Figure 3. A SNAP25 intramolecular FRET... | Rockefeller University Press

Figure 3.

$A SNAP25 intramolecular FRET probe signals the formation of the acceptor complex in the plasma membrane ∼100 ms before fusion.(A) Rationale of the use of the SNAP25 FRET probe to examine structural changes in SNAP25 upon interaction with syntaxin. Cerulean (Cer) and citrine (Cit) were used as the donor and acceptor fluorophores in the experiments of Wang et al. (2008). The orange rectangle in the N-terminal domain of the linker reflects palmitoylated cysteines. CFP and Venus were used as the donor and acceptor fluorophores in the experiments of Zhao et al. (2013). (B) The SNAP25 FRET probe detects interaction with syntaxin in biological membranes (Wang et al., 2008). HEK293T cells were transfected with a plasmid encoding the SNAP-25 FRET probe alone or together with a plasmid encoding syntaxin. Shown are the emission spectra of a suspended membrane fraction excited at 435 nm. The increased emission at 526 nm upon coexpression of syntaxin reflects a conformational change that brings the N termini of SN1 and SN2 closer together. From Wang et al. (2008). (C) FRET increases at the fusion site ∼100 ms before fusion pore formation (Zhao et al., 2013). Chromaffin cells were infected with virus to express the SNAP25 FRET probe. The time and location of fusion events in single cells were detected using a four-electrode electrochemical array to measure catecholamine release from individual granules. Total internal reflection fluorescence microscopy was used to detect local FRET changes. FRET changes from many hundreds of events were aligned to the beginning of the amperometric spike. A method was devised to account for the random timing of shutter opening relative to the fusion event. The timing of the half-maximal FRET change (dashed horizontal line) reflects the average time of the FRET change relative to fusion pore formation. It occurred 90 ms before amperometric spikes without a prespike foot, and 120 ms before amperometric spikes with a prespike foot. Fig. 3 is reprinted with permission from Proc. Natl. Acad. Sci. USA.$

A SNAP25 intramolecular FRET probe signals the formation of the acceptor complex in the plasma membrane ∼100 ms before fusion. (A) Rationale of the use of the SNAP25 FRET probe to examine structural changes in SNAP25 upon interaction with syntaxin. Cerulean (Cer) and citrine (Cit) were used as the donor and acceptor fluorophores in the experiments of Wang et al. (2008). The orange rectangle in the N-terminal domain of the linker reflects palmitoylated cysteines. CFP and Venus were used as the donor and acceptor fluorophores in the experiments of Zhao et al. (2013). (B) The SNAP25 FRET probe detects interaction with syntaxin in biological membranes (Wang et al., 2008). HEK293T cells were transfected with a plasmid encoding the SNAP-25 FRET probe alone or together with a plasmid encoding syntaxin. Shown are the emission spectra of a suspended membrane fraction excited at 435 nm. The increased emission at 526 nm upon coexpression of syntaxin reflects a conformational change that brings the N termini of SN1 and SN2 closer together. From Wang et al. (2008). (C) FRET increases at the fusion site ∼100 ms before fusion pore formation (Zhao et al., 2013). Chromaffin cells were infected with virus to express the SNAP25 FRET probe. The time and location of fusion events in single cells were detected using a four-electrode electrochemical array to measure catecholamine release from individual granules. Total internal reflection fluorescence microscopy was used to detect local FRET changes. FRET changes from many hundreds of events were aligned to the beginning of the amperometric spike. A method was devised to account for the random timing of shutter opening relative to the fusion event. The timing of the half-maximal FRET change (dashed horizontal line) reflects the average time of the FRET change relative to fusion pore formation. It occurred 90 ms before amperometric spikes without a prespike foot, and 120 ms before amperometric spikes with a prespike foot. Fig. 3 is reprinted with permission from Proc. Natl. Acad. Sci. USA.