Photoswitching fingerprint analysis bypasses the 10-nm resolution barrier.

Advances in super-resolution microscopy have demonstrated single-molecule localization precisions of a few nanometers. However, translation of such high localization precisions into sub-10-nm spatial resolution in biological samples remains challenging. Here we show that resonance energy transfer between fluorophores separated by less than 10 nm results in accelerated fluorescence blinking and consequently lower localization probabilities impeding sub-10-nm fluorescence imaging.

Genetic Code Expansion and Click-Chemistry Labeling to Visualize GABA-A Receptors by Super-Resolution Microscopy.

Fluorescence labeling of difficult to access protein sites, e.g., in confined compartments, requires small fluorescent labels that can be covalently tethered at well-defined positions with high efficiency.

Bioorthogonal labeling of transmembrane proteins with non-canonical amino acids unveils masked epitopes in live neurons.

Progress in biological imaging is intrinsically linked to advances in labeling methods. The explosion in the development of high-resolution and super-resolution imaging calls for new approaches to label targets with small probes. These should allow to faithfully report the localization of the target within the imaging resolution - typically nowadays a few nanometers - and allow access to any epitope of the target, in the native cellular and tissue environment.

Tethered agonist exposure in intact adhesion/class B2 GPCRs through intrinsic structural flexibility of the GAIN domain.

Adhesion G protein-coupled receptors (aGPCRs)/family B2 GPCRs execute critical tasks during development and the operation of organs, and their genetic lesions are associated with human disorders, including cancers. Exceptional structural aGPCR features are the presence of a tethered agonist (TA) concealed within a GPCR autoproteolysis-inducing (GAIN) domain and their non-covalent heteromeric two-subunit layout. How the TA is poised for activation while maintaining this delicate receptor architecture is central to conflicting signaling paradigms that either involve or exclude aGPCR heterodimer separation.

Bioorthogonal labeling with tetrazine-dyes for super-resolution microscopy.

Genetic code expansion (GCE) technology allows the specific incorporation of functionalized noncanonical amino acids (ncAAs) into proteins. Here, we investigated the Diels-Alder reaction between trans-cyclooct-2-ene (TCO)-modified ncAAs, and 22 known and novel 1,2,4,5-tetrazine-dye conjugates spanning the entire visible wavelength range. A hallmark of this reaction is its fluorogenicity - the tetrazine moiety can elicit substantial quenching of the dye.

The Neutral Sphingomyelinase 2 Is Required to Polarize and Sustain T Cell Receptor Signaling.

By promoting ceramide release at the cytosolic membrane leaflet, the neutral sphingomyelinase 2 (NSM) is capable of organizing receptor and signalosome segregation. Its role in T cell receptor (TCR) signaling remained so far unknown. We now show that TCR-driven NSM activation is dispensable for TCR clustering and initial phosphorylation, but of crucial importance for further signal amplification.