmosome tension through the use of desmoglein-2 and -3 FRET- based tension sensors. This novel approach will provide significant insight into the magnitude and regulation of desmosome forces, the ability of the IF cytoskeleton to transmit and apply mechanical force, and the role of desmosome forces in skin physiology and pathology. In Aim 1, the dynamics of desmosome tension will be examined during the process of desmosome formation, and regulators of desmosome tension will be identified. Additionally, changes in desmosome tension will be measured in cells subjected to cyclic stretch. In Aim 2, desmosome tension will be measured during the process of wound healing and also in epithelial to mesenchymal transition. In Aim 3, in vitro models of pemphigus vulgaris and epidermolysis bullosa simplex will be used to determine if desmosome tension is altered in skin blistering diseases. We will address a significant gap in the understanding the role of tissue strength in diseases with skin blistering and wounding diseases caused by mutations in or autoimmunity to desmosomes or keratin IFs. Namely, we will address if the principal role of desmosomes is mechanical or signaling. These basic mechanobiology studies will provide significant insight into the dynamics of mechanical forces across desmosomes under conditions of normal homeostasis and disease, and are essential to the identification of new therapeutic targets for wound healing and desmosome-related skin pathologies. Additionally, the role of desmosomes in force transmission may also provide mechanistic insight into why desmosome-associated diseases also frequently present as cardiomyopathies. Lastly, our understanding of the ability of IF to transmit mechanical forces is lacking. As a result, these studies will fundamentally impact the understanding of cellular biomechanics.