Mechanisms of Steady-State Nuclear Size Regulation in Xenopus

非洲爪蟾稳态核尺寸调节机制

基本信息

批准号：
8496932
负责人：
Daniel Leon Levy
金额：
$ 31.06万
依托单位：
UNIVERSITY OF WYOMING
依托单位国家：
美国
项目类别：
财政年份：
2013
资助国家：
美国
起止时间：
2013-09-01 至 2017-08-31
项目状态：
已结题

项目摘要

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.

描述（由申请人提供）：细胞大小在不同的细胞类型和生物之间，尤其是在表征早期发育的还原分裂期间。一个基本问题是，相对于细胞大小，细胞器的大小如何受到适当调节。核是一个细胞器，在发育过程和物种之间表现出精致的尺寸尺寸。在癌症和其他疾病状态中，核与细胞大小之间的正常关系通常被废除，但是调节核大小的机制在很大程度上尚不清楚，并且可能直接导致癌症进展。确定稳态核大小的确定程度很低。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，核功能和亚核组织。该应用的目的是确定调节核扩展和收缩的分子机制，并证明这些机制如何控制体内的核大小。中心假设是稳态核大小取决于平衡的核增长和缩小活性，这将通过追求以下三个特定目标来测试。 1）确定调节核扩张的机制：在两个不同大小的爪蟾物种的卵提取物中重构的核表现出差异的核生长速率。通过这些提取物和显微镜的生化表征，将证明核进口碳对核扩展种间差异的贡献。 2）确定调节核收缩的机制：早期的异爪蟾胚胎含有比后期胚胎更大的细胞和核，并且从早期胚胎中分离出的大核时，当从晚期胚胎中孵育在细胞提取物中时，会变小。实时的时光显微镜将用于表征这种新活性的动力学，生化方法将确定负责核收缩的因素。 3）证明核缩放因素的体内活性：某些控制核大小的因素是已知的，而其他因素将在目标1和2中确定。将通过mRNA微注射和对核大小和动力学的影响在Xenopus胚胎中操纵核缩放活性体内将通过活细胞显微镜检查。核缩放因子也将在萌芽的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控制机制，为测试核大小调节核组织和功能的新假设提供了基础。细胞器缩放对于细胞平衡至关重要，但是在细胞中保持大小比的机制在很大程度上未知。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缩放的理解。