VPS72/YL1 function in nuclear re-assembly

在有丝分裂期间，细胞核在结构和功能上都发生了相当大的重组。在动物细胞中，核膜在有丝分裂开始时破裂，染色质大量凝聚成个性化的染色体，这些染色体被纺锤体捕获并分离-这一过程已经并仍在许多实验室进行深入研究。我们更不知道在有丝分裂结束时，细胞核的间期状态是如何重新建立的，能够胜任它的多种功能。为此，高度浓缩的有丝分裂染色体被解压。围绕着去凝集的染色质，核膜重新组装，核仁改革和核运输等核亚结构重新建立分区。这些过程对于重新启动转录和基因组信息的永久化是必不可少的，因此在细胞生命周期中具有核心重要性。尽管它对基础研究以及潜在的医学意义具有重要意义，但有丝分裂退出时的核改革在分子方面仍然没有明确的定义。我们已经确定VPS72/YL1，一个组蛋白变异体H_2A.Z的伴侣，及其保守的YL-1结构域是核重组的关键因素：在非洲爪哇卵提取液中，核重组可以方便地重组，VPS72的缺失阻碍了H_2A.Z整合到染色质中，导致核结构缺陷。染色质看起来杂乱无章，核膜不规则，延伸到通常由染色质占据的区域。反过来，YL-1结构域并不是装载大量H2A.Z染色质所必需的，但在核重组中具有特定的、尚不清楚的功能。有趣的是，这个结构域与主轴组件检查点组件MAD2和P31以及一个定义较少的因子WDR83相互作用。P31和MAD2的缺失损害了VPS72的核结构，表明了检查点蛋白P31和MAD2的非规范功能。在这里，我们将使用非洲爪哇卵提取液和细胞分析来定义YL-1结构域及其结合伙伴MAD2、P31和WDR83在核重组中的功能。第二种包括基于CRISPR/CAS的敲除或诱导组织培养细胞中目标蛋白的降解，结合生命细胞成像。我们将确定VPS72/MAD2/P31/WDR83核组装相互作用网络的功能，并确定如果这些组件缺失，核改革的哪些方面会受到影响。我们将检验YL-1结构域需要将H2A.Z加载到或识别特定染色质基因座的假设，这反过来对正确的核重组至关重要。为此，我们在非洲爪哇卵提取液和组织培养细胞中进行了芯片实验，该实验将在该系统中建立。通过结合无细胞和细胞分析，我们将阐明在有丝分裂结束时一个定义不清但重要的细胞生物学过程，这对重建核结构和功能至关重要。

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 that have been and still are intensively studied in many laboratories. We know much less 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. Around the decondensing chromatin the nuclear envelope re-assembles, nuclear substructures like nucleoli reform and nuclear transport re-establishes compartmentalization. These processes are 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, nuclear reformation during mitotic exit is in molecular terms still ill defined. We have identified VPS72/YL1, a chaperone of the histone variant H2A.Z, and its conserved YL-1 domain as a crucial factor for nuclear re-assembly: In Xenopus egg extracts, where nuclear reformation can be conveniently reconstituted, depletion of VPS72 blocks H2A.Z integration into chromatin and leads to nuclear structure defects. The chromatin appears disorganized and the nuclear envelope is irregular and reaches into areas usually occupied by chromatin. The YL-1 domain, in turn, is not required for bulk H2A.Z chromatin loading but has a specific, yet unknown, function in nuclear re-assembly. Interestingly, this domain interacts with the spindle assembly checkpoint components MAD2 and p31 and a less defined factor, WDR83. Depletion of p31 and MAD2 compromises the nuclear structure as VPS72 depletion indicating a non-canonical function of the checkpoint proteins p31 and MAD2. Here, we will define the function of the YL-1 domain and its binding partners MAD2, p31 and WDR83 in nuclear re-assembly using Xenopus egg extracts and cellular assays. The second includes CRISPR/CAS based knockouts or inducible degradation of target proteins in tissue culture cells combined with life cell imaging. We will characterize the function of the VPS72/MAD2/p31/WDR83 interaction network for nuclear assembly and define which aspects of nuclear reformation are compromised if these components are missing. We will test the hypothesis that the YL-1 domain is required to load H2A.Z to or recognize specific chromatin loci, which in turn are crucial for proper nuclear re-assembly. For this, we use CHIP experiments in Xenopus egg extracts, to be established in this system, and in tissue culture cells. By a combination of cell-free and cellular assays, we will shed light on an ill-defined but important cell biological process at the end of mitosis vital to reestablish nuclear structure and function.