Periodic Reporting for period 4 - MemDense (Cellular control of membrane protein density in the endoplasmic reticulum via the unfolded protein response)
Reporting period: 2024-10-01 to 2025-03-31
The MemDense project aims to identify how eukaryotic cells control the ratio of proteins and lipid in their membranes. Despite the enormous relevance of these biomolecules for virtually every cellular function, it remains unknown how cells balance the production of proteins and lipids. It has been known for decades that the different organelles (e.g. the endoplasmic reticulum (ER) or mitochondria) feature remarkably distinct protein-to-lipid ratios. Barely anything is known about, how different organelles control the exchange and flux of lipids for maintaining these largely distinct packing densities, and if an overcrowding of proteins in the membrane induces cellular stress.
We pursue the hypothesis that the protein-to-lipid ratio is sensed via the unfolded protein response (UPR), a conserved pathway, which controls the relative rate of lipid and protein production and which serves as a diagnostic marker for ER stress. We study this mechanistic connection in two model systems: The baker's yeast Saccharomyces cerevisiae and cultured, mammalian cells. We aim to understand, how communication and exchange of lipid metabolites between organelles support cell functions during acute, prolonged, and chronic ER stresses.
The MemDense project is important for society, because a large number of chronic inflammatory and complex metabolic diseases, such as type II diabetes and non-alcoholic fatty liver disease have been firmly associated with chronic ER stress, while the molecular basis of ER stress remains unresolved. The MemDense project establishes new experimental pipelines to test the contribution of the ER membrane and its composition to ER stress and UPR activation in health and disease.
Our objectives are
1) Isolating organelles from cells with unprecedented purity,
2) Establishing lipid fingerprints of ER stress (or: What are the characteristic lipid compositions of the stressed ER membrane?),
3) Studying the cooperation of cellular organelles, e.g. the ER, mitochondria, and the vacuole, in dealing with ER stress, and
4) Figuring out how the protein-to-lipid ratio is balanced in healthy cells and how membrane protein overcrowding causes chronic ER stress.
We find that acute and prolonged ER stress signaling is regulated by aberrant biophysical properties of the ER membrane. Surprisingly, we find that negatively charged lipids play a key role as modulators of ER stress. We establish lipid finger prints of ER stress in both yeast and mammalian cells and find that a decreased membrane compressibility is a major determinant of ER stress signaling by UPR transducers from yeast an mammals. We developed new experimental pipelines to quantitatively analyze the protein and lipid composition in ultra-pure organelle-derived preparations. By establishing new paradigms of modulating and exchanging membrane lipid compositions, we can now address - for the first time - the lipid dependency of membrane protein insertion, topology and folding by mammalian membrane protein insertion machineries. By addressing fundamental questions in membrane cell biology, we work towards lipid metabolic perturbations, which may pave the way to new preventive and therapeutic interventions to fight diseases associated with aberrant metabolism, infection, chronic ER stress, and disrupted (membrane) protein folding.
We pursue the hypothesis that the protein-to-lipid ratio is sensed via the unfolded protein response (UPR), a conserved pathway, which controls the relative rate of lipid and protein production and which serves as a diagnostic marker for ER stress. We study this mechanistic connection in two model systems: The baker's yeast Saccharomyces cerevisiae and cultured, mammalian cells. We aim to understand, how communication and exchange of lipid metabolites between organelles support cell functions during acute, prolonged, and chronic ER stresses.
The MemDense project is important for society, because a large number of chronic inflammatory and complex metabolic diseases, such as type II diabetes and non-alcoholic fatty liver disease have been firmly associated with chronic ER stress, while the molecular basis of ER stress remains unresolved. The MemDense project establishes new experimental pipelines to test the contribution of the ER membrane and its composition to ER stress and UPR activation in health and disease.
Our objectives are
1) Isolating organelles from cells with unprecedented purity,
2) Establishing lipid fingerprints of ER stress (or: What are the characteristic lipid compositions of the stressed ER membrane?),
3) Studying the cooperation of cellular organelles, e.g. the ER, mitochondria, and the vacuole, in dealing with ER stress, and
4) Figuring out how the protein-to-lipid ratio is balanced in healthy cells and how membrane protein overcrowding causes chronic ER stress.
We find that acute and prolonged ER stress signaling is regulated by aberrant biophysical properties of the ER membrane. Surprisingly, we find that negatively charged lipids play a key role as modulators of ER stress. We establish lipid finger prints of ER stress in both yeast and mammalian cells and find that a decreased membrane compressibility is a major determinant of ER stress signaling by UPR transducers from yeast an mammals. We developed new experimental pipelines to quantitatively analyze the protein and lipid composition in ultra-pure organelle-derived preparations. By establishing new paradigms of modulating and exchanging membrane lipid compositions, we can now address - for the first time - the lipid dependency of membrane protein insertion, topology and folding by mammalian membrane protein insertion machineries. By addressing fundamental questions in membrane cell biology, we work towards lipid metabolic perturbations, which may pave the way to new preventive and therapeutic interventions to fight diseases associated with aberrant metabolism, infection, chronic ER stress, and disrupted (membrane) protein folding.
The MemDense project emphasizes the impact of macromolecular crowding in biological membranes, creating resources for membrane biochemistry, biophysics, and cell biology through innovative preparative and analytical methods.
Our team has made significant strides towards the project's objectives. We've developed techniques to characterize ER stress signatures in organellar membranes and track subtle changes in protein-to-lipid ratios under stress. Our publications address a wide array of methodological advances and insights into how celllular membranes change upon ER stress, and how these changes manifest in cellular adjustments and adaptations.
We exploit our experimental platforms and insights for addressing new cutting-edge questions in membrane biology:
1) How is the maintenace of different biophysical membrane properties balanced during metabolic stress?
2) How do lipids affect the insertion, topology, folding of membrane proteins in mammalian cells?
3) What is the relevance of lipid heterogeneity? How do closely related yeast strains with vastly distinct lipid compositions cope with acute and chronic ER stress?
4) What constitutes the diffusion barrier between the ER and the nuclear envelope and how is it regulated during ER stress.
5) What are the lipid fingerprint of cell health and cell stress and their therapeutic implications?
The MemDense team has disseminated its findings at prestigious, international scientific conferences with posters, invited talks, and by organizing scientific meetings. The scope of dissemination broadened continuously covering organelle biology, biophyiscs, pharmaceutical sciences, and integrative cell biology. T
Our team has made significant strides towards the project's objectives. We've developed techniques to characterize ER stress signatures in organellar membranes and track subtle changes in protein-to-lipid ratios under stress. Our publications address a wide array of methodological advances and insights into how celllular membranes change upon ER stress, and how these changes manifest in cellular adjustments and adaptations.
We exploit our experimental platforms and insights for addressing new cutting-edge questions in membrane biology:
1) How is the maintenace of different biophysical membrane properties balanced during metabolic stress?
2) How do lipids affect the insertion, topology, folding of membrane proteins in mammalian cells?
3) What is the relevance of lipid heterogeneity? How do closely related yeast strains with vastly distinct lipid compositions cope with acute and chronic ER stress?
4) What constitutes the diffusion barrier between the ER and the nuclear envelope and how is it regulated during ER stress.
5) What are the lipid fingerprint of cell health and cell stress and their therapeutic implications?
The MemDense team has disseminated its findings at prestigious, international scientific conferences with posters, invited talks, and by organizing scientific meetings. The scope of dissemination broadened continuously covering organelle biology, biophyiscs, pharmaceutical sciences, and integrative cell biology. T
The newly developed MemPrep technology opens new horizons in cell biology, by 1) enabling the quantitative analysis of organelle membranes and organelle subdomains with respect to lipids and proteins, 2) by comprehensively tracing the lipid composition of organelles with functional and dysfunctional lipid exchange mechanisms, and 3) identifying the spectrum of clients for inter-organelle protein transport (e.g. from the ER to mitochondria or from the ER to the Golgi) by quantitative proteomics.
The MemDense team identified a critical role of anionic lipids as negative modulators of UPR signaling in both yeast and mammalian cells. Presumably, this has wide implications for diseases associated with chronic ER stress and aberrant protein folding. Going far beyond the expected results of the proposed project, we have established new reconstitution schemes and lipid-exchange protocols, which represent important steps towards the functional and structural analysis of transmembrane proteins in naive-like, protein-rich, complex, asymmetric, sterol-rich membrane environments.
Our serendipitous observation that lipid bilayer stress disrupts a diffusion barrier between the nuclear envelope and the ER, opens new opportunities to understand the role of diffusion barriers in membrane protein compartmentalization, nuclear integrity and aging.
By optimizing a protocol to modulate and rebuild the lipidome of isolated membrane vesciles isolated from cells, we can now systematically dissect the lipid dependency of membrane protein insertion, topology, and folding with microsomes isolated from mammalian cells. This exciting new avenue of research would not have been made possible without the technological advances of the MemDense project.
The MemDense team identified a critical role of anionic lipids as negative modulators of UPR signaling in both yeast and mammalian cells. Presumably, this has wide implications for diseases associated with chronic ER stress and aberrant protein folding. Going far beyond the expected results of the proposed project, we have established new reconstitution schemes and lipid-exchange protocols, which represent important steps towards the functional and structural analysis of transmembrane proteins in naive-like, protein-rich, complex, asymmetric, sterol-rich membrane environments.
Our serendipitous observation that lipid bilayer stress disrupts a diffusion barrier between the nuclear envelope and the ER, opens new opportunities to understand the role of diffusion barriers in membrane protein compartmentalization, nuclear integrity and aging.
By optimizing a protocol to modulate and rebuild the lipidome of isolated membrane vesciles isolated from cells, we can now systematically dissect the lipid dependency of membrane protein insertion, topology, and folding with microsomes isolated from mammalian cells. This exciting new avenue of research would not have been made possible without the technological advances of the MemDense project.