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- Bäckström, Anna, et al.
(författare)
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A Sample Preparation Protocol for High Throughput Immunofluorescence of Suspension Cells on an Adherent Surface
- 2020
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Ingår i: Journal of Histochemistry and Cytochemistry. - : SAGE Publications. - 0022-1554 .- 1551-5044. ; 68:7, s. 473-489
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Tidskriftsartikel (refereegranskat)abstract
- Imaging is a powerful approach for studying protein expression and has the advantage over other methodologies in providing spatial informationin situat single cell level. Using immunofluorescence and confocal microscopy, detailed information of subcellular distribution of proteins can be obtained. While adherent cells of different tissue origin are relatively easy to prepare for imaging applications, non-adherent cells from hematopoietic origin, present a challenge due to their poor attachment to surfaces and subsequent loss of a substantial fraction of the cells. Still, these cell types represent an important part of the human proteome and express genes that are not expressed in adherent cell types. In the era of cell mapping efforts, overcoming the challenge with suspension cells for imaging applications would enable systematic profiling of hematopoietic cells. In this work, we successfully established an immunofluorescence protocol for preparation of suspension cell lines, peripheral blood mononucleated cells (PBMC) and human platelets on an adherent surface. The protocol is based on a multi-well plate format with automated sample preparation, allowing for robust high throughput imaging applications. In combination with confocal microscopy, the protocol enables systematic exploration of protein localization to all major subcellular structures.
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| 4. |
- Chang, Yun Chien, et al.
(författare)
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Decrypting lysine deacetylase inhibitor action and protein modifications by dose-resolved proteomics
- 2024
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Ingår i: Cell Reports. - : Elsevier BV. - 2211-1247. ; 43:6
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Tidskriftsartikel (refereegranskat)abstract
- Lysine deacetylase inhibitors (KDACis) are approved drugs for cutaneous T cell lymphoma (CTCL), peripheral T cell lymphoma (PTCL), and multiple myeloma, but many aspects of their cellular mechanism of action (MoA) and substantial toxicity are not well understood. To shed more light on how KDACis elicit cellular responses, we systematically measured dose-dependent changes in acetylation, phosphorylation, and protein expression in response to 21 clinical and pre-clinical KDACis. The resulting 862,000 dose-response curves revealed, for instance, limited cellular specificity of histone deacetylase (HDAC) 1, 2, 3, and 6 inhibitors; strong cross-talk between acetylation and phosphorylation pathways; localization of most drug-responsive acetylation sites to intrinsically disordered regions (IDRs); an underappreciated role of acetylation in protein structure; and a shift in EP300 protein abundance between the cytoplasm and the nucleus. This comprehensive dataset serves as a resource for the investigation of the molecular mechanisms underlying KDACi action in cells and can be interactively explored online in ProteomicsDB.
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| 5. |
- Cho, Nathan H., et al.
(författare)
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OpenCell : Endogenous tagging for the cartography of human cellular organization
- 2022
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Ingår i: Science. - : American Association for the Advancement of Science (AAAS). - 0036-8075 .- 1095-9203. ; 375:6585, s. 1143-
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Tidskriftsartikel (refereegranskat)abstract
- Elucidating the wiring diagram of the human cell is a central goal of the postgenomic era. We combined genome engineering, confocal live-cell imaging, mass spectrometry, and data science to systematically map the localization and interactions of human proteins. Our approach provides a data-driven description of the molecular and spatial networks that organize the proteome. Unsupervised clustering of these networks delineates functional communities that facilitate biological discovery. We found that remarkably precise functional information can be derived from protein localization patterns, which often contain enough information to identify molecular interactions, and that RNA binding proteins form a specific subgroup defined by unique interaction and localization properties. Paired with a fully interactive website (opencell.czbiohub.org), our work constitutes a resource for the quantitative cartography of human cellular organization.
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| 7. |
- Gnann, Christian, et al.
(författare)
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An image-based map of the mitochondrial proteome reveals widespread metabolic heterogeneity
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Annan publikation (övrigt vetenskapligt/konstnärligt)abstract
- Mitochondria are involved in a wide range of cellular functions beyond their role in energy metabolism. Defining the human mitochondrial proteome is crucial to understand the mitochondria’s diverse functions and role in disease. Here, we present an image-based map of the human mitochondrial proteome containing 1,121 proteins with subcellular resolution. Our analysis shows that 48.3% (n=542) of the proteins localize to additional cellular compartments, further contributing to the diverse cellular functions of mitochondria and connectivity to other organelles. Furthermore, the mitochondrial proteome reveals tissue specific clustering, suggesting tissue specific functions and physiology. Strikingly, the single cell resolution of our dataset revealed extensive heterogeneity for as much as 33.5% (n=376) of the mitochondrial proteome which could not be explained by cell cycle progression. By performing a high throughput immunofluorescence screen, we conclude that heterogeneity in mitochondria protein expression can establish metabolic states in cell populations. This map of the mitochondrial proteome, part of the Human Protein Atlas database (www.proteinatlas.org), provides a valuable knowledge resource for studies of mitochondria function, dysfunction and disease.
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| 8. |
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| 9. |
- Gnann, Christian, 1994-
(författare)
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Finding order in chaos : Dissecting single-cell heterogeneity in space and time
- 2024
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Doktorsavhandling (övrigt vetenskapligt/konstnärligt)abstract
- The cell is the smallest unit of life and contains DNA, RNA, proteins and a variety of other macromolecules. In recent years, technological advances in the field of single cell biology have revealed a staggering amount of phenotypic heterogeneity between cells in a population, which were previously considered homogenous. Previous work has largely been focused on studies of RNA. As proteins however are the ultimate effectors of genetic information, this thesis aims to provide a protein-centered view on cellular heterogeneity, particularly focusing on cell cycle and cellular metabolism.Most of my work has been performed within the framework of the Human Protein Atlas project. In the context of this project, we mapped the spatial distribution of more than 13.000 human proteins with subcellular resolution and found that around a quarter of all human proteins exhibit protein expression heterogeneity.In Paper I, we hypothesized that a majority of the observed cellular heterogeneity can be explained by differences in cell cycle progression. Therefore, we generated a map of proteomic and transcriptomic heterogeneity at subcellular resolution, which we precisely aligned to the cell cycle position of individual cells. This approach allowed us to identify hundreds of previously unknown cell cycle-related proteins. With sustained proliferative signaling representing a hallmark of cancer, novel cell-cycle proteins could serve as potential new drug targets against cancer. We further show that a large part of cell cycle dependent proteome variability is not established by transcriptomic cycling. This suggests that post-translational modifications are a major contributor to the regulation of cell cycle dependent protein level changes. Therefore, in Paper II, we carried out a deep phosphoproteome mass spectrometry profiling of the same cellular model as in Paper I and identified almost 5,000 cell cycle dependent phosphosites on over 2,000 proteins. The unprecedented scale of our phosphoproteomic data allows us to link cell cycle dependent protein expression dynamics to phosphorylation events. Furthermore, we identify a large set of proteins with stable expression levels and fluctuating phosphorylation patterns along cell cycle progression that likely alters protein function.Despite identifying hundreds of novel cell cycle dependent proteins in paper I, we observed that the majority of heterogeneously expressed proteins display variable expression independent of cell cycle progression, among them a large number of metabolic enzymes. Thus, we sought to describe the extent of subcellular metabolic complexity in human cells and tissues in Paper III. While we confirm metabolic compartmentalization in our dataset, we show that around 50% of metabolic enzymes localize to multiple cellular compartments. By integrating public protein-protein interaction data with our subcellular location information, we identify several enzymes with novel compartment-specific functions. Additionally, we observe a strongly elevated number of heterogeneously expressed enzymes compared to the background of the human proteome that is largely independent of cell cycle progression. We show that this heterogeneity can be manifested in the lineage of a single cell and is conserved in situ. To conclude, we suggest that the extensive metabolic heterogeneity can establish functional metabolic states in a population of human cells.Finally, in Paper IV, we assessed the heterogeneity of the mitochondrial proteome as they are metabolic powerhouses containing an elevated number of cell cycle independent variably expressed proteins. In this study, we correlated the variable expression of over 400 mitochondrial proteins to the expression of rate limiting enzymes in important mitochondrial pathways; such as the TCA cycle and ROS metabolism. We show that enzymes in the same pathways often correlate in their expression, indicating that their expression variability may contribute to the establishment of metabolic states.Altogether, the thesis illuminates the spatiotemporal complexity of the human proteome established by protein multilocalization and expression heterogeneity as fundamental non-genetic means of functional cell regulation.
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| 10. |
- Gnann, Christian, et al.
(författare)
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Illuminating Non-genetic Cellular Heterogeneity with Imaging-Based Spatial Proteomics
- 2021
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Ingår i: Trends in cancer. - : Elsevier BV. - 2405-8025 .- 2405-8033. ; 7:4, s. 278-282
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Tidskriftsartikel (övrigt vetenskapligt/konstnärligt)abstract
- Cellular heterogeneity is an important biological phenomenon observed across space and time in human tissues. Imaging-based spatial proteomic technologies can provide fruitful new readouts of phenotypic states for individual cells at subcellular resolution, which may help unravel the roles of non-genetic cellular heterogeneity in tumorigenesis and drug resistance.
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