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| 2. |
- Lork, Alicia, et al.
(författare)
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Subcellular protein turnover in human neural progenitor cells revealed by correlative electron microscopy and nanoscale secondary ion mass spectrometry imaging
- 2024
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Ingår i: CHEMICAL SCIENCE. - 2041-6520 .- 2041-6539.
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Tidskriftsartikel (refereegranskat)abstract
- Protein turnover is a critical process for accurate cellular function, in which damaged proteins in the cells are gradually replaced with newly synthesized ones. Many previous studies on cellular protein turnover have used stable isotopic labelling by amino acids in cell culture (SILAC), followed by proteomic bulk analysis. However, this approach does not take into account the heterogeneity observed at the single-cell and subcellular levels. To address this, we investigated the protein turnover of neural progenitor cells at the subcellular resolution, using correlative TEM and NanoSIMS imaging, relying on a pulse-chase analysis of isotopically-labelled protein precusors. Cellular protein turnover was found significantly heterogenous across individual organelles, which indicates a possible relation between protein turnover and subcellular activity. In addition, different isotopically-labelled amino acids provided different turnover patterns, in spite of all being protein precursors, suggesting that they undergo distinct protein synthesis and metabolic pathways at the subcellular level. Protein turnover is a critical process for accurate cellular function, in which damaged proteins in the cells are gradually replaced with newly synthesized ones.
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| 3. |
- Nguyen, Tho D. K., et al.
(författare)
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Quantitative Nanoscale Secondary Ion Mass Spectrometry (NanoSIMS) Imaging of Individual Vesicles to Investigate the Relation between Fraction of Chemical Release and Vesicle Size
- 2023
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Ingår i: Angewandte Chemie. - 0044-8249 .- 1521-3757. ; 62:28
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Tidskriftsartikel (refereegranskat)abstract
- We used correlative transmission electron microscopy (TEM) and nanoscale secondary ion mass spectrometry (NanoSIMS) imaging to quantify the contents of subvesicular compartments, and to measure the partial release fraction of C-13-dopamine in cellular nanovesicles as a function of size. Three modes of exocytosis comprise full release, kiss-and-run, and partial release. The latter has been subject to scientific debate, despite a growing amount of supporting literature. We tailored culturing procedures to alter vesicle size and definitively show no size correlation with the fraction of partial release. In NanoSIMS images, vesicle content was indicated by the presence of isotopic dopamine, while vesicles which underwent partial release were identified by the presence of an I-127-labelled drug, to which they were exposed during exocytosis allowing entry into the open vesicle prior to its closing again. Demonstration of similar partial release fractions indicates that this mode of exocytosis is predominant across a wide range of vesicle sizes.
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| 4. |
- Rabasco, Stefania, et al.
(författare)
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Characterization of Stress Granule Protein Turnover in Neuronal Progenitor Cells Using Correlative STED and NanoSIMS Imaging
- 2023
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Ingår i: International Journal of Molecular Sciences. - : MDPI AG. - 1661-6596 .- 1422-0067. ; 24
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Tidskriftsartikel (refereegranskat)abstract
- Stress granules (SGs) are stress-induced biomolecular condensates which originate primarily from inactivated RNA translation machinery and translation initiation factors. SG formation is an important defensive mechanism for cell survival, while its dysfunction has been linked to neurodegenerative diseases. However, the molecular mechanisms of SG assembly and disassembly, as well as their impacts on cellular recovery, are not fully understood. More thorough investigations into the molecular dynamics of SG pathways are required to understand the pathophysiological roles of SGs in cellular systems. Here, we characterize the SG and cytoplasmic protein turnover in neuronal progenitor cells (NPCs) under stressed and non-stressed conditions using correlative STED and NanoSIMS imaging. We incubate NPCs with isotopically labelled (15N) leucine and stress them with the ER stressor thapsigargin (TG). A correlation of STED and NanoSIMS allows the localization of individual SGs (using STED), and their protein turnover can then be extracted based on the 15N/14N ratio (using NanoSIMS). We found that TG-induced SGs, which are highly dynamic domains, recruit their constituents predominantly from the cytoplasm. Moreover, ER stress impairs the total cellular protein turnover regimen, and this impairment is not restored after the commonly proceeded stress recovery period.
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| 5. |
- Rabasco, Stefania
(författare)
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Correlative Chemical Imaging of Nanoscale Subcellular Structures
- 2023
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Doktorsavhandling (övrigt vetenskapligt/konstnärligt)abstract
- Chemical imaging can elucidate complex mechanisms, relationships, and components of biological samples. For example, it can reveal properties such as chemical composition, chemical structure, reactivity, and topography. Several imaging techniques exist, each providing different types of information. Yet, no single technique can comprehensively characterize a sample. Having a holistic profile often requires correlating complementary methods; for example, scanning electron microscopy (SEM) can be combined with secondary ion mass spectrometry (SIMS) imaging to obtain insights on both the physical topography (via SEM) and the chemical composition (via SIMS) of a sample surface. This approach is referred to as correlative imaging. Correlative chemical imaging is applicable in many scientific fields, such as biology, chemistry, geology, and material science. Among the wide variety of modern imaging techniques that exist, nanoscale SIMS (NanoSIMS) emerges as a powerful tool, having seen growing applications, especially in biochemistry and cell biology. To this end, it can be used for the detection of isotopically labeled material in a sample and provides the chemical composition of the sample surface with high lateral resolution (down to 50 nm), sensitivity (ppm-ppb range), and mass resolution (up to 10000). By using an isotopic label, target molecules in the sample can be studied, although unlabeled samples can be used in some cases. NanoSIMS presents some limitations; for example, it usually cannot discern the ultrastructure of very small, intricate sample details (e.g., subcellular ultrastructure). Therefore, NanoSIMS is often correlated with additional imaging techniques, such as microscopy, to push its capabilities and overcome its shortcomings. In the papers which are part of this thesis, NanoSIMS imaging was correlated with either electron or light microscopy to address different biological questions. To discern nanoscale subcellular ultrastructures, transmission electron microscopy (TEM) was employed, and to localize an organelle labeled with an antibody and a fluorescent tag, STED microscopy was used. In paper I, NanoSIMS was employed to detect 13C-dopamine in PC12 cells, and the images correlated with TEM to localize the dopamine within large dense core vesicles (LDCVs). In paper II, NanoSIMS was correlated with stimulated emission-depletion (STED) microscopy to localize endoplasmic reticulum stress-induced stress granules (SGs) in neuronal progenitor cells (NPCs) incubated with an isotopically labeled amino acid, and to characterize their protein turnover by changes in isotopic enrichment. In paper III, I investigated the role of vesicle size in the dynamics of partial release exocytosis events of PC12 cells by correlating TEM and NanoSIMS imaging data. In paper IV, NanoSIMS and TEM were correlated to look at the subcellular protein turnover in NPCs using different isotopically labeled amino acids and time-points. Overall, these studies demonstrate the importance of adequate correlative imaging strategies, and the variety of biological aims that can be achieved through different correlative chemical imaging approaches.
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| 6. |
- Rabasco, Stefania, et al.
(författare)
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Localization and absolute quantification of dopamine in discrete intravesicular compartments using nanoSIMS imaging
- 2022
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Ingår i: International Journal of Molecular Sciences. - : MDPI AG. - 1661-6596 .- 1422-0067. ; 23:1
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Tidskriftsartikel (refereegranskat)abstract
- The absolute concentration and the compartmentalization of analytes in cells and organelles are crucial parameters in the development of drugs and drug delivery systems, as well as in the fundamental understanding of many cellular processes. Nanoscale secondary ion mass spectrometry (NanoSIMS) imaging is a powerful technique which allows subcellular localization of chemical species with high spatial and mass resolution, and high sensitivity. In this study, we combined NanoSIMS imaging with spatial oversampling with transmission electron microscopy (TEM) imaging to discern the compartments (dense core and halo) of large dense core vesicles in a model cell line used to study exocytosis, and to localize13C dopamine enrichment following 4–6 h of 150 μM13C L-3,4-dihydroxyphenylalanine (L-DOPA) incubation. In addition, the absolute concentrations of13C dopamine in distinct vesicle domains as well as in entire single vesicles were quantified and validated by comparison to electrochemical data. We found concentrations of 87.5 mM, 16.0 mM and 39.5 mM for the dense core, halo and the whole vesicle, respectively. This approach adds to the potential of using combined TEM and NanoSIMS imaging to perform absolute quantification and directly measure the individual contents of nanometer-scale organelles.
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