Analysis of SNARE-mediated Membrane Fusion Using an Enzymatic Cell Fusion Assay

Summary

We have developed a cell fusion assay that quantifies SNARE-mediated membrane fusion events by activated expression of β-galactosidase.

Abstract

The interactions of SNARE (soluble N-ethylmaleimide-sensitive factor attachment protein receptor) proteins on vesicles (v-SNAREs) and on target membranes (t-SNAREs) catalyze intracellular vesicle fusion^1-4. Reconstitution assays are essential for dissecting the mechanism and regulation of SNARE-mediated membrane fusion⁵. In a cell fusion assay^6,7, SNARE proteins are expressed ectopically at the cell surface. These "flipped" SNARE proteins drive cell-cell fusion, demonstrating that SNAREs are sufficient to fuse cellular membranes. Because the cell fusion assay is based on microscopic analysis, it is less efficient when used to analyze multiple v- and t-SNARE interactions quantitatively.

Here we describe a new assay⁸ that quantifies SNARE-mediated cell fusion events by activated expression of β-galactosidase. Two components of the Tet-Off gene expression system⁹ are used as a readout system: the tetracycline-controlled transactivator (tTA) and a reporter plasmid that encodes the LacZ gene under control of the tetracycline-response element (TRE-LacZ). We transfect tTA into COS-7 cells that express flipped v-SNARE proteins at the cell surface (v-cells) and transfect TRE-LacZ into COS-7 cells that express flipped t-SNARE proteins at the cell surface (t-cells). SNARE-dependent fusion of the v- and t-cells results in the binding of tTA to TRE, the transcriptional activation of LacZ and expression of β-galactosidase. The activity of β-galactosidase is quantified using a colorimetric method by absorbance at 420 nm.

The vesicle-associated membrane proteins (VAMPs) are v-SNAREs that reside in various post-Golgi vesicular compartments^10-15. By expressing VAMPs 1, 3, 4, 5, 7 and 8 at the same level, we compare their membrane fusion activities using the enzymatic cell fusion assay. Based on spectrometric measurement, this assay offers a quantitative approach for analyzing SNARE-mediated membrane fusion and for high-throughput studies.

Protocol

1. Cell Culture and Transfection

1. COS-7 cells are cultured in Dulbecco Modified Eagle's Medium (DMEM) supplemented with 4.5 g/l glucose and 10% fetal bovine serum (FBS).
2. Plasmid transfection is done with Lipofectamine according to the manufacturer's instructions (Invitrogen).

2. Fluorescence Microscopic Analysis of Cell Surface SNARE Expression

1. The day before transfection, 3×10⁴ COS-7 cells are seeded on sterile 12-mm glass coverslips (Fisher Scientific) contained in 24-well plates.
2. For v-cells in each well, 0.25 μg of the plasmid that encodes tTA (pTet-Off, CLONTECH) is ....

Results

To develop a quantitative cell fusion assay, we take advantage of the strong transcriptional activation by the binding of tTA to TRE. In the absence of tTA, transcription of the LacZ gene in TRE-LacZ is silent. When tTA is present, it binds to the TRE and activates the transcription of LacZ. Figure 1 shows a flowchart of the enzymatic cell fusion assay. tTA is transfected into v-cells that express v-SNARE proteins at the cell surface, and TRE-LacZ is transfected into t.......

Discussion

The original cell fusion assay⁶ determines SNARE-mediated cell fusion events by fluorescence microscopy. Here we describe an innovative assay that quantifies SNARE-mediated cell fusion events by activated expression of β-galactosidase and spectrometric measurement. Using this assay, we routinely analyze 15 - 20 v- and t-SNARE combinations in a single experiment. Using flow cytometry to measure SNARE expression at the cell surface, we titrate the expression levels of VAMPs and compare their membrane.......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
DMEM	Invitrogen	12800-017
COS-7 cells	ATCC	CRL-1651
tunicamycin	Sigma-Aldrich	T7765-5MG
pTet-Off	Clontech	K1620-A
pBI-G	Clontech	6150-1
lipofectamine	Invitrogen	18324-012
Enzyme-free cell dissociation solution	Invitrogen	13151-014
Rabbit anti-TET Repressor polyclonal antibody	Millipore	AB3541
FITC-conjugated donkey anti-mouse IgG (H+L)	Jackson ImmunoResearch	715-095-150
Laser scanning confocal microscope	Olympus	FV1000
FACSCalibur flow cytometer	BD Biosciences
β-Galactosidase Enzyme Assay System with Reporter Lysis Buffer	Promega	E2000
Model 100-40 UV-VIS spectrophotometer	Hitachi	C740843