Mechanisms of Steady-State Nuclear Size Regulation in Xenopus

描述（由申请人提供）：细胞大小在不同细胞类型和生物体之间差异很大，尤其是在表征早期发育的还原分裂期间。一个基本的问题是细胞器的大小如何相对于细胞大小进行适当的调节。细胞核是一种细胞器，在发育过程中和物种之间都表现出精致的尺寸缩放。核和细胞大小之间的正常关系在癌症和其他疾病状态中通常被废除，但调节核大小的机制在很大程度上是未知的，可能直接导致癌症进展。如何确定稳定状态的核大小是知之甚少。这种知识差距使我们无法理解核大小如何影响染色质组织，基因表达和细胞功能。长期目标是阐明核大小调节的机制，以了解核大小如何影响细胞和核功能以及亚核组织。本申请的目的是确定调节核膨胀和收缩的分子机制，并证明这些机制如何在体内控制核大小。核心假设是，稳态核大小是由平衡的核生长和收缩活动决定的，这将通过追求以下三个具体目标来检验。1)确定机制，调节核扩张：细胞核重建卵提取物从两个不同大小的非洲爪蟾蛙物种表现出不同的核生长速率。通过这些提取物和显微镜的生化特性，核输入货物的核扩张种间差异的贡献将被证明。2)识别调节核收缩的机制：早期非洲爪蟾胚胎含有比晚期胚胎更大的细胞和核，从早期胚胎分离的大核在晚期胚胎的细胞提取物中孵育时变得更小。实时延时显微镜将被用来表征这种新的活动和生化方法的动态将确定负责核收缩的因素。3)证明核比例因子的体内活性：一些控制核大小的因子是已知的，其他因子将在目标1和2中确定。核缩放活动将操纵非洲爪蟾胚胎的mRNA显微注射和核的大小和动力学在体内的影响将通过活细胞显微镜检查。核比例因子也将在芽殖酵母中进行遗传调节，以确定它们的功能是否保守。预期的结果是阐明核大小的控制机制，提供基础来测试新的假设，核大小调节核的组织和功能。细胞器缩放对于细胞平衡是必不可少的，然而维持细胞中大小比例的机制在很大程度上是未知的。因此，这项研究将显着影响我们对生物发生和生长过程中如何调节规模的理解。

DESCRIPTION (provided by applicant): Cell size varies greatly among different cell types and organisms, and especially during the reductive divisions that characterize early development. A fundamental question is how organelle size is appropriately regulated relative to cell size. The nucleus is one organelle that exhibits exquisite size scaling both during development and between species. The normal relationship between nuclear and cell size is often abrogated in cancers and other disease states, yet mechanisms that regulate nuclear size are largely unknown and may directly contribute to cancer progression. How steady-state nuclear size is determined is poorly understood. This knowledge gap prevents us from understanding how nuclear size impacts chromatin organization, gene expression, and cell function. The long-term goal is to elucidate mechanisms of nuclear size regulation to understand how nuclear size impacts cell and nuclear function and sub-nuclear organization. The objective of this application is to identify the molecular mechanisms that regulate nuclear expansion and shrinking and to demonstrate how these mechanisms control nuclear size in vivo. The central hypothesis is that steady-state nuclear size is determined by balanced nuclear growth and shrinking activities, which will be tested by pursuing the following three specific aims. 1) Identify mechanisms that regulate nuclear expansion: Nuclei reconstituted in egg extracts from two different size Xenopus frog species exhibit differential nuclear growth rates. Through biochemical characterization of these extracts and microscopy, the contribution of nuclear import cargos to interspecies differences in nuclear expansion will be demonstrated. 2) Identify mechanisms that regulate nuclear shrinking: Early stage Xenopus embryos contain larger cells and nuclei than later stage embryos, and large nuclei isolated from early stage embryos become smaller when incubated in cell extract from late stage embryos. Live time-lapse microscopy will be used to characterize the dynamics of this novel activity and biochemical approaches will identify factors responsible for nuclear shrinking. 3) Demonstrate the in vivo activities of nuclear scaling factors: Some factors that control nuclear size are known and others will be identified in Aims 1 and 2. Nuclear scaling activities will be manipulated in Xenopus embryos by mRNA microinjection and effects on nuclear size and dynamics in vivo will be examined by live cell microscopy. Nuclear scaling factors will also be genetically modulated in budding yeast to determine if their function is conserved. The expected outcome is elucidation of nuclear size control mechanisms, providing the foundation to test the novel hypothesis that nuclear size regulates nuclear organization and function. Organellar scaling is essential to cellular balance, yet mechanisms that maintain size ratios in a cell are largely unknown. This research will thus significantly impact our understanding of how scaling is regulated during biogenesis and growth.