| 1. |
- Lange, Felix, et al.
(författare)
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Correlative fluorescence microscopy, transmission electron microscopy and secondary ion mass spectrometry (CLEM-SIMS) for cellular imaging
- 2021
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Ingår i: PLoS ONE. - 1932-6203. ; 16
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Tidskriftsartikel (refereegranskat)abstract
- Electron microscopy (EM) has been employed for decades to analyze cell structure. To also analyze the positions and functions of specific proteins, one typically relies on immuno-EM or on a correlation with fluorescence microscopy, in the form of correlated light and electron microscopy (CLEM). Nevertheless, neither of these procedures is able to also address the isotopic composition of cells. To solve this, a correlation with secondary ion mass spectrometry (SIMS) would be necessary. SIMS has been correlated in the past to EM or to fluorescence microscopy in biological samples, but not to CLEM. We achieved this here, using a protocol based on transmission EM, conventional epifluorescence microscopy and nanoSIMS. The protocol is easily applied, and enables the use of all three technologies at high performance parameters. We suggest that CLEM-SIMS will provide substantial information that is currently beyond the scope of conventional correlative approaches.
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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. |
- Meschkat, Martin, et al.
(författare)
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White matter integrity in mice requires continuous myelin synthesis at the inner tongue
- 2022
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Ingår i: NATURE COMMUNICATIONS. - : Springer Science and Business Media LLC. - 2041-1723. ; 13:1
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Tidskriftsartikel (refereegranskat)abstract
- Myelin is formed of proteins of long half-lives. The mechanisms of renewal of such a stable structure are unclear. Here, the authors show that myelin integrity requires continuous myelin synthesis at the inner tongue, contributing to the maintenance of a functional axon-myelin unit. Myelin, the electrically insulating sheath on axons, undergoes dynamic changes over time. However, it is composed of proteins with long lifetimes. This raises the question how such a stable structure is renewed. Here, we study the integrity of myelinated tracts after experimentally preventing the formation of new myelin in the CNS of adult mice, using an inducible Mbp null allele. Oligodendrocytes survive recombination, continue to express myelin genes, but they fail to maintain compacted myelin sheaths. Using 3D electron microscopy and mass spectrometry imaging we visualize myelin-like membranes failing to incorporate adaxonally, most prominently at juxta-paranodes. Myelinoid body formation indicates degradation of existing myelin at the abaxonal side and the inner tongue of the sheath. Thinning of compact myelin and shortening of internodes result in the loss of about 50% of myelin and axonal pathology within 20 weeks post recombination. In summary, our data suggest that functional axon-myelin units require the continuous incorporation of new myelin membranes.
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| 4. |
- Richter, Katharina N., et al.
(författare)
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Glyoxal as an alternative fixative to formaldehyde in immunostaining and super-resolution microscopy
- 2018
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Ingår i: EMBO Journal. - : WILEY. - 0261-4189 .- 1460-2075. ; 37:1, s. 139-159
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Tidskriftsartikel (refereegranskat)abstract
- Paraformaldehyde (PFA) is the most commonly used fixative for immunostaining of cells, but has been associated with various problems, ranging from loss of antigenicity to changes in morphology during fixation. We show here that the small dialdehyde glyoxal can successfully replace PFA. Despite being less toxic than PFA, and, as most aldehydes, likely usable as a fixative, glyoxal has not yet been systematically tried in modern fluorescence microscopy. Here, we tested and optimized glyoxal fixation and surprisingly found it to be more efficient than PFA-based protocols. Glyoxal acted faster than PFA, cross-linked proteins more effectively, and improved the preservation of cellular morphology. We validated glyoxal fixation in multiple laboratories against different PFA-based protocols and confirmed that it enabled better immunostainings for a majority of the targets. Our data therefore support that glyoxal can be a valuable alternative to PFA for immunostaining.
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| 5. |
- Richter, Katharina N., et al.
(författare)
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Review of combined isotopic and optical nanoscopy
- 2017
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Ingår i: Neurophotonics. - 2329-4248 .- 2329-423X. ; 4:2, s. 020901-
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Forskningsöversikt (refereegranskat)abstract
- Investigating the detailed substructure of the cell is beyond the ability of conventional optical microscopy. Electron microscopy, therefore, has been the only option for such studies for several decades. The recent implementation of several super-resolution optical microscopy techniques has rendered the investigation of cellular substructure easier and more efficient. Nevertheless, optical microscopy only provides an image of the present structure of the cell, without any information on its long-Temporal changes. These can be investigated by combining super-resolution optics with a nonoptical imaging technique, nanoscale secondary ion mass spectrometry, which investigates the isotopic composition of the samples. The resulting technique, combined isotopic and optical nanoscopy, enables the investigation of both the structure and the "history" of the cellular elements. The age and the turnover of cellular organelles can be read by isotopic imaging, while the structure can be analyzed by optical (fluorescence) approaches. We present these technologies, and we discuss their implementation for the study of biological samples. We conclude that, albeit complex, this type of technology is reliable enough for mass application to cell biology.
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| 6. |
- Roeder, Sebastian S., et al.
(författare)
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Tracking cell turnover in human brain using 15N-thymidine imaging mass spectrometry
- 2023
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Ingår i: Frontiers in Neuroscience. - 1662-4548. ; 17
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Tidskriftsartikel (refereegranskat)abstract
- Microcephaly is often caused by an impairment of the generation of neurons in the brain, a process referred to as neurogenesis. While most neurogenesis in mammals occurs during brain development, it thought to continue to take place through adulthood in selected regions of the mammalian brain, notably the hippocampus. However, the generality of neurogenesis in the adult brain has been controversial. While studies in mice and rats have provided compelling evidence for neurogenesis occurring in the adult rodent hippocampus, the lack of applicability in humans of key methods to demonstrate neurogenesis has led to an intense debate about the existence and, in particular, the magnitude of neurogenesis in the adult human brain. Here, we demonstrate the applicability of a powerful method to address this debate, that is, the in vivo labeling of adult human patients with 15N-thymidine, a non-hazardous form of thymidine, an approach without any clinical harm or ethical concerns. 15N-thymidine incorporation into newly synthesized DNA of specific cells was quantified at the single-cell level with subcellular resolution by Multiple-isotype imaging mass spectrometry (MIMS) of brain tissue resected for medical reasons. Two adult human patients, a glioblastoma patient and a patient with drug-refractory right temporal lobe epilepsy, were infused for 24 h with 15N-thymidine. Detection of 15N-positive leukocyte nuclei in blood samples from these patients confirmed previous findings by others and demonstrated the appropriateness of this approach to search for the generation of new cells in the adult human brain. 15N-positive neural cells were easily identified in the glioblastoma tissue sample, and the range of the 15N signal suggested that cells that underwent S-phase fully or partially during the 24 h in vivo labeling period, as well as cells generated therefrom, were detected. In contrast, within the hippocampus tissue resected from the epilepsy patient, none of the 2,000 dentate gyrus neurons analyzed was positive for 15N-thymidine uptake, consistent with the notion that the rate of neurogenesis in the adult human hippocampus is rather low. Of note, the likelihood of detecting neurogenesis was reduced because of (i) the low number of cells analyzed, (ii) the fact that hippocampal tissue was explored that may have had reduced neurogenesis due to epilepsy, and (iii) the labeling period of 24 h which may have been too short to capture quiescent neural stem cells. Yet, overall, our approach to enrich NeuN-labeled neuronal nuclei by FACS prior to MIMS analysis provides a promising strategy to quantify even low rates of neurogenesis in the adult human hippocampus after in vivo15N-thymidine infusion. From a general point of view and regarding future perspectives, the in vivo labeling of humans with 15N-thymidine followed by MIMS analysis of brain tissue constitutes a novel approach to study mitotically active cells and their progeny in the brain, and thus allows a broad spectrum of studies of brain physiology and pathology, including microcephaly.
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| 7. |
- Szegő, Éva M., et al.
(författare)
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Cytosolic Trapping of a Mitochondrial Heat Shock Protein Is an Early Pathological Event in Synucleinopathies
- 2019
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Ingår i: Cell Reports. - : Elsevier BV. - 2211-1247. ; 28:1, s. 6-77
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Tidskriftsartikel (refereegranskat)abstract
- Alpha-synuclein (aSyn) accumulates in intracellular inclusions in synucleinopathies, but the molecular mechanisms leading to disease are unclear. We identify the 10 kDa heat shock protein (HSP10) as a mediator of aSyn-induced mitochondrial impairments in striatal synaptosomes. We find an age-associated increase in the cytosolic levels of HSP10, and a concomitant decrease in the mitochondrial levels, in aSyn transgenic mice. The levels of superoxide dismutase 2, a client of the HSP10/HSP60 folding complex, and synaptosomal spare respiratory capacity are also reduced. Overexpression of HSP10 ameliorates aSyn-associated mitochondrial dysfunction and delays aSyn pathology in vitro and in vivo. Altogether, our data indicate that increased levels of aSyn induce mitochondrial deficits, at least partially, by sequestering HSP10 in the cytosol and preventing it from acting in mitochondria. Importantly, these alterations manifest first at presynaptic terminals. Our study not only provides mechanistic insight into synucleinopathies but opens new avenues for targeting underlying cellular pathologies. Szegő et al. identify HSP10 as a modulator of alpha-synuclein-induced mitochondrial impairment in striatal synaptosomes. Age-associated increase in the cytosolic and decrease in mitochondrial levels of HSP10 results in a reduction in the levels of SOD2 and of synaptosomal ATP production on demand. HSP10 overexpression delays alpha-synuclein pathology both in vitro and in vivo.
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