In project 1, we investigate from a structural perspective how the cellular translation machinery is remodeled in response to proteotoxic stress originating from either a systemic heat-shock or impaired protein folding in the Endoplasmic reticulum (ER). We have collected extended cellular cryo-electron tomography datasets at various time points after stress induction, complemented by control conditions. Using advanced subtomogram analysis, we obtained and analysed high-resolution structures of various active and hibernating ribosome states coexisting in the imaged cells and quantified their relative abundancies between control and stress conditions. This work is providing detailed insights into stress-induced changes of translational activity and fidelity, the adaptation of translational mechanisms and alterations of cell architecture in response to stress.
In project 2, we extend our analysis to cellular stress originating from persistent translational stalling and ribosome collision, and how these events trigger pathways of translational quality control, including ribosome-associated quality control (RQC). In extended cryo-electron tomography datasets of cells experiencing elevated translational stalling, we observed strong effects on polyribosome organization and the formation of structurally defined collided disomes. These data are illuminating which structural features distinguish collided disomes from translationally active polyribosome configurations, enabling their specific recognition by translational quality control pathways.
In the framework of our ERC project, we have also developed and optimized various computational approaches for the analysis of supramolecular ribosome organization in cellular cryo-electron tomography data.
