Activation of autophagy in response to dynamic proteome changes due to aneuploidy
Zuzana Storchova - Markus Räschle - Timo Mühlhaus - Jan Hauth/ Andreas Wirsen
Upon chromosome missegregation, cells with abnormal chromosome numbers arise. This so called aneuploidy is largely detrimental for human cells and leads either to cell death or global changes in cell physiology. On organismal level, aneuploidy is associated with multiple pathologies, such as in Down’s syndrome (trisomy of chromosome 21) or with cancer, as more than 70 % all tumors contain abnormal aneuploid chromosomal content. Upon chromosome gain, the extra chromosomes are transcribed and translated, which leads to a marked protein imbalance and defects in maintenance of protein homeostasis. We and others have recently shown that autophagy is strongly activated by aneuploidy and that aneuploid cells are hypersensitive to inhibitors of the autophagy pathway. The current hypothesis proposes that autophagy is activated in response to aneuploidy to counteract the protein imbalance by either specifically degrading the excess proteins expressed from the extra chromosome or by mechanisms that generally reduce protein synthesis. Yet, no systematic data that would support this hypothesis are available. Specifically, it is unclear how autophagy is activated and what cellular components are degraded. Using our model system of aneuploid human cells we will address the following questions:
1. How is autophagy activated in aneuploid cells? To address this question, we will use live cell imaging of autophagic components following chromosome missegregation. We will determine the flux of autophagosomes in normal and aneuploid cells using live cell spinning disc confocal microscopy. Autophagosomes and autophagolysosome can be visualized by double-tagged RFP-GFP-LC3, which becomes integrated into the autophagosome membrane upon lipidation. Due to different folding kinetics and pH stability of GFP and RFP, this system allows to distinguish between newly formed autophagosomes and late autophagolysosomes. Using this well-established system, we will quantify the numbers of autophagosomes and autophagolysosome at specific time points after chromosome missegregation to obtain quantitative data on autophagy dynamics in normal and aneuploid cells. Additionally, we will use inhibitors which block or activate autophagy at distinct stages (wortmannin: blocking the activation of autophagy, chloroquine, bafilomycin: blocking autophagosome-lysosome fusion, rapamycin: inducing autophagy) and determine the alteration of autophagy flux in diploid cells. By comparing this data with the data obtained in aneuploid cells, we will identify the steps of autophagy that are specifically altered in response to aneuploidy. If necessary, we will use mathematical modelling of flux through the autophagosome-lysosome system in both healthy and abnormal conditions to determine the affected steps.
2. What is the function of increased autophagy in aneuploid cells? We will tackle this question by analyzing the molecules that are targeted for degradation via autophagy. We will purify autophagosomes from aneuploid cells using established protocols to determine their protein content by quantitative mass spectrometry and nucleic acids content by next generation sequencing. Treatments with autophagy inhibitors and optimization of the protocol may be required to enrich autophagosomes as a distinct subcellular compartment. Taking advantage of the extensive characterization of our defined aneuploid cells at the genome, transcriptome and proteome level will allow us to determine whether autophagy specifically degrades the proteins expressed from the extra chromosome or whether it serves to restore the protein balance by other mechanisms, such as a global repression of protein expression. This might globally attenuate protein expression, which we recently discovered as a frequent response of human cells to aneuploidy. The proteomic data may also be used to generate “organellar maps”, which have proven powerful for the global analysis of subcellular compartments and small vesicles in particular (www.MapOfTheCell.org).
The project will profit from the expertise of other BioComp members, such as Torsten Möhlmann (purification and analysis of autophagosomes or lysosomes), Michael Schroda, Markus Räschle and Timo Mühlhaus (proteomics and bioinformatics) and the ITWM (bioinformatics, modelling support). We expect that the developed methods will also be applicable in the future for the analysis of subproteomes of plants and yeast.