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

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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.