Pharmacological Inhibition of the Vacuolar ATPase in Bloodstream-Form Trypanosoma brucei Rescues Genetic Knockdown of Mitochondrial Gene Expression

Schaffner-Barbero, Claudia; Miskinyte, Migla; Grewal, Jaspreet Singh; Schnaufer, Achim

doi:10.1128/aac.02268-17

Cited by 5 publications

(5 citation statements)

References 20 publications

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“…( C ) To obtain additional evidence for the specificity of the probe sets, we used chemically fixed BSF cells that lacked kDNA and therefore cannot produce mitochondrial transcripts. Such akinetoplastic trypanosomes can be produced by treatment with 10 nM ethidium bromide for three days ( 42 , 43 ) and, if cells carry the compensating mutation L262P in their ATPase gamma subunit, they can survive this treatment ( 44 , 45 ). Control cells and akinetoplastic trypanosomes were probed for edited and unedited A6 , and as a control, for the cytoplasmic mRNA FUTSCH .…”

Section: Resultsmentioning

confidence: 99%

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

Krämer

Meyer-Natus

Stigloher

et al. 2020

Nucleic Acids Research

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Single mRNA molecules are frequently detected by single molecule fluorescence in situ hybridization (smFISH) using branched DNA technology. While providing strong and background-reduced signals, the method is inefficient in detecting mRNAs within dense structures, in monitoring mRNA compactness and in quantifying abundant mRNAs. To overcome these limitations, we have hybridized slices of high pressure frozen, freeze-substituted and LR White embedded cells (LR White smFISH). mRNA detection is physically restricted to the surface of the resin. This enables single molecule detection of RNAs with accuracy comparable to RNA sequencing, irrespective of their abundance, while at the same time providing spatial information on RNA localization that can be complemented with immunofluorescence and electron microscopy, as well as array tomography. Moreover, LR White embedding restricts the number of available probe pair recognition sites for each mRNA to a small subset. As a consequence, differences in signal intensities between RNA populations reflect differences in RNA structures, and we show that the method can be employed to determine mRNA compactness. We apply the method to answer some outstanding questions related to trans-splicing, RNA granules and mitochondrial RNA editing in single-cellular trypanosomes and we show an example of differential gene expression in the metazoan Caenorhabditis elegans.

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Section: Resultsmentioning

confidence: 99%

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

Krämer

Meyer-Natus

Stigloher

et al. 2020

Nucleic Acids Research

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“…Subsequently, 47.5 μl of parasite culture in complete HMI-9 medium ( 29 ), supplemented with 20% (vol/vol) fetal calf serum, was seeded at 50 cells per well, giving a total volume of 50 μl with 1 × 10 3 cells/ml and a final compound concentration of 10 μM. The plates were incubated under an atmosphere of 5% CO 2 at 37°C for 4 days ( 30 ). Following incubation, the cells were stained first with the cytoplasmic viability stain 5(6)-carboxyfluorescein diacetate succinimidyl ester (CFDA-SE; CAS no.…”

Section: High-throughput Screen (Hts) Design and Image Analysismentioning

confidence: 99%

A Novel High-Content Phenotypic Screen To Identify Inhibitors of Mitochondrial DNA Maintenance in Trypanosomes

Miskinyte

Dawson

Makda

et al. 2022

Antimicrob Agents Chemother

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Kinetoplastid parasites cause diverse neglected diseases in humans and livestock, with an urgent need for new treatments. Survival of kinetoplastids depends on their uniquely structured mitochondrial genome (kDNA), the eponymous kinetoplast. Here we report development of a high-content screen for pharmacologically induced kDNA loss, based on specific staining of parasites and automated image analysis. As proof-of-concept we screened a diverse set of ∼14,000 small molecules and exemplify a validated hit as a novel kDNA-targeting compound.

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“…Both mRNAs are pan-edited 39,41 and of sufficiently large size to allow the design of specific probe sets against both the edited and unedited version, despite the presence of low complexity sequences that in particular edited mRNAs in particular are enriched for (Supplementary Figure S5, Figure 7A). through ethidium bromide treatment [42][43][44][45] . Both the edited and the unedited A6 signal were at least 13 times reduced, while a cytoplasmic control mRNA was only mildly affected (less than 2-fold reduction in dot number) ( Figure 7C and Supplementary Figure S7).…”

Section: Combination With Immunofluorescence and Electron Microscopymentioning

confidence: 99%

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

Krämer

Meyer-Natus

Thoma

et al. 2020

Preprint

Self Cite

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Single mRNA molecules are frequently detected by single molecule fluorescence in situ hybridisation (smFISH) using branched DNA technology. While providing strong and background-reduced signals, the method is inefficient in detecting mRNAs within dense structures, in monitoring mRNA compactness and in quantifying abundant mRNAs.To overcome these limitations, we have hybridised slices of high pressure frozen, LR White embedded cells (LR White smFISH). mRNA detection is physically restricted to the surface of the resin. This enables single molecule detection of RNAs with accuracy comparable to RNA sequencing, irrespective of their abundance, while at the same time providing spatial information on RNA localisation that can be complemented with immunofluorescence and electron microscopy, as well as electron tomography. Moreover, LR White embedding restricts the number of available probe pair recognition sites for each mRNA to a small subset. As a consequence, differences in signal intensities between RNA populations reflect differences in RNA tertiary structures, and we show that the method can be employed to probe for mRNA compactness. We apply LR White smFISH to answer some outstanding questions related to trans-splicing, RNA granules and mitochondrial RNA editing, using trypanosomes and their versatile RNA biology as a model system.

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Pharmacological Inhibition of the Vacuolar ATPase in Bloodstream-Form Trypanosoma brucei Rescues Genetic Knockdown of Mitochondrial Gene Expression

Cited by 5 publications

References 20 publications

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

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

A Novel High-Content Phenotypic Screen To Identify Inhibitors of Mitochondrial DNA Maintenance in Trypanosomes

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

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