Periodic Reporting for period 2 - MemDense (Cellular control of membrane protein density in the endoplasmic reticulum via the unfolded protein response)
Periodo di rendicontazione: 2021-10-01 al 2023-03-31
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 goals are
1) Isolating organelles from cells with unprecedented purity
2) Establishing lipid fingerprints of ER stress, in other words: 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
4) Figuring out how the protein-to-lipid ratio is balanced in healthy cells and how membrane protein overcrowding causes chronic ER 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 goals are
1) Isolating organelles from cells with unprecedented purity
2) Establishing lipid fingerprints of ER stress, in other words: 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
4) Figuring out how the protein-to-lipid ratio is balanced in healthy cells and how membrane protein overcrowding causes chronic ER stress
The MemDense projects places a particular emphasis on the role of macromolecular crowding in biological membranes. By developing new preparative and analytical pipelines, we establish valuable resources for the fields of membrane biochemistry, biophysics, and cell biology.
The team has been progressing continuously towards the goals of the project. Several publications (and pre-prints) have already resulted from this work. We have developed tools to characterize the fingerprints of ER stress in organellar membrane preparations, to follow even most subtle changes of the protein-to-lipid ratio in stressed cellular membranes, obtained unprecedented insight into how membrane stiffness is sensed by the UPR-transducer IRE1, and uncovered the molecular basis for micrometer-sized membrane phase separation phenomena in living yeasts.
PUBLICATION 1: An important baseline for the MemDense project is a careful analysis of the connections between protein folding, cellular growth, and lipid metabolism. Our rigorously controlled study "A quantitative analysis of cellular lipid compositions during acute proteotoxic ER stress reveals specificity in the production of asymmetric lipids" in Frontiers in Developmental and Cell Biology dissected, on the cellular level, the distinct impact of two proteotoxic agents on lipid metabolism (doi.org/10.3389/fcell.2020.00756).
PUBLICATION 2: For balancing the protein-to-lipid ratio, the UPR must integrate proteotoxic and lipotoxic signals. It was unclear if proteotoxic and lipotoxic signals trigger different structures and/or distinct functional modalities in UPR transducers. Our publication "Cysteine cross-linking in native membranes establishes the transmembrane architecture of Ire1" in JCB revealed that different forms of ER stress converge in a single, signaling-active configuration of the ER stress sensor IRE1 (doi.org/10.1083/jcb.202011078). This study provides strong support for a hydrophobic mismatch-based oligomerization of IRE1 in the stressed ER.
PUBLICATION 3: We summarized the current knowledge on the mechanism of UPR activation by proteins and lipid bilayer stress in a comprehensive review article "The Unfolded Protein Response as a Guardian of the Secretory Pathway" published in Cells (doi.org/10.3390/cells10112965). We focus particularly on the role of the UPR as a guardian of the secretory pathway and elaborate a conceptual, cell biological framework for how UPR transducers might monitor protein (over)crowding and ER membrane stiffness as a proxy for the workload of the secretory pathway.
PUBLICATION 4,5 (PRE-PRINTs): A crucial milestone for the MemDense project is establishing new methodologies for isolating organelle membranes from stressed and unstressed cells. We have succeeded in isolating the ER and the vacuolar membranes by establishing the MemPrep technology ("A new technology for isolating organellar membranes provides fingerprints of lipid bilayer stress"; doi.org/10.1101/2022.09.15.508072). Our findings have important implications for understanding the role of lipids in membrane protein insertion, folding, and their sorting along the secretory pathway. Application of the combined preparative and analytical platform to acutely stressed cells reveals dynamic ER membrane remodeling and establishes molecular fingerprints of ER stress.
PUBLICATION 6: The MemDense team has contributed to a major effort designed to systematically identify new membrane contact sites. We contributed our expertise on lipid cell biology and lipidomic analyses for the publication "Systematic analysis of membrane contact sites in Saccharomyces cerevisiae uncovers modulators of cellular lipid distribution" in eLife (doi.org/10.7554/eLife.74602).
PUBLICATION 7: PUBLICATION 5 (PRE-PRINT) has been published in the Biophysical Journal yielding PUBLICATION 7.
The team has been progressing continuously towards the goals of the project. Several publications (and pre-prints) have already resulted from this work. We have developed tools to characterize the fingerprints of ER stress in organellar membrane preparations, to follow even most subtle changes of the protein-to-lipid ratio in stressed cellular membranes, obtained unprecedented insight into how membrane stiffness is sensed by the UPR-transducer IRE1, and uncovered the molecular basis for micrometer-sized membrane phase separation phenomena in living yeasts.
PUBLICATION 1: An important baseline for the MemDense project is a careful analysis of the connections between protein folding, cellular growth, and lipid metabolism. Our rigorously controlled study "A quantitative analysis of cellular lipid compositions during acute proteotoxic ER stress reveals specificity in the production of asymmetric lipids" in Frontiers in Developmental and Cell Biology dissected, on the cellular level, the distinct impact of two proteotoxic agents on lipid metabolism (doi.org/10.3389/fcell.2020.00756).
PUBLICATION 2: For balancing the protein-to-lipid ratio, the UPR must integrate proteotoxic and lipotoxic signals. It was unclear if proteotoxic and lipotoxic signals trigger different structures and/or distinct functional modalities in UPR transducers. Our publication "Cysteine cross-linking in native membranes establishes the transmembrane architecture of Ire1" in JCB revealed that different forms of ER stress converge in a single, signaling-active configuration of the ER stress sensor IRE1 (doi.org/10.1083/jcb.202011078). This study provides strong support for a hydrophobic mismatch-based oligomerization of IRE1 in the stressed ER.
PUBLICATION 3: We summarized the current knowledge on the mechanism of UPR activation by proteins and lipid bilayer stress in a comprehensive review article "The Unfolded Protein Response as a Guardian of the Secretory Pathway" published in Cells (doi.org/10.3390/cells10112965). We focus particularly on the role of the UPR as a guardian of the secretory pathway and elaborate a conceptual, cell biological framework for how UPR transducers might monitor protein (over)crowding and ER membrane stiffness as a proxy for the workload of the secretory pathway.
PUBLICATION 4,5 (PRE-PRINTs): A crucial milestone for the MemDense project is establishing new methodologies for isolating organelle membranes from stressed and unstressed cells. We have succeeded in isolating the ER and the vacuolar membranes by establishing the MemPrep technology ("A new technology for isolating organellar membranes provides fingerprints of lipid bilayer stress"; doi.org/10.1101/2022.09.15.508072). Our findings have important implications for understanding the role of lipids in membrane protein insertion, folding, and their sorting along the secretory pathway. Application of the combined preparative and analytical platform to acutely stressed cells reveals dynamic ER membrane remodeling and establishes molecular fingerprints of ER stress.
PUBLICATION 6: The MemDense team has contributed to a major effort designed to systematically identify new membrane contact sites. We contributed our expertise on lipid cell biology and lipidomic analyses for the publication "Systematic analysis of membrane contact sites in Saccharomyces cerevisiae uncovers modulators of cellular lipid distribution" in eLife (doi.org/10.7554/eLife.74602).
PUBLICATION 7: PUBLICATION 5 (PRE-PRINT) has been published in the Biophysical Journal yielding PUBLICATION 7.
The MemPrep technology (PRE-PRINT PUBLICATIONS 4) opens new horizons in cell biology, by providing the possibility for the first time, to 1) quantitatively study the composition of organelle subdomains, 2) study comprehensively the lipid composition of organelles with functional and dysfunctional lipid exchange mechanisms, and 3) identify 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.
At the EMBO workshop "Endoplasmic reticulum: The master regulator of membrane trafficking" in Lucca, Italy (2022) the presentation "Fingerprints of the stressed ER" received tremendous attention. Consequently, we initiated several collaborations on the MemPrep technology to pursue important proof-of-concept experiments on the above-mentioned applications with leading experts in organelle biology, lipid metabolism, DNA quality control, membrane biochemistry and biophysics.
After optimizing the MemPrep technology for organelle isolations from baker's yeast, we were surprised how readily our protocols can be modified and optimized for isolating ER membrane subdomains from cultured mammalian cells. This success goes well beyond the expected results and opens new horizons for understanding the role of organelle homeostasis in mammalian cells.
At the EMBO workshop "Endoplasmic reticulum: The master regulator of membrane trafficking" in Lucca, Italy (2022) the presentation "Fingerprints of the stressed ER" received tremendous attention. Consequently, we initiated several collaborations on the MemPrep technology to pursue important proof-of-concept experiments on the above-mentioned applications with leading experts in organelle biology, lipid metabolism, DNA quality control, membrane biochemistry and biophysics.
After optimizing the MemPrep technology for organelle isolations from baker's yeast, we were surprised how readily our protocols can be modified and optimized for isolating ER membrane subdomains from cultured mammalian cells. This success goes well beyond the expected results and opens new horizons for understanding the role of organelle homeostasis in mammalian cells.