This work is supported by startup funds from the University of Louisville and CA135123 from the National Institutes of Health (to C.H.).

1. Cell Culture and Transfection
<ol>
 <li>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).</li>
 <li>Plasmid transfection is done with Lipofectamine according to the manufacturer's instructions (Invitrogen).</li>
</ol>
2. Fluorescence Microscopic Analysis of Cell Surface SNARE Expression
<ol>
 <li>The day before transfection, 3&times;104 COS-7 cells are seeded on sterile 12-mm glass coverslips (F...

<ol>
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No conflicts of interest declared.

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 &beta;-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...

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

analysis of snare-mediated membrane fusion using an enzymatic cell fusion assay

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 fusion1-4. Reconstitution assays are essential for dissecting the mechanism and regulation of SNARE-mediated membrane fusion5. In a cell fusion assay6,7, 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 &beta;-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 &beta;-galactosidase. The activity of &beta;-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 compartments10-15. 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.

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...

Watch this Scientific Journal Video about Analysis of SNARE-mediated Membrane Fusion Using an Enzymatic Cell Fusion Assay at JoVE.com

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

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

The interactions of SNARE (soluble N-ethylmaleimide-sensitive factor attachment protein receptor)  proteins on vesicles (v-SNAREs) and on target membranes ...

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13.9K visualizações.  University of Louisville School of Medicine. Nós desenvolvemos um ensaio de fusão de células que quantifica SNARE mediadas por eventos de fusão da membrana por expressão activada de β-galactosidase.