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 癌细胞以倍性无关的方式不适当地放大，这是病理学家在 癌症诊断和分期。目前尚不清楚癌症中核大小的变化是癌症的原因还是结果 由于我们对调节核大小的机制的了解存在差距而导致疾病。我的实验室地址 关于使用生化可处理的细胞质提取物调节核大小的基本问题 重建核组装和非洲爪蟾胚胎，以便进行体内功能测试。 (1) 什么 控制核大小的机制？我的实验室的最新进展揭示了核进口和核 核纤层蛋白有助于调节核大小。补充识别核的候选方法 尺寸效应器，进行了基于成像的 RNAi 筛选。此屏幕的结果将用于剖析 使用非洲爪蟾卵提取物和胚胎控制核大小的新机制，重点是富集的命中 屏幕中：核结构蛋白、组蛋白和 DNA 甲基化调节剂以及囊泡运输 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 将非洲爪蟾提取物封装在指定大小和形状的液滴中，我的实验室最近证明，限制 组蛋白伴侣的数量有助于核大小的发育调节。 (3) 哪些是 推动核增长的物理力量？确定了染色质结构的多个调节因子 核尺寸效应器，我们假设施加到核膜的核内推力允许 用于蛋白质掺入核层，从而促进核生长。利用多种体外 方法，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 以及核内推动力是否足以驱动核扩张。 (4) 阐述 微流控提取物封装方法，我们将介绍 f-肌动蛋白、自然细胞循环和修饰 液滴皮质。这种自下而上的方法产生越来越复杂和天然的合成细胞 属性将使我们能够解决尺寸控制、细胞骨架组织和 细胞周期计时。 (5) 细胞核大小在发育和分化过程中是如何调节的？扩展我们的工作 关于非洲爪蟾向哺乳动物细胞的发育，我们已经启动了人类诱导多能干细胞的研究 细胞（iPSC）。我们发现 iPSC 过程中核形态和核纤层蛋白动力学发生显着变化 分化，我们将利用从非洲爪蟾获得的信息来研究潜在的机制 系统。我们的工作得到了持续富有成效的合作的支持，这些合作采用了不同的跨学科 技术包括高分辨率显微镜、RNAi 筛选、微流体、蛋白质组学和 RNA 测序。最终，从这项工作中获得的机制信息将使实验能够解决 核大小如何在发育、分化和癌症的背景下影响细胞和核功能。