Mechanisms of nuclear size regulation

项目概述：细胞器的大小控制是一个基本的细胞生物学问题，核的大小往往是 在癌细胞中以不依赖于倍性的方式不适当地扩大，病理学家使用这种变化， 癌症诊断和分期。目前尚不清楚癌症中细胞核大小的变化是癌症的原因还是结果。 疾病，因为我们对调节核大小的机制的知识存在差距。我的实验室地址 关于使用生化易处理的细胞质提取物调节核大小的基本问题， 重组核组件和非洲爪蟾胚胎，允许体内功能测试。(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武器 核纤层蛋白有助于调节核大小。补充查明核武器的备选办法， 大小效应物，进行基于成像的RNAi筛选。该屏幕的结果将用于解剖 使用非洲爪蟾卵提取物和胚胎控制核大小的新机制，重点关注富集的命中 在屏幕上：核结构蛋白，组蛋白和DNA甲基化的调节剂，和囊泡运输 proteins. (2)细胞质体积如何影响细胞核大小？利用微流体技术， 将非洲爪蟾提取物封装在确定大小和形状的液滴中，我的实验室最近证明， 组蛋白伴侣蛋白的量有助于核大小的发育调节。(3)有哪些 推动核增长的物理力量在鉴定了多种染色质结构调节因子后， 核大小效应，我们假设，核内推力施加到核膜允许 用于蛋白质掺入核纤层，从而促进核生长。使用多种体外 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 以及核内推力是否足以驱动核扩张。(4)详细阐述 微流控提取物包封方法，我们将介绍f-肌动蛋白，自然细胞周期，和修改， 液滴皮层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在大小控制，细胞骨架组织， 细胞周期定时(5)在发育和分化过程中，细胞核的大小是如何调节的？为了扩展我们的工作 在非洲爪蟾发育为哺乳动物细胞方面，我们已经开始了人类诱导多能干细胞的研究。 细胞（iPSC）。我们发现，核形态和核纤层蛋白动力学在iPSC过程中发生了显著变化， 分化，我们将调查的基本机制，利用信息从非洲爪蟾 系统我们的工作得到了持续的富有成效的合作的支持， 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械信息将使实验能够解决 核大小如何影响细胞和核功能在发展，分化和癌症的背景下。

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.