Molecular-biological studies about the fate of micronuclei: underlying mechanisms and toxicological relevance

不同类型的突变在癌症的发生中起主要作用，例如染色体突变，其特征是染色体结构的改变。显性染色体突变的一种形式是所谓的微核，它是由染色体碎片或错误分离的染色体形成的。人们对微核的形成进行了深入的研究，并对其潜在机制有了很好的了解。然而，微核是否在细胞内持续存在，或者是否存在任何特定的消除机制，目前还不太清楚。原则上，可能有四种可能性：在细胞内持续存在、降解、从细胞中挤出或重新融入主核。在先前的项目中，首次系统地讨论了微核的命运及其与整个细胞的相关性。在现在提出的项目中，将识别和表征潜在的机制。在先前的项目中，建立了活细胞显微镜系统。在初步测试中，确定了合适的实验条件，如微核诱导物、浓度、处理次数、成像条件和评价标准。对细胞进行96小时的跟踪和评估。总体而言，大多数细胞持续存在，没有任何变化。一些微核重新聚集到主核中。降解和挤压是罕见的事件。没有观察到物质之间的差异。在这里提出的项目中，应该调查潜在的机制。为此，将讨论微核的内容是否对特定的命运负责的问题。在这方面，人们特别感兴趣的是微核是否含有完整的染色体或片段。为了研究微核的降解，将分析可能的解离途径，包括自噬和溶酶体降解。为了分析细胞的再结合和排出，将研究细胞骨架结构蛋白的参与。这将有助于阐明细胞骨架在微核定位及其转运到主核(表示重新结合)或细胞膜(表示挤出)中的作用。这些实验将有助于理解微核不同命运的机制背景。

Different types of mutations play a major role in carcinogenesis, e. g. chromosomal mutations, which are characterized by modifications of the chromosome structure. One form of manifested chromosomal mutations are so-called micronuclei that are formed from chromosomal fragments or missegregated chromosomes. The formation of micronuclei has been investigated thoroughly and the underlying mechanisms are well understood. However, it is less clear whether micronuclei persist within the cell or whether there are any specific mechanisms of elimination. In principle, there could be four possibilities: persistence in the cell, degradation, extrusion from the cell or reintegration into the main nucleus. In the previous project, the fate of micronuclei and the relevance for the whole cell was addressed systematically for the first time. In the project now proposed, the underlying mechanisms shall be identified and characterized.In the previous project, a system for live cell microscopy was established. In preliminary tests, suitable experimental conditions were identified, such as micronucleus-inducing substances, concentrations, treatment times, imaging conditions, and evaluation criteria. Cells were followed for 96 hours and evaluated. Overall, most cells persisted without any change. Some micronuclei were reincorporated into the main nucleus. Degradation and extrusion were rare events. Differences between the substances were not observed.In the project proposed here, the underlying mechanisms shall be investigated. To this end, the question whether the content of a micronucleus is responsible for a specific fate will be addressed. In this context, it is of particular interest whether micronuclei contain whole chromosomes or fragments. For the investigation of the degradation of micronuclei, putative dissociation pathways will be analysed, including autophagy and lysosomal degradation. For the analysis of reincorporation and extrusion from the cell, the involvement of structural proteins of the cytoskeleton will be investigated. This will help elucidating the role of the cytoskeleton for the localisation of micronuclei and for their transport to the main nucleus (indicating reincorporation) or to the cell membrane (indicating extrusion).These experiments will help understanding the mechanistic backgrounds of the different fates of micronuclei.