RuvBL1/2 function in mitotic chromatin decondensation

RuvBL1/2 在有丝分裂染色质解浓缩中的功能

• 批准号: 107479298
• 负责人: Professor Dr. Wolfram Antonin
• 金额: --
• 依托单位: Institut für Biochemie und Molekulare Zellbiologie
• 依托单位国家: 德国
• 项目类别: Research Grants
• 财政年份: 2009
• 资助国家: 德国
• 起止时间: 2008-12-31 至 2022-12-31
• 项目状态: 已结题
• 来源: https://gepris.dfg.de/gepris/projekt/107479298?language=en
• 关键词: RuvBL1; function; mitotic; chromatin; decondensation

The nucleus reorganizes structurally and functionally considerably during mitosis. In animal cells, the nuclear envelope breaks down at the beginning of mitosis, the chromatin massively condenses to individualized chromosomes which are captured and segregated by the spindle apparatus - processes which have been and still are intensively studied in many laboratories. Much less is known how at the end of mitosis the interphase state of the nucleus is reestablished, competent for its manifold functions. For this, the highly condensed mitotic chromosomes are decompacted, a process indispensable for reinitiating transcription and the perpetuation of genomic information and thus of central importance in the cellular life cycle. Despite its significance to basic research as well as its potential medical implications, mitotic chromatin decondensation is still ill-defined. It has been well and early described cytologically, but we are largely ignorant of the underlying molecular events and mechanisms.We have previously identified and characterized the ATPase complex RuvBL1/2 as a crucial chromatin decondensation factor. By SILAC based mass spectrometry from cells exiting mitosis we now identify eIF4A1, PKP1 and JUP as RuvBL1/2 interactors, which bind specifically during the time of chromatin decondensation. Indeed, downregulation or depletion of the proteins in cells or in biochemical assays impairs chromatin decondensation. eIF4A1 is a RNA helicase with a function in translation initiation but we propose an additional role in chromatin decondensation independent from its role in protein synthesis. PKP1 and JUP are desmosome components with an additional localization in the nucleus but their precise function in this compartment is largely unclear. Here, we will analyze how eIF4A1, PKP1 and JUP precisely function in chromatin decondensation using cellular assays including life cell imaging and biochemical methods like a cell free reconstitution assay of mitotic chromatin decondensation. We will determine whether eIF4A1, PKP1 and JUP form a single chromatin decondensation complex involving RuvBL1/2 or whether they are part of different RuvBL1/2 containing complexes mediating distinguishable aspects of chromatin decondensation. We will analyze whether the chromatin decondensation complex(es) localize to chromatin during mitotic exit similar to RuvBL1/2 and if so determine which components of the complex(es) define chromatin binding. By depleting the crucial decondensation components in tissue culture cells and the in vitro decondensation assay we will analyze which aspects of mitotic exit (chromatin compaction, nuclear envelope reformation, reassembly of subnuclear structures etc.) are affected and determine how the involved proteins act in the processes. We will shed light on an ill-defined but important cell biological process at the end of mitosis indispensable to reestablish nuclear structure and function.

在有丝分裂过程中，细胞核在结构和功能上进行了相当大的重组。在动物细胞中，核膜在有丝分裂开始时破裂，染色质大量浓缩成个体化的染色体，这些染色体被纺锤体捕获并分离-许多实验室已经并仍然在深入研究这些过程。在有丝分裂结束时，细胞核的间期状态是如何重新建立的，并能胜任其多种功能的，我们所知甚少。为此，高度浓缩的有丝分裂染色体被解压缩，这是重新启动转录和基因组信息永久化所不可或缺的过程，因此在细胞生命周期中至关重要。尽管它的重要性，基础研究以及其潜在的医学意义，有丝分裂染色质去凝聚仍然是不明确的。它已被很好地和早期的细胞学描述，但我们在很大程度上是无知的潜在的分子事件和mechanism.We以前确定和表征的ATP酶复合体RuvBL 1/2作为一个关键的染色质解凝聚因子。通过来自退出有丝分裂的细胞的基于SILAC的质谱，我们现在鉴定eIF 4A 1、PKP 1和JUP为RuvBL 1/2相互作用物，其在染色质解凝聚期间特异性结合。事实上，细胞中或生物化学测定中蛋白质的下调或耗尽损害染色质去浓缩。eIF 4A 1是一种RNA解旋酶，具有翻译起始功能，但我们提出了一个独立于其在蛋白质合成中的作用的染色质解凝聚的额外作用。PKP 1和JUP是桥粒组分，在细胞核中具有额外的定位，但它们在该隔室中的确切功能在很大程度上尚不清楚。在这里，我们将分析eIF 4A 1，PKP 1和JUP如何精确地使用细胞测定，包括生命细胞成像和生物化学方法，如有丝分裂染色质去凝聚的无细胞重建测定在染色质去凝聚中发挥作用。我们将确定eIF 4A 1、PKP 1和JUP是否形成涉及RuvBL 1/2的单个染色质去凝聚复合物，或者它们是否是不同的含有RuvBL 1/2的复合物的一部分，所述复合物介导染色质去凝聚的可区分方面。我们将分析染色质解凝聚复合物是否在有丝分裂退出期间类似于RuvBL 1/2定位于染色质，如果是，则确定复合物的哪些组分定义染色质结合。通过消耗组织培养细胞中的关键解凝聚组分和体外解凝聚测定，我们将分析有丝分裂退出的哪些方面（染色质压实、核膜重组、亚核结构的重新组装等）。并决定参与的蛋白质如何在过程中发挥作用。我们将阐明在有丝分裂结束时一个不明确但重要的细胞生物学过程，这是重建核结构和功能所不可或缺的。