Stable SNARE Complex Prior to Evoked Synaptic Vesicle Fusion Revealed by Fluorescence Resonance Energy Transfer

Zongping Xia,Qiong Zhou,Jialing Lin,Yuechueng Liu

doi:10.1074/jbc.m008741200

Abstract

Although it is clear that soluble N-ethylmaleimide-sensitive factor (NSF) attachment protein receptor (SNARE) complex plays an essential role in synaptic vesicle fusion, the dynamics of SNARE assembly during vesicle fusion remain to be determined. In this report, we employ fluorescence resonance energy transfer technique to study the formation of SNARE complexes. Donor/acceptor pair variants of green fluorescent protein (GFP), cyan fluorescent protein (CFP), and yellow fluorescent protein (YFP) are fused with the N termini of SNAP-25 and synaptobrevin, respectively. In vitro assembly of SNARE core complex in the presence of syntaxin shows strong fluorescence resonance energy transfer (FRET) between the CFP-SNAP-25 and YFP-synaptobrevin. Under the same conditions, CFP fused to the C terminus of SNAP-25, and YFP- synaptobrevin have no FRET. Adenovirus-mediated gene transfer is used to express the fusion proteins in PC12 cells and cultured rat cerebellar granule cells. Strong FRET is associated with neurite membranes and vesicular structures in PC12 cells co-expressing CFP-SNAP-25 and YFP-synaptobrevin. In cultured rat cerebellar granule cells, FRET between CFP-SNAP-25 and YFP-synaptobrevin is mostly associated with sites presumed to be synaptic junctions. Neurosecretion in PC12 cells initiated by KCl depolarization leads to an increase in the extent of FRET. These results demonstrate that significant amounts of stable SNARE complex exist prior to evoked synaptic vesicle fusion and that the assembly of SNARE complex occurs during vesicle docking/priming stage. Moreover, it demonstrates that FRET can be used as an effective tool for investigating dynamic SNARE interactions during synaptic vesicle fusion.

Highlights

The importance of SNARE complex in neurosecretion and the basic structure of the SNARE core complex have been well studied and established, the molecular mechanisms underlying its involvement in the membrane fusion process are less clear
It is clear that soluble N-ethylmaleimidesensitive factor (NSF) attachment protein receptor (SNARE) complex plays an essential role in synaptic vesicle fusion, the dynamics of SNARE assembly during vesicle fusion remain to be determined
1 The abbreviations used are: SNARE, soluble N-ethylmaleimidesensitive factor attachment protein receptor; SNAP-25, synaptosomal associated protein of 25 kDa; m.o.i., multiplicity of infection; green fluorescent protein (GFP), green fluorescence protein; CFP, cyan fluorescent protein; YFP, yellow fluorescent protein; C-SN, CFP fused to the N terminus of SNAP-25; SN-C, CFP fused to the C terminus of SNAP-25; Y-SB, YFP fused to the N terminus of synaptobrevin; FRET, fluorescence resonance energy transfer; NSF, N-ethylmaleimide-sensitive factor; PMA, phorbol 12-myristate 13-acetate; NGF, nerve growth factor; ROI, region of interest; GST, consists of target SNARE syntaxin and SNAP-25 and vesicle SNARE synaptobrevin/VAMP [1,2,3]

Summary

EXPERIMENTAL PROCEDURES

Mutagenesis and Preparation of GFP Fusion Proteins—A mouse cDNA encoding synaptobrevin-2 was purchased from the I.M.A.G.E. The following primary antibodies were used: the HPC-1 monoclonal anti-syntaxin (1: 1,000), a goat anti-SNAP-25 antibody (1:10,000) [27], and a goat antisynaptobrevin (1:5,000) raised against a GST-synaptobrevin fusion protein. In Vitro SNARE Core Complex Formation and Spectrofluorometer Assay—COS-7 cells were infected with recombinant adenoviruses to express C-SN, SN-C, and Y-SB. Microscopy, Image Analysis, and FRET Calculation—PC12 cells or primary cerebellar neurons were infected with adenoviruses expressing ECFP and EYFP fusion proteins. For detection of ECFP, cells were viewed with an inverted fluorescence microscope (Leica DMIL) under a filter set with an excitation filter of 440/21 nm, a dichroic beam splitter of 455, and an emission filter of 480/30 nm. EYFP expressing cells were viewed under a filter set with an excitation filter of 500/25 nm, a dichroic beam splitter of 525 nm, and an emission filter of 545/35 nm. The images were adjusted pixel-by-pixel using a reference channel of ROI

RESULTS

DISCUSSION

Granule cellsb