Development of pan-Expansion Microscopy to reveal mechanisms underlying epidermal differentiation

摘要 越来越多的人认识到机械信号在组织动态平衡中起着关键作用。这个项目 旨在定义通过链接器将力传递到细胞核的细胞和分子机制 核骨架和细胞骨架(LINC)复合体以皮肤为模型系统调节分化， 对细胞生物学和机械生物学中的基本机制有深入的了解。这项提议的核心是 需要能够1)分辨特定标记的核特征的成像技术(例如 基因组位置、特定的组蛋白修饰或感兴趣的蛋白质)，2)在周围核的上下文中 形态，3)分辨率接近核小体大小(~10纳米)。然而，由于没有现成的 目前可获取的显微镜方法满足了所有这些要求，迫切需要新的成像方法 允许研究人员在无偏见的纳米尺度环境中可视化感兴趣的特定结构的技术 周围的核组织。我们最近开发了泛膨胀显微镜(PAN-EXM)，它 使用标准共聚焦显微镜提供等同于EM重金属染色的光学对比度，但仍 与免疫标记等分子技术完全兼容。在这个项目中，我们将1)，验证结构 在使用PAN-EXM方案时，通过研究 DNA的完整性、基因组的折叠和核小体结构；以及2)应用泛exm来研究 LINC复合体消融影响表皮分化过程中的核重塑。对于后者，我们将测试 “表皮分化复合体”(EDC)这一专门化区域的折叠和区划 在包含60多个表皮分化基因的基因组中，在没有LINC复合体的情况下会发生变化， 并探索表皮分化中的关键染色质因子如何被招募到EDC中(或从EDC中移除 在表皮分化过程中。我们的项目将在两个方面产生重大影响：它将为 机械信号如何被翻译来改变表皮中的基因表达的根本问题， 从而为更大规模的研究计划奠定了基础。其次，泛EXM代表了一种新的工具，它将 加速研究，特别是通过向任何感兴趣的人传播在表皮中验证的方案 研究实验室，从而提供了一个急需的研究工具，使研究人员能够系统地 探讨正常表皮分化和正常皮肤组织中染色质结构和功能的关系 在疾病的背景下也是如此。

Summary There is an increasing appreciation that mechanical signals play key roles in tissue homeostasis. This project seeks to define the cellular and molecular mechanisms by which force transmission to the nucleus through Linker of Nucleoskeleton and Cytoskeleton (LINC) complexes regulates differentiation using skin as a model system, yielding insights into fundamental mechanisms in cell biology and mechanobiology. Central to this proposal is the need for an imaging technology that is capable of 1) resolving specifically labeled nuclear features (e.g. a genomic locus, specific histone modification, or proteins of interest), 2) in the context of the surrounding nuclear morphology, 3) at a resolution approaching the size of nucleosomes (~10 nanometers). However, as no readily accessible microscopy method currently meets all of these requirements, there is an urgent need for new imaging techniques that allow researchers to visualize specific structures of interest in the unbiased nanoscale context of the surrounding nuclear organization. We recently developed pan-Expansion Microcopy (pan-ExM), which provides optical contrast equivalent to EM heavy-metal stains using standard confocal microscopy but remains fully compatible with molecular techniques such as immunolabeling. In this project, we will 1), validate structural preservation of the expanded nucleus in the epidermis when using the pan-ExM protocol by investigating the integrity of the DNA, folding of the genome and nucleosome structure; and 2), apply pan-ExM to investigate how LINC complex ablation impacts nuclear remodeling during epidermal differentiation. For the latter, we will test how the folding and compartmentalization of the “epidermal differentiation complex” (EDC), a specialized region of the genome housing over 60 epidermal differentiation genes, are altered in the absence of LINC complexes, and explore how key chromatin factors in epidermal differentiation are recruited to (or removed from) the EDC during epidermal differentiation. Our project will have high impact in two aspects: it will provide answers to the fundamental question of how mechanical cues can be translated to alter gene expression in the epidermis, thereby laying the groundwork for a larger research program. Second, pan-ExM represents a novel tool that will accelerate research, particularly by dissemination of protocols validated in the epidermis to any interested research lab, thereby providing a much-needed research tool that will allow researchers to systematically investigate the connection between chromatin structure and function both in normal epidermal differentiation and also in disease contexts.