Periodic Reporting for period 1 - RiboStress (Stress-induced structural and organizational adaptations of the cellular translation machinery)
Período documentado: 2023-01-01 hasta 2025-06-30
Many cellular and extracellular events cause perturbations of protein homeostasis by affecting either de novo protein folding or by destabilizing already folded proteins. Under such proteotoxic stress conditions, cells engage in various strategies to avoid further damage to the proteome, e.g. by timely modulation of translation activity and specificity, or by resolving the underlying events.
The main goal in this project is to dissect from a unique structural angle how such damage avoidance strategies impact on the structure and molecular organization of the translation machinery by directly imaging their effects on ribosome structure, supramolecular organization and distribution in a cellular context with cryo-electron tomography (cryo-ET), an innovative imaging approach unique in its capability to provide highly detailed three-dimensional structural information on macromolecular complexes in their cellular environment. Building on pioneering work in the field of cryo-ET and integrating novel image processing solutions that have recently marked a breakthrough in the field, this project will elucidate at unprecedented resolution how the cellular translation machinery is remodeled after a general heat-shock, during the Endoplasmic Reticulum unfolded protein response and during persistent translational stalling triggering ribosome-associated quality control.
This project will provide detailed structural and mechanistic insights into how cells counteract an imbalance of protein homeostasis - a hallmark of neurodegenerative diseases. Thus, key concepts emerging from our work will likely have direct implications on mechanistic understanding of central pathological principles underlying these diseases.
The main goal in this project is to dissect from a unique structural angle how such damage avoidance strategies impact on the structure and molecular organization of the translation machinery by directly imaging their effects on ribosome structure, supramolecular organization and distribution in a cellular context with cryo-electron tomography (cryo-ET), an innovative imaging approach unique in its capability to provide highly detailed three-dimensional structural information on macromolecular complexes in their cellular environment. Building on pioneering work in the field of cryo-ET and integrating novel image processing solutions that have recently marked a breakthrough in the field, this project will elucidate at unprecedented resolution how the cellular translation machinery is remodeled after a general heat-shock, during the Endoplasmic Reticulum unfolded protein response and during persistent translational stalling triggering ribosome-associated quality control.
This project will provide detailed structural and mechanistic insights into how cells counteract an imbalance of protein homeostasis - a hallmark of neurodegenerative diseases. Thus, key concepts emerging from our work will likely have direct implications on mechanistic understanding of central pathological principles underlying these diseases.
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.
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.
This project will provide a novel structural angle on how cells counteract an imbalance of protein homeostasis - a hallmark of neurodegenerative diseases. Thus, key concepts emerging from our work may have direct implications for mechanistic understanding of central pathological principles underlying these diseases.