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. 2016 Jun 30;8:16. doi: 10.3389/fnsyn.2016.00016

Figure 1.

Figure 1

Scheme of single component synapse detectors. (A) Graphical depiction of the conventional green fluorescent protein (GFP) tagging scheme and summary of the expressed carrier-sensor construct. GFP (green circle) and other fluorescent proteins (FPs) are directly tagged to synaptic proteins in the presynaptic terminal or postsynaptic spines. Major targets for tagging include presynaptic vesicle proteins (e.g., vesicle-associated membrane protein 2 (VAMP2) and synaptophysin), postsynaptic receptors (e.g., α-amino-hydroxy-5-methyl-4-isoxazolepropionic acid receptor, AMPAR) and postsynaptic density protein-95 (PSD-95). (B) pH-sensitive FP mutants are fused to synaptic vesicle membrane proteins, such as VAMP2, to visualize vesicle secretion/recycling and neurotransmission. A pH-sensitive GFP variant, pHluorin does not fluoresce (gray circle) when inside the acidic chemical environment of the synaptic vesicle, but becomes highly fluorescent (green circle) when the vesicle is released and exposed to the neutral extracellular environment. (C) Inhibition of Synaptic Release with CALI (InSynC): attached to target SNARE proteins, molecular actuators such as mini small singlet oxygen generator (miniSOG; light blue filled-in circle) selectively inactivate specific synaptic proteins that regulate vesicle release and other synaptic events. When illuminated with blue light, miniSOG stimulates generation of reactive oxygen species (small, dark blue filled-in circles), which then oxidizes susceptible amino acid residues in target vesicle proteins and deactivates protein functions. (D) TimeSTAMP effectively tracks spatiotemporally controlled protein synthesis and trafficking in living neurons. In the presence of a membrane-permeable protease inhibitor, NS3 protease (gray oval) activity is inhibited, and Venus C-terminus (Venus CT) and Venus N-terminus (Venus NT) reconstitute as fluorescent Venus (yellow circle). Reconstituted Venus accumulates in the postsynaptic spine, the trafficking destination of the fused PSD-95. When the protease inhibitor is present, however, NS3 protease cleaves the protease target sites (gray circles flanking NS3), preventing Venus reconstitution.