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

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

doi:10.1101/2020.07.14.203562

Cited by 3 publications

(1 citation statement)

References 61 publications

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“…For example, investigating all three Plasmodium RNA polymerases (I, II and III) and assessing their spatiotemporal localization dynamics in multinucleated cells will be informative ( Zhao et al., 2016 ). Moreover, single-molecule fluorescence in situ hybridization (smFISH) of different RNA transcripts (e.g., ‘housekeeping’ genes and stage specific genes) can provide high-resolution snapshots of the transcription and localization of different RNAs at defined time points ( Kramer et al., 2020 ). Integrating such data with RNA species quantification in blood parasites and also liver and mosquito stages, will shed light on the potential mechanisms that are in play to buffer against the increasing DNA content.…”

Section: Increased Dna Content and Expression Homeostasismentioning

confidence: 99%

Plasmodium Reproduction, Cell Size, and Transcription: How to Cope With Increasing DNA Content?

Machado

Steinke

Ganter

2021

Front. Cell. Infect. Microbiol.

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Plasmodium, the unicellular parasite that causes malaria, evolved a highly unusual mode of reproduction. During its complex life cycle, invasive or transmissive stages alternate with proliferating stages, where a single parasite can produce tens of thousands of progeny. In the clinically relevant blood stage of infection, the parasite replicates its genome up to thirty times and forms a multinucleated cell before daughter cells are assembled. Thus, within a single cell cycle, Plasmodium develops from a haploid to a polypoid cell, harboring multiple copies of its genome. Polyploidy creates several biological challenges, such as imbalances in genome output, and cells can respond to this by changing their size and/or alter the production of RNA species and protein to achieve expression homeostasis. However, the effects and possible adaptations of Plasmodium to the massively increasing DNA content are unknown. Here, we revisit and embed current Plasmodium literature in the context of polyploidy and propose potential mechanisms of the parasite to cope with the increasing gene dosage.

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Section: Increased Dna Content and Expression Homeostasismentioning

confidence: 99%

Plasmodium Reproduction, Cell Size, and Transcription: How to Cope With Increasing DNA Content?

Machado

Steinke

Ganter

2021

Front. Cell. Infect. Microbiol.

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High-Resolution Chemical Mapping and Microbial Identification of Rhizosphere using Correlative Microscopy

Bandara

Schmidt

Davoudpour

et al. 2021

Preprint

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During the past decades, several stand-alone and combinatory 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 capable of characterizing soil-root-microbe interactions simultaneously in their spatial arrangement. Here we present a novel approach that allows chemical and microbial identification 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. Embedding quality was analyzed by Helium Ion Microscopy (HIM). Bacteria in the embedded soil was identified by Catalyzed Reporter Deposition-Fluorescence In Situ Hybridization (CARD-FISH) to avoid interferences from high levels of auto fluorescence 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), nanoSIMS and 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 chemically map the rhizosphere and identify spatial organization of bacteria. 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 ample understanding of the rhizosphere processes and their ecological significance.

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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 mRNA abundance, localisation and compactness with correlative single molecule FISH on LR White embedded samples

Cited by 3 publications

References 61 publications

Plasmodium Reproduction, Cell Size, and Transcription: How to Cope With Increasing DNA Content?

Plasmodium Reproduction, Cell Size, and Transcription: How to Cope With Increasing DNA Content?

High-Resolution Chemical Mapping and Microbial Identification of Rhizosphere using Correlative Microscopy

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

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