Homodimeric class I cytokine receptors are assumed to exist as preformed dimers that are activated by ligand-induced conformational changes. We quantified the dimerization of three prototypic class I cytokine receptors in the plasma membrane of living cells by single-molecule fluorescence microscopy. Spatial and spatiotemporal correlation of individual receptor subunits showed ligand-induced dimerization and revealed that the associated Janus kinase 2 (JAK2) dimerizes through its pseudokinase domain. Oncogenic receptor and hyperactive JAK2 mutants promoted ligand-independent dimerization, highlighting the formation of receptor dimers as the switch responsible for signal activation. Atomistic modeling and molecular dynamics simulations based on a detailed energetic analysis of the interactions involved in dimerization yielded a mechanistic blueprint for homodimeric class I cytokine receptor activation and its dysregulation by individual mutations.

Type IV pili are ubiquitous bacterial motors that power surface motility. In peritrichously piliated species, it is unclear how multiple pili are coordinated to generate movement with directional persistence. Here we use a combined theoretical and experimental approach to test the hypothesis that multiple pili of Neisseria gonorrhoeae are coordinated through a tug-of-war. Based on force-dependent unbinding rates and pilus retraction speeds measured at the level of single pili, we build a tug-of-war model. Whereas the one-dimensional model robustly predicts persistent movement, the two-dimensional model requires a mechanism of directional memory provided by re-elongation of fully retracted pili and pilus bundling. Experimentally, we confirm memory in the form of bursts of pilus retractions. Bursts are seen even with bundling suppressed, indicating re-elongation from stable core complexes as the key mechanism of directional memory. Directional memory increases the surface range explored by motile bacteria and likely facilitates surface colonization.

In various bacterial species surface motility is mediated by cycles of type IV pilus motor elongation, adhesion, and retraction, but it is unclear whether bacterial movement follows a random walk. Here we show that the correlation time of persistent movement in Neisseria gonorrhoeae increases with the number of pili. The unbinding force of individual pili from the surface F=10 pN was considerably lower than the stalling force F>100 pN, suggesting that density, force, and adhesive properties of the pilus motor enable a tug-of-war mechanism for bacterial movement.

Niewidok et al. analyze the distribution and dynamics of RNA-binding proteins (RBPs) with single-molecule resolution in living neuronal cells, providing direct support for liquid droplet behavior of stress granules in living cells and revealing transient binding of RBPs in nanocores.

Upconversion nanoparticles (UCNPs) convert near-infrared into visible light at much lower excitation densities than those used in classic two-photon absorption microscopy. Here, we engineered <50 nm UCNPs for application as efficient lanthanide resonance energy transfer (LRET) donors inside living cells. By optimizing the dopant concentrations and the core-shell structure for higher excitation densities, we observed enhanced UCNP emission as well as strongly increased sensitized acceptor fluorescence. For the application of these UCNPs in complex biological environments, we developed a biocompatible surface coating functionalized with a nanobody recognizing green fluorescent protein (GFP). Thus, rapid and specific targeting to GFP-tagged fusion proteins in the mitochondrial outer membrane and detection of protein interactions by LRET in living cells was achieved.