Mechanisms of nuclear size regulation

核大小调节机制

• 批准号: 10319561
• 负责人: Daniel Leon Levy
• 金额: $ 34.79万
• 依托单位: UNIVERSITY OF WYOMING
• 依托单位国家: 美国
• 财政年份: 2020
• 资助国家: 美国
• 起止时间: 2020-01-01 至 2024-12-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10319561
• 关键词: Address; Affect; Biochemical; Biological; Cell Cycle; Cell Differentiation process; Cells; Chromatin Structure; Collaborations; Complement; Complex; Cytoskeletal Modeling; DNA Methylation; Development; Diagnosis; Diagnostic Neoplasm Staging; Disease; Embryo; Embryonic Development; Encapsulated; F-Actin; Growth; Histones; Homeostasis; Human; Image; In Vitro; Knowledge; Lamins; Light; Malignant Neoplasms; Mammalian Cell; Microfluidics; Microscopy; Modification; Molecular Chaperones; Morphology; Nuclear; Nuclear Envelope; Nuclear Import; Nuclear Lamin; Nuclear Lamina; Organelles; Pathologist; Ploidies; Proteins; Proteomics; RNA interference screen; Regulation; Resolution; Screening Result; Shapes; Structural Protein; System; Techniques; Technology; Testing; Therapeutic; Vesicular Transport Proteins; Work; Xenopus; base; cancer cell; cancer diagnosis; cancer therapy; carcinogenesis; egg; experimental study; histone methylation; in vivo; induced pluripotent stem cell; novel; novel strategies; reconstitution; stem cell differentiation; transcriptome sequencing; xenopus development

Project Summary: Organelle size control is a fundamental cell biological problem, and nuclear size is often inappropriately enlarged in cancer cells in a ploidy-independent manner, a change used by pathologists in cancer diagnosis and staging. It is not known if nuclear size changes in cancer are a cause or consequence of disease due to a gap in our knowledge of the mechanisms that regulate nuclear size. My lab addresses fundamental questions about nuclear size regulation using biochemically tractable cytoplasmic extracts that reconstitute nuclear assembly and Xenopus embryos that allow for in vivo functional testing. (1) What mechanisms control nuclear size? Recent progress from my lab has revealed how nuclear import and nuclear lamins contribute to the regulation of nuclear size. To complement candidate approaches to identifying nuclear size effectors, an imaging-based RNAi screen was performed. Results from this screen will be used to dissect novel mechanisms of nuclear size control using Xenopus egg extracts and embryos, focusing on hits enriched in the screen: nuclear structural proteins, regulators of histone and DNA methylation, and vesicular transport proteins. (2) How does cytoplasmic volume influence nuclear size? Using microfluidic-based technologies to encapsulate Xenopus extract in droplets of defined size and shape, my lab recently demonstrated that limiting amounts of a histone chaperone contribute to developmental regulation of nuclear size. (3) What are the physical forces that drive nuclear growth? Having identified multiple regulators of chromatin structure as nuclear size effectors, we hypothesize that intranuclear pushing forces applied to the nuclear envelope allow for protein incorporation into the nuclear lamina, thereby promoting nuclear growth. Using a variety of in vitro approaches, we will test the relative contributions of chromatin structure and nuclear f-actin to nuclear growth and whether intranuclear pushing forces are sufficient to drive nuclear expansion. (4) Elaborating on the microfluidic extract encapsulation approach, we will introduce f-actin, natural cell cycling, and modifications to the droplet cortex. This bottom-up approach to generating synthetic cells with increasingly complex and native attributes will allow us to address questions at the intersection of size control, cytoskeletal organization, and cell cycle timing. (5) How is nuclear size regulated during development and differentiation? To extend our work on Xenopus development to mammalian cells, we have initiated studies with human induced pluripotent stem cells (iPSCs). We find that nuclear morphology and lamin dynamics change significantly during iPSC differentiation, and we will investigate the underlying mechanisms using information gained from the Xenopus system. Our work is bolstered by ongoing productive collaborations that employ diverse interdisciplinary techniques including high-resolution microscopy, RNAi screening, microfluidics, proteomics, and RNA sequencing. Ultimately, the mechanistic information gained from this work will enable experiments to address how nuclear size impacts cell and nuclear function in the context of development, differentiation, and cancer.

项目摘要：细胞器大小控制是一个基本的细胞生物学问题，核大小通常是 以倍性非依赖性的方式在癌细胞中不当放大，病理学家在 癌症诊断和分期。尚不清楚癌症的核大小是否是导致的原因或结果 疾病由于我们对调节核大小的机制的了解而引起的疾病。我的实验室地址 关于核大小调节的基本问题，使用可生化的细胞质提取物，这些提取物 重建核总成和爪蟾胚胎，可进行体内功能测试。 （1）什么 机制控制核大小？我实验室的最新进展揭示了核进口和核进口 粘液素有助于调节核大小。补充候选方法来识别核 尺寸效应器，基于成像的RNAi屏幕。该屏幕的结果将用于剖析 使用爪蟾鸡蛋提取物和胚胎的核大小控制的新型机制，重点是富含命中 在屏幕上：核结构蛋白，组蛋白和DNA甲基化的调节剂以及囊泡转运 蛋白质。 （2）细胞质体积如何影响核大小？使用基于微流体的技术来 我的实验室最近证明了局限性的Xenopus提取物在定义的大小和形状的液滴中 组蛋白伴侣的量有助于核大小的发展调节。 （3）什么是 驱动核生长的身体力量？已经确定了染色质结构的多个调节剂 核尺寸效应子，我们假设应用于核包膜的核内推力允许 将蛋白质掺入核薄片中，从而促进核生长。使用各种体外 方法是，我们将测试染色质结构和核F-肌动蛋白对核生长的相对贡献 以及核内推力是否足以驱动核扩展。 （4）详细说明 微流体提取物封装方法，我们将引入F-肌动蛋白，天然细胞循环和修饰 液滴皮质。这种自下而上的方法是生成越来越复杂和天然的合成细胞 属性将使我们能够在大小控制，细胞骨架组织和 细胞周期时机。 （5）在开发和分化过程中如何调节核大小？扩展我们的工作 关于对哺乳动物细胞的爪蟾发育，我们已经开始对人类诱导的多能茎进行研究 细胞（IPSC）。我们发现在IPSC期间，核形态和层粘连动力学发生了显着变化 差异化，我们将使用从爪蟾中获得的信息研究基本机制 系统。我们的工作得到了持续的生产合作的支持，这些合作采用多样化的跨学科 包括高分辨率显微镜，RNAi筛选，微流体，蛋白质组学和RNA在内的技术 测序。最终，从这项工作中获得的机械信息将使实验能够解决 在发育，分化和癌症的背景下，核大小如何影响细胞和核功能。