Mechanisms of Steady-State Nuclear Size Regulation in Xenopus

描述（由申请人提供）：细胞大小在不同的细胞类型和生物体之间差异很大，特别是在早期发育的还原性分裂期间。一个基本的问题是，细胞器的大小是如何相对于细胞大小进行适当调节的。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大小缩放。在癌症和其他疾病状态中，核和细胞大小之间的正常关系经常被取消，然而调节核大小的机制在很大程度上是未知的，可能直接导致癌症的进展。人们对核的稳态大小是如何确定的知之甚少。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。本应用程序的目的是确定调节核扩张和收缩的分子机制，并证明这些机制如何控制体内核的大小。核心假设是，稳定状态的核规模是由平衡的核增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)确定调节核扩张的机制：两种不同大小的爪蟾卵提取物重建的细胞核表现出不同的核生长速率。通过这些提取物的生化表征和显微镜，将证明核进口货物对核扩张的种间差异的贡献。2)确定细胞核收缩的调节机制：早期爪蟾胚胎的细胞和细胞核比后期胚胎大，从早期胚胎分离的大细胞核在后期胚胎的细胞提取物中孵育后变小。实时延时显微镜将用于表征这种新活动的动态和生化方法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素是已知的，其他因素将在目标1和目标2中确定。将通过mRNA微注射控制爪蟾胚胎的核缩放活动，并通过活细胞显微镜检查对体内核大小和动力学的影响。核比例因子也将在出芽酵母中进行遗传调节，以确定其功能是否保守。预期的结果是阐明核大小的控制机制，为验证核大小调节核组织和功能的新假设提供基础。细胞器结垢对细胞平衡至关重要，但维持细胞大小比例的机制在很大程度上是未知的。因此，这项研究将显著影响我们对生物发生和生长过程中如何调节结垢的理解。

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.