Investigating the Role of Novel Nuclear Envelope-Associated Plant Proteins in Nuclear Dynamics

研究新型核膜相关植物蛋白在核动力学中的作用

• 批准号: 0079577
• 负责人: Iris Meier
• 金额: --
• 依托单位: Ohio State University Research Foundation -DO NOT USE
• 依托单位国家: 美国
• 项目类别: Continuing grant
• 财政年份: 2001
• 资助国家: 美国
• 起止时间: 2001-03-15 至 2005-02-28
• 项目状态: 已结题
• 来源: https://www.nsf.gov/awardsearch/showAward?AWD_ID=0079577&HistoricalAwards=false
• 关键词: Investigating; Role; Novel; Nuclear; Envelope

ABSTRACTA striking feature of the nucleus of multicellular eukaryotes is its ability to completely disassemble and re-assemble during the course of cell division. This process may allow higher organisms to have a structurally highly complex nuclear interior during interphase, but not have such structures interfere with the condensation and partitioning of chromosomes during cell division. Such nuclear dynamics require the regulated reversibility of interactions between chromatin and proteins of the nuclear envelope and probably involve dynamic changes of nucleoskeletal structures. Since plants and animals are most likely derived from different unicellular ancestors that underwent intranuclear mitosis, one expects that nuclear assembly and disassembly have evolved twice. Presently, their molecular mechanisms are poorly understood in any system.The long-term goal of this research is to understand the mechanism, the function in interphase, and the dynamics during cell cycle of chromatin-nuclear envelope interactions in plants. Two novel, interacting plant proteins (MFP 1 and MAF 1) have been identified that are associated both with a filamentous nuclear framework called the nuclear matrix and with the nuclear envelope. MFP 1 is a plant-specific, filament-like protein, which binds specifically to matrix attachment regions (MARs), specific DNA segments which have been proposed to be involved in chromatin organization. In vitro, its MAR-binding activity is regulated by phosphorylation. The hypothesis is that MFP1 is involved in attaching chromatin to the plant interphase nuclear envelope and that MAF 1 is involved in this process through interaction with MFP 1.The specific questions asked in this project are:1) How does the localization of MFP 1 and MAF 1 change during cell cycle? The strategy employs dual-color imaging of co-expressed MFP 1 and MAF 1 fusion proteins with GFP spectral variants in synchronized cell cultures. The results will show whether the localization of the two proteins functionally correlates with nuclear envelope dynamics.2) How does the depletion of MFP 1 and MAF 1 alter nuclear, cell, and plant phenotypes? To this end, insertional knock-out mutations of MFP 1 and MAF 1 will be identified in Arabidopsis. They are expected to cause informative cellular and possibly developmental phenotypes, which will allow conclusions about the in vivo function of the proteins.3) Is phosphorylation of MFP1 involved in reversible regulation of DNA-binding during cell cycle? The pattern of phosphorylated MFP1 isoforms and their MAR-binding activity will be compared at different cell-cycle stages. The effect of overexpressing a constitutively DNA-binding MFP 1 isoform on cellular and plant phenotypes will be investigated. These data will indicate whether reversible MFP1 phosphorylation is a means to regulate the interaction of chromatin with the nuclear envelope.Together, the answers to these questions will allow the evaluation of the role of MFP 1 and MAF 1 in chromatin-nuclear envelope interactions in plants. Because of the possibility that nuclear dissociation and re-association have evolved twice in plants and animals, any molecular information about the plant nucleus is likely to add novel concepts to our understanding of a process which is currently enigmatic in any organism. In addition, the discovery of plant-specific aspects of cellular organization could lead to the development of new classes of broad-spectrum herbicides.

摘要多细胞真核生物细胞核的一个显著特征是在细胞分裂过程中能够完全解体和重新组装。这一过程可能允许高等生物在间期期间具有结构高度复杂的核内部，但这种结构不会干扰细胞分裂期间染色体的浓缩和分配。这样的核动力学需要调控的可逆性之间的相互作用的染色质和蛋白质的核膜，并可能涉及核骨架结构的动态变化。由于植物和动物很可能来自不同的单细胞祖先，这些祖先经历了核内有丝分裂，因此人们认为核组装和拆卸已经进化了两次。目前，它们在任何系统中的分子机制都不清楚，本研究的长期目标是了解植物染色质与核膜相互作用的机制、在细胞分裂间期的功能以及在细胞周期中的动态变化。已经鉴定出两种新的相互作用的植物蛋白质（MFP 1和MAF 1），它们与称为核基质的丝状核框架和核膜都相关。MFP 1是一种植物特异性的、类似于蛋白质的蛋白质，其特异性地结合到基质附着区（MAR），已经提出参与染色质组织的特异性DNA片段。在体外，其MAR结合活性受磷酸化调节。本研究假设MFP 1参与了染色质附着在植物间期核膜上的过程，而MAF 1通过与MFP 1相互作用参与了这一过程。本研究的具体问题是：1）MFP 1和MAF 1在细胞周期中的定位是如何变化的？该策略采用在同步细胞培养物中共表达的MFP 1和MAF 1融合蛋白与GFP光谱变体的双色成像。结果将显示这两种蛋白质的定位是否与核膜动力学功能相关。2）MFP 1和MAF 1的缺失如何改变核、细胞和植物表型？为此，将在拟南芥中鉴定MFP 1和MAF 1的插入敲除突变。预计它们会引起信息丰富的细胞表型和可能的发育表型，这将有助于得出有关蛋白质体内功能的结论。3）MFP 1的磷酸化是否参与细胞周期期间DNA结合的可逆调节？在不同的细胞周期阶段比较磷酸化MFP 1亚型的模式及其MAR结合活性。将研究过表达组成型DNA结合MFP 1同种型对细胞和植物表型的影响。这些数据将表明可逆的MFP 1磷酸化是否是调节染色质与核膜相互作用的一种手段，这些问题的答案将允许评估MFP 1和MAF 1在植物染色质-核膜相互作用中的作用。由于植物和动物的核解离和再结合可能经历了两次进化，因此任何关于植物核的分子信息都可能为我们理解目前在任何生物体中都是谜一样的过程增添新的概念。此外，发现植物特有的细胞组织方面可能导致开发新类别的广谱除草剂。