Parallel monitoring of RNA abundance, localization and compactness with correlative single molecule FISH on LR White embedded samples

Krämer, Susanne; Meyer-Natus, Elisabeth; Stigloher, Christian; Thoma, Hanna; Schnaufer, Achim; Engstler, Markus

doi:10.1093/nar/gkaa1142

Cited by 8 publications

(7 citation statements)

References 57 publications

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“…Overall, it is important to note that the fixation and resin embedding steps have to be carefully selected depending on the specific analysis planned. The soft LR white resin we choose is used to embed and preserve microbial cells in various tissues, the environmental samples in correlation with FISH and TEM to study functions of microbes in natural systems (Knierim et al, 2012;Schimak et al, 2012;Susanne Kramer et al, 2020). However, LR white has not yet been used for soils or rhizosphere samples in high resolution workflows where bacteria-plantorganic-inorganic interphases are correlatively characterized.…”

Section: Resultsmentioning

confidence: 99%

Microbial Identification, High-Resolution Microscopy and Spectrometry of the Rhizosphere in Its Native Spatial Context

et al. 2021

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During the past decades, several stand-alone and combinatorial methods have been developed to investigate the chemistry (i.e., mapping of elemental, isotopic, and molecular composition) and the role of microbes in soil and rhizosphere. However, none of these approaches are currently applicable to characterize soil-root-microbe interactions simultaneously in their spatial arrangement. Here we present a novel approach that allows for simultaneous microbial identification and chemical analysis of the rhizosphere at micro− to nano-meter spatial resolution. Our approach includes (i) a resin embedding and sectioning method suitable for simultaneous correlative characterization of Zea mays rhizosphere, (ii) an analytical work flow that allows up to six instruments/techniques to be used correlatively, and (iii) data and image correlation. Hydrophilic, immunohistochemistry compatible, low viscosity LR white resin was used to embed the rhizosphere sample. We employed waterjet cutting and avoided polishing the surface to prevent smearing of the sample surface at nanoscale. The quality of embedding was analyzed by Helium Ion Microscopy (HIM). Bacteria in the embedded soil were identified by Catalyzed Reporter Deposition-Fluorescence in situ Hybridization (CARD-FISH) to avoid interferences from high levels of autofluorescence emitted by soil particles and organic matter. Chemical mapping of the rhizosphere was done by Scanning Electron Microscopy (SEM) with Energy-dispersive X-ray analysis (SEM-EDX), Time-of-Flight Secondary Ion Mass Spectrometry (ToF-SIMS), nano-focused Secondary Ion mass Spectrometry (nanoSIMS), and confocal Raman spectroscopy (μ-Raman). High-resolution correlative characterization by six different techniques followed by image registration shows that this method can meet the demanding requirements of multiple characterization techniques to identify spatial organization of bacteria and chemically map the rhizosphere. Finally, we presented individual and correlative workflows for imaging and image registration to analyze data. We hope this method will be a platform to combine various 2D analytics for an improved understanding of the rhizosphere processes and their ecological significance.

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Section: Resultsmentioning

confidence: 99%

Microbial Identification, High-Resolution Microscopy and Spectrometry of the Rhizosphere in Its Native Spatial Context

et al. 2021

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“…Another important evidence parting eIF4E3 and PABP1 functions is the localization of their mRNAs and proteins associated in starvation stress granules (SG). mRNAs encoding ribosomal proteins are underrepresented from SG [83], [84]. T. brucei PABP2, eIF4E3, eIF4G4 and most of PABP2 interaction partners localize to SG, where PABP1, eIF4E4, eIF4A1 and the other PABP1 partners are largely absent [36], [85].…”

Section: Discussionmentioning

confidence: 99%

Two distinctTrypanosomaeIF4F complexes co-exist, bind different mRNAs and are regulated during nutritional stress

Gabiatti,

Freire,

da Costa

et al. 2024

Preprint

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Many eIF4F subunits and PABP paralogues are found in trypanosomes: six eIF4E, five eIF4G, one eIF4A and two PABPs. They are expressed simultaneously and assemble into different complexes, contrasting the situation in metazoans that use distinct complexes in different cell types or developmental stages. Each eIF4F complex has its own proteins, mRNAs and, consequently, a distinct function. We set out to study the function and regulation of the two major eIF4F complexes of Trypanosoma cruzi. We identified the associated proteins and mRNAs of eIF4E3 and eIF4E4 in cells in exponential growth and in nutritional stress. Upon stress, eIF4G/eIF4A and PABP remain associated to the eIF4E, but the association with other 43S pre-initiation factors decreases, indicating that ribosome attachment is impaired. Most eIF4E3-associated mRNAs encode for metabolic proteins, while eIF4E4 associate to mRNAs encoding ribosomal proteins. Interestingly, for both eIF4E3/4, more mRNAs were associated in stressed cells than in non-stressed cells, even though these mRNAs have lower translational efficiencies in stress. In summary, trypanosomes have two co-existing eIF4F complexes involved in translation of distinct mRNA cohorts important for growth. Under stress conditions, both complexes exit translation but remain bound to their mRNA targets.

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“…Presentations are either as single plane or as Z-stack projection (sum slices), as indicated. Scanning electron microscopy and correlation with light microscopy was done as previously described (Kramer et al, 2020).…”

Section: Microscopymentioning

confidence: 99%

Beyond BioID: Streptavidin outcompetes antibody fluorescence signals in protein localization and readily visualises targets evading immunofluorescence detection

Odenwald,

Gabiatti,

Braune

et al. 2023

Preprint

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Immunofluorescence is a common method to localise proteins within their cellular context via fluorophore labelled antibodies and for some applications without alternative. However, some protein targets evade detection due to low protein abundance or accessibility issues. In addition, some imaging methods require a massive reduction in antigen density thus impeding detection of even medium-abundant proteins.Here, we show that the fusion of the target protein to TurboID, a biotin ligase labelling lysine residues in close proximity, and subsequent detection of biotinylation by fluorescent streptavidin offers an “all in one” solution to the above-mentioned restrictions. For a wide range of target proteins tested, the streptavidin signal was significantly stronger than an antibody signal, markedly improving the imaging sensitivity in expansion microscopy and correlative light and electron microscopy, with no loss in resolution. Importantly, proteins within phase-separated regions, such as the central channel of the nuclear pores, the nucleolus or RNA granules, were readily detected with streptavidin, while most antibodies fail to label proteins in these environments. When TurboID is used in tandem with an HA epitope tag, co-probing with streptavidin and anti-HA can be used to map antibody-accessibility to certain cellular regions. As a proof of principle, we mapped antibody access to all trypanosome nuclear pore proteins (NUPs) and found restricted antibody labelling of all FG NUPs of the central channel that are known to be phase-separated, while most non-FG Nups could be labelled. Lastly, we show that streptavidin imaging can resolve dynamic, temporally and spatially distinct sub-complexes and, in specific cases, reveal a history of dynamic protein interaction.In conclusion, streptavidin imaging has major advantages for the detection of lowly abundant or inaccessible proteins and in addition, can provide information on protein interactions and biophysical environment.

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Parallel monitoring of RNA abundance, localization and compactness with correlative single molecule FISH on LR White embedded samples

Cited by 8 publications

References 57 publications

Microbial Identification, High-Resolution Microscopy and Spectrometry of the Rhizosphere in Its Native Spatial Context

Microbial Identification, High-Resolution Microscopy and Spectrometry of the Rhizosphere in Its Native Spatial Context

Two distinctTrypanosomaeIF4F complexes co-exist, bind different mRNAs and are regulated during nutritional stress

Beyond BioID: Streptavidin outcompetes antibody fluorescence signals in protein localization and readily visualises targets evading immunofluorescence detection

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