QuasAr Odyssey: the origin of fluorescence and its voltage sensitivity in microbial rhodopsins.

Rhodopsins had long been considered non-fluorescent until a peculiar voltage-sensitive fluorescence was reported for archaerhodopsin-3 (Arch3) derivatives. These proteins named QuasArs have been used for imaging membrane voltage changes in cell cultures and small animals, but they could not be applied in living rodents. To develop the next generation of sensors, it is indispensable to first understand the molecular basis of the fluorescence and its modulation by the membrane voltage. Based on spectroscopic studies of fluorescent Arch3 derivatives, we propose a unique photo-reaction scheme with extended excited-state lifetimes and inefficient photoisomerization. Molecular dynamics simulations of Arch3, of the Arch3 fluorescent derivative Archon1, and of several its mutants have revealed different voltage-dependent changes of the hydrogen-bonding networks including the protonated retinal Schiff-base and adjacent residues. Experimental observations suggest that under negative voltage, these changes modulate retinal Schiff base deprotonation and promote a decrease in the populations of fluorescent species. Finally, we identified molecular constraints that further improve fluorescence quantum yield and voltage sensitivity.

Split-HaloTag imaging assay for sophisticated microscopy of protein-protein interactions in planta.

An ever-increasing number of intracellular multi-protein networks have been identified in plant cells. Split-GFP-based protein-protein interaction assays combine the advantages of in vivo interaction studies in a native environment with additional visualization of protein complex localization. Because of their simple protocols, they have become some of the most frequently used methods. However, standard fluorescent proteins present several drawbacks for sophisticated microscopy. With the HaloTag system, these drawbacks can be overcome, as this reporter forms covalent irreversible bonds with synthetic photostable fluorescent ligands. Dyes can be used in adjustable concentrations and are suitable for advanced microscopy methods. Therefore, we have established the Split-HaloTag imaging assay in plants, which is based on the reconstitution of a functional HaloTag protein upon protein-protein interaction and the subsequent covalent binding of an added fluorescent ligand. Its suitability and robustness were demonstrated using a well-characterized interaction as an example of protein-protein interaction at cellular structures: the anchoring of the molybdenum cofactor biosynthesis complex to filamentous actin. In addition, a specific interaction was visualized in a more distinctive manner with subdiffractional polarization microscopy, Airyscan, and structured illumination microscopy to provide examples of sophisticated imaging. Split-GFP and Split-HaloTag can complement one another, as Split-HaloTag represents an alternative option and an addition to the large toolbox of in vivo methods. Therefore, this promising new Split-HaloTag imaging assay provides a unique and sensitive approach for more detailed characterization of protein-protein interactions using specific microscopy techniques, such as 3D imaging, single-molecule tracking, and super-resolution microscopy.