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 cotransfected with 0.25 μg of the plasmids that express flipped VAMPs.
3. For t-cells in each well, 0.25 μg of the plasmid encoding TRE-LacZ (pBI-G, CLONTECH) is cotransfected with 0.25 μg each of the plasmids that express flipped SNAP-25 and syntaxins 1 or 4.
4. 24 hr after transfection, cells are fixed with 4% paraformaldehyde in PBS++ (PBS supplemented with 0.1 g/l CaCl₂ and 0.1 g/l MgCl₂) for 10 min, and then blocked in 10% FBS in PBS++ for 30 min.
5. The cells are incubated for 1.5 hr with the anti-Myc monoclonal antibody 9E10 (neat hybridoma culture supernatant).
6. After four washes with PBS++, the cells are incubated for 1 hr with FITC-conjugated secondary antibodies (Jackson Immunoresearch Laboratories) at a dilution of 1:500.
7. After four washes with PBS++, the labeled cells are mounted in Prolong Gold antifade reagent (Invitrogen).
8. Confocal images are collected on an Olympus laser scanning confocal microscope. The images are processed with the Adobe Photoshop software.

3. FACS Analysis of Cell Surface SNARE Expression

1. The day before transfection, 2×10⁵ COS-7 cells are seeded in each well of 6-well plates.
2. 24 hr after transfection with the flipped SNARE, pTet-Off and pBI-G plasmids, cells are fixed with 1% paraformaldehyde in PBS++ for 15 min, and then blocked in 10% FBS in PBS++ for 15 min.
3. The cells are incubated with the anti-Myc monoclonal antibody 9E10 for 60 min.
4. After three washes with PBS++, the cells are labeled with FITC-conjugated secondary antibodies (1:200 dilution) for 45 min.
5. After three washes with PBS++, the cells are scraped off the plates with a cell scraper.
6. 15,000 cells are analyzed using a FACSCalibur flow cytometer (BD Biosciences). The mean fluorescence intensity of each sample is obtained using the CellQuest Pro software.

4. Enzymatic Cell Fusion Assay

1. The day before transfection, 1.2×10⁶ COS-7 cells are seeded in each 100-mm tissue culture dish, and 2×10⁵ COS-7 cells are seeded in each well of 6-well plates.
2. For v-cells, 0.5 - 5 μg each of flipped VAMP plasmid is cotransfected with 5 μg of pTet-Off into the cells in each 100-mm culture dish. Control cells are cotransfected with the empty vector pcDNA3.1(+) and pTet-Off. To prevent the N-glycosylation of VAMPs 1, 4, 5, 7 and 8, tunicamycin (10 μg/ml) is included in cell culture medium during transfection.
3. For t-cells in each well of the 6-well plates, 1 μg of flipped SNAP-25 plasmid and pBI-G are cotransfected with 0.5 μg of flipped syntaxin1 plasmid or 0.05 μg of flipped syntaxin4 plasmid.
4. 24 hr after transfection, the v-cells are detached from culture dishes with the Enzyme-free Cell Dissociation Buffer (Invitrogen). Detached cells are counted with a hemacytometer and resuspended in HEPES-buffered DMEM supplemented with 10% FBS, 6.7 μg/ml tunicamycin and 0.67 mM DTT.
5. 4.8×10⁵ resuspended v-cells are added to each well already containing the t-cells.
6. After 6, 12 or 24 hr rat 37 °C in 5% CO₂, the expression of β-galactosidase is measured using the β-Galactosidase Enzyme Assay System with Reporter Lysis Buffer according to the manufacturer's instructions (Promega). Briefly, the cells are washed twice with PBS, and then lysed in the Reporter Lysis Buffer. Cell lysates are mixed with equal volume of Assay 2× Buffer. As a blank control, the Reporter Lysis Buffer is mixed with the Assay 2× Buffer.
7. After 90 min, the colorimetric reaction is stopped by adding 1 M sodium carbonate.
8. Absorbance at 420 nm is measured using a HITACHI 100-40 spectrophotometer.

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

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