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Single-molecule super-resolution microscopy allows imaging of fluorescently-tagged proteins in live cells with a precision well below that of the diffraction limit. Here, we demonstrate 3D sectioning with single-molecule super-resolution microscopy by making use of the fitting information that is usually discarded to reject fluorophores that emit from above or below a virtual-'light-sheet', a thin volume centred on the focal plane of the microscope. We describe an easy-to-use routine (implemented as an open-source ImageJ plug-in) to quickly analyse a calibration sample to define and use such a virtual light-sheet. In addition, the plug-in is easily usable on almost any existing 2D super-resolution instrumentation. This optical sectioning of super-resolution images is achieved by applying well-characterised width and amplitude thresholds to diffraction-limited spots that can be used to tune the thickness of the virtual light-sheet. This allows qualitative and quantitative imaging improvements: by rejecting out-of-focus fluorophores, the super-resolution image gains contrast and local features may be revealed; by retaining only fluorophores close to the focal plane, virtual-'light-sheet' single-molecule localisation microscopy improves the probability that all emitting fluorophores will be detected, fitted and quantitatively evaluated.
Prostaglandin E2 (PGE2) favors multiple aspects of tumor development and immune evasion. Therefore, microsomal prostaglandin E synthase (mPGES-1/-2), is a potential target for cancer therapy. We explored whether inhibiting mPGES-1 in human and mouse models of breast cancer affects tumor-associated immunity. A new model of breast tumor spheroid killing by human PBMCs was developed. In this model, tumor killing required CD80 expression by tumor-associated phagocytes to trigger cytotoxic T cell activation. Pharmacological mPGES-1 inhibition increased CD80 expression, whereas addition of PGE2, a prostaglandin E2 receptor 2 (EP2) agonist, or activation of signaling downstream of EP2 reduced CD80 expression. Genetic ablation of mPGES-1 resulted in markedly reduced tumor growth in PyMT mice. Macrophages of mPGES-1-/- PyMT mice indeed expressed elevated levels of CD80 compared to their wildtype counterparts. CD80 expression in tumor-spheroid infiltrating mPGES-1-/- macrophages translated into antigen-specific cytotoxic T cell activation. In conclusion, mPGES-1 inhibition elevates CD80 expression by tumor-associated phagocytes to restrict tumor growth. We propose that mPGES-1 inhibition in combination with immune cell activation might be part of a therapeutic strategy to overcome the immunosuppressive tumor microenvironment.
African trypanosomes cause a parasitic disease known as sleeping sickness. Mitochondrial transcript maturation in these organisms requires a RNA editing reaction that is characterized by the insertion and deletion of U-nucleotides into otherwise non-functional mRNAs. Editing represents an ideal target for a parasite-specific therapeutic intervention since the reaction cycle is absent in the infected host. In addition, editing relies on a macromolecular protein complex, the editosome, that only exists in the parasite. Therefore, all attempts to search for editing interfering compounds have been focused on molecules that bind to proteins of the editing machinery. However, in analogy to other RNA-driven biochemical pathways it should be possible to stall the reaction by targeting its substrate RNAs. Here we demonstrate inhibition of editing by specific aminoglycosides. The molecules bind into the major groove of the gRNA/pre-mRNA editing substrates thereby causing a stabilization of the RNA molecules through charge compensation and an increase in stacking. The data shed light on mechanistic details of the editing process and identify critical parameters for the development of new trypanocidal compounds.
In eukaryotic cells, mitochondria host ancient essential bioenergetic and biosynthetic pathways. LYR (leucine/tyrosine/arginine) motif proteins (LYRMs) of the Complex1_LYR-like superfamily interact with protein complexes of bacterial origin. Many LYR proteins function as extra subunits (LYRM3 and LYRM6) or novel assembly factors (LYRM7, LYRM8, ACN9 and FMC1) of the oxidative phosphorylation (OXPHOS) core complexes. Structural insights into complex I accessory subunits LYRM6 and LYRM3 have been provided by analyses of EM and X-ray structures of complex I from bovine and the yeast Yarrowia lipolytica, respectively. Combined structural and biochemical studies revealed that LYRM6 resides at the matrix arm close to the ubiquinone reduction site. For LYRM3, a position at the distal proton-pumping membrane arm facing the matrix space is suggested. Both LYRMs are supposed to anchor an acyl-carrier protein (ACPM) independently to complex I. The function of this duplicated protein interaction of ACPM with respiratory complex I is still unknown. Analysis of protein-protein interaction screens, genetic analyses and predicted multi-domain LYRMs offer further clues on an interaction network and adaptor-like function of LYR proteins in mitochondria.