RuvBL1/2 function in mitotic chromatin decondensation

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，核包膜在有丝分裂开始时被破坏，染色质大量凝聚成个体化的染色体，这些染色体被纺锤体捕获并分离，这一过程已经并且仍然在许多实验室中被深入研究。在有丝分裂结束时，细胞核的间期状态是如何重建的，如何胜任其多种功能的，我们所知甚少。为此，高度浓缩的有丝分裂染色体被分解，这是重新启动转录和基因组信息延续所必不可少的过程，因此在细胞生命周期中具有中心重要性。尽管有丝分裂染色质去浓缩对基础研究和潜在的医学意义具有重要意义，但它仍然是不明确的。它已经很好和早期的细胞学描述，但我们很大程度上是无知的潜在的分子事件和机制。我们之前已经确定并表征了atp酶复合物RuvBL1/2作为一个关键的染色质去浓缩因子。通过基于SILAC的质谱分析，我们现在鉴定出eIF4A1， PKP1和JUP是RuvBL1/2相互作用体，它们在染色质去浓缩时特异性结合。事实上，细胞或生化分析中蛋白质的下调或耗竭会损害染色质的去浓缩。eIF4A1是一种具有翻译起始功能的RNA解旋酶，但我们认为它在染色质去浓缩中具有独立于蛋白质合成的作用。PKP1和JUP是桥粒成分，在细胞核中有一个额外的定位，但它们在这个隔室中的确切功能在很大程度上是不清楚的。在这里，我们将分析eIF4A1， PKP1和JUP如何精确地在染色质去浓缩中发挥作用，使用细胞分析，包括生命细胞成像和生化方法，如有丝分裂染色质去浓缩的细胞自由重建分析。我们将确定eIF4A1， PKP1和JUP是否形成涉及RuvBL1/2的单个染色质去浓缩复合体，或者它们是否属于不同的RuvBL1/2含复合体的一部分，介导染色质去浓缩的不同方面。我们将分析染色质去浓缩复合体（es）是否在有丝分裂退出时定位于染色质上，类似于RuvBL1/2，如果是这样，则确定复合体的哪些组分（es）定义染色质结合。通过消耗组织培养细胞中关键的去浓缩成分和体外去浓缩实验，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、亚核结构重组等）受到影响，并确定相关蛋白质在该过程中的作用。我们将阐明在有丝分裂结束时重建细胞核结构和功能不可或缺的一个不明确但重要的细胞生物学过程。

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.