Bright ligand-activatable fluorescent protein for high-quality multicolor live-cell super-resolution microscopy

We introduce UnaG as a green-to-dark photoswitching fluorescent protein capable of high-quality super-resolution imaging with photon numbers equivalent to the brightest photoswitchable red protein. UnaG only fluoresces upon binding of a fluorogenic metabolite, bilirubin, enabling UV-free reversible photoswitching with easily controllable kinetics and low background under Epi illumination. The on- and off-switching rates are controlled by the concentration of the ligand and the excitation light intensity, respectively, where the dissolved oxygen also promotes the off-switching. The photo-oxidation reaction mechanism of bilirubin in UnaG suggests that the lack of ligand-protein covalent bond allows the oxidized ligand to detach from the protein, emptying the binding cavity for rebinding to a fresh ligand molecule. We demonstrate super-resolution single-molecule localization imaging of various subcellular structures genetically encoded with UnaG, which enables facile labeling and simultaneous multicolor imaging of live cells. UnaG has the promise of becoming a default protein for high-performance super-resolution imaging. Photoconvertible proteins occupy two color channels thereby limiting multicolour localisation microscopy applications. Here the authors present UnaG, a new green-to-dark photoswitching fluorescent protein for super-resolution imaging, whose activation is based on a noncovalent binding with bilirubin

Interaction between the vesicular glutamate transporter type 1 and endophilin A1, a protein essential for endocytosis

In the nerve terminal, neurotransmitter is actively packaged into synaptic vesicles before its release by Ca2+-dependent exocytosis. The three vesicular glutamate transporters (VGLUT1, -2 and -3) are highly conserved proteins that display similar bioenergetic and pharmacological properties but are expressed in different brain areas. We used the divergent C-terminus of VGLUT1 as a bait in a yeast two-hybrid screen to identify and map the interaction between a proline-rich domain of VGLUT1 and the Src homology domain 3 (SH3) domain of endophilin. We further confirmed this interaction by using different glutathione-S-transferase-endophilin fusion proteins to pull down VGLUT1 from rat brain extracts. The expression profiles of the two genes and proteins were compared on rat brain sections, showing that endophilin is most highly expressed in regions and cells expressing VGLUT1. Double immunofluorescence in the rat cerebellum shows that most VGLUT1-positive terminals co-express endophilin, whereas VGLUT2-expressing terminals are often devoid of endophilin. However, neither VGLUT1 transport activity, endophilin enzymatic activity nor VGLUT1 synaptic targeting were altered by this interaction. Overall, the discovery of endophilin as a partner for VGLUT1 in nerve terminals strongly suggests the existence of functional differences between VGLUT1 and -2 terminals in their abilities to replenish vesicle pools