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

开发泛膨胀显微镜以揭示表皮分化的机制

• 批准号: 10539999
• 负责人: Joerg Bewersdorf
• 金额: $ 22.11万
• 依托单位: YALE UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2022
• 资助国家: 美国
• 起止时间: 2022-07-15 至 2024-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10539999
• 关键词: Ablation; Address; Benchmarking; Biological; Biological Models; Biology; Cell Adhesion; Cell Fate Control; Cell Line; Cell Nucleus; Cell-Matrix Junction; Cells; Cellular biology; Characteristics; Chromatin; Chromatin Structure; Complex; Confocal Microscopy; Cues; Cytoskeleton; DNA; DNA Folding; Data; Development; Differentiated Gene; Dimensions; Disease; Electron Microscopy; Environment; Epidermis; Epigenetic Process; Fluorescence Microscopy; Fluorescent in Situ Hybridization; Gene Expression; Genes; Genetic Transcription; Genome; Health; Heavy Metals; Heterochromatin; Histones; Homeostasis; Housing; Image; Imaging Techniques; Imaging technology; In Situ Hybridization; Label; Light; Light Microscope; Methods; Microscopy; Modeling; Modification; Molecular; Morphology; Nuclear; Nuclear Envelope; Nuclear Lamina; Nuclear Structure; Nucleic Acids; Nucleosomes; Optics; Output; Pattern; Phase; Play; Proteins; Protocols documentation; Research; Research Personnel; Resolution; Risk; Sampling; Series; Skin; Stains; Stratification; Structure; Techniques; Technology; Testing; Three-Dimensional Imaging; Tissue imaging; Tissues; Translating; Up-Regulation; Validation; Variant; genomic locus; histone modification; insight; instrumentation; interest; keratinocyte; light microscopy; mechanical force; mechanical signal; mechanotransduction; nanometer; nanoscale; new technology; novel; preservation; programs; promoter; recruit; tool; transmission process

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.

总结 越来越多的人认识到机械信号在组织稳态中起着关键作用。这个项目 试图定义力通过连接体传递到细胞核的细胞和分子机制， 核骨架和细胞骨架（LINC）复合物的调节分化使用皮肤作为模型系统， 从而深入了解细胞生物学和机械生物学的基本机制。这项建议的核心是 需要一种成像技术，其能够1）分辨特异性标记的核特征（例如， 基因组基因座、特异性组蛋白修饰或感兴趣的蛋白质），2）在周围核的背景下， 形态学，3）在接近核小体的大小（~10纳米）的分辨率。然而，由于没有现成的 一种可访问的显微镜方法目前满足所有这些要求，迫切需要新的成像 技术，使研究人员能够在无偏见的纳米级背景下可视化感兴趣的特定结构 周边的核组织。我们最近开发了泛扩展显微镜（pan-ExM）， 使用标准共聚焦显微镜提供与EM重金属染色剂相当的光学对比度， 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 当使用pan-ExM方案时，通过研究表皮中的 DNA的完整性，基因组的折叠和核小体结构;和2），应用泛ExM来研究如何 LINC复合体消融影响表皮分化期间的核重塑。对于后者，我们将测试 “表皮分化复合体”（EDC）的折叠和区室化， 包含超过60个表皮分化基因的基因组，在不存在LINC复合物的情况下被改变， 并探索表皮分化中的关键染色质因子如何被招募到EDC（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很大的影响：它将提供答案， 机械信号如何被翻译以改变表皮中的基因表达的基本问题， 从而为更大的研究计划奠定基础。其次，泛ExM代表了一种新颖的工具， 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 研究染色质结构和功能之间的联系，在正常的表皮分化， 也在疾病的背景下。