Mechanisms of nuclear size regulation

项目摘要：细胞器大小控制是一个基本的细胞生物学问题，而细胞核大小往往是 以一种与倍体无关的方式在癌细胞中不适当地放大，这是病理学家在 癌症诊断和分期。目前尚不清楚癌症中的核大小变化是导致还是导致 这种疾病是由于我们对调节核大小的机制的了解不足所致。我的实验室地址 使用生物化学易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哇胚胎，以便进行体内功能测试。(1)什么 控制核大小的机制？我实验室的最新进展揭示了核进口和核 板层有助于调节核的大小。以补充识别核的候选方法 大小效应器，进行基于成像的RNAi筛选。此屏幕的结果将用于解剖 利用非洲爪哇卵提取液和胚胎控制核大小的新机制，专注于丰富的HITS 在屏幕上：核结构蛋白，组蛋白和DNA甲基化的调节器，以及囊泡运输 蛋白质。(2)胞质体积如何影响核的大小？使用基于微流控的技术 将非洲爪哇提取物包裹在定义的大小和形状的液滴中，我的实验室最近证明了限制 大量的组蛋白伴侣有助于核大小的发育调节。(3)什么是 推动核增长的物理力量？已确定染色质结构的多个调节因子为 核大小效应器，我们假设施加在核包层上的核内推力允许 使蛋白质进入核层，从而促进核生长。使用各种体外培养技术 方法，我们将测试染色质结构和核功能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张。(四)阐述 微流控抽提物包封法，我们将介绍f-肌动蛋白、自然细胞周期和修饰 液滴皮质。这种自下而上的方法生成具有日益复杂和本机的合成细胞 属性将允许我们在大小控制、细胞骨架组织和 细胞周期计时。(5)核的大小在发育和分化过程中是如何调节的？为了扩展我们的工作 关于非洲爪哇向哺乳动物细胞的发育，我们已经启动了人类诱导的多能干细胞的研究 细胞(IPSCs)。我们发现，在IPSC过程中，核的形态和层蛋白动力学发生了显著的变化 分化，我们将利用从非洲爪哇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的富有成效的合作的支持，这些合作采用了不同的跨学科 包括高分辨率显微镜、RNAi筛选、微流体、蛋白质组学和RNA等技术 测序。最终，从这项工作中获得的机械性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。

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.