Name: Analysis of SNARE-mediated Membrane Fusion Using an Enzymatic Cell Fusion Assay (Video) | JoVE Journal
Uploaded: 2012-10-19T13:00:00.000+00:00
Duration: 9 min 19 s
Description: Learn to quantify SNARE-mediated membrane fusion using an enzymatic cell fusion assay with β-galactosidase reporter. This method enables quantitative analysis of multiple SNARE combinations in COS-7 cells.

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5 minutes

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 (Fisher Scientific) contained in 24-well plates.</li>
 <li>For v-cells in each well, 0.25 &mu;g of the plasmid that encodes tTA (pTet-Off, CLONTECH) is cotransfected with 0.25 &mu;g of the plasmids that express flipped VAMPs.</li>
 <li>For t-cells in each well, 0.25 &mu;g of the plasmid encoding TRE-LacZ (pBI-G, CLONTECH) is cotransfected with 0.25 &mu;g each of the plasmids that express flipped SNAP-25 and syntaxins 1 or 4.</li>
 <li>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.</li>
 <li>The cells are incubated for 1.5 hr with the anti-Myc monoclonal antibody 9E10 (neat hybridoma culture supernatant).</li>
 <li>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.</li>
 <li>After four washes with PBS++, the labeled cells are mounted in Prolong Gold antifade reagent (Invitrogen).</li>
 <li>Confocal images are collected on an Olympus laser scanning confocal microscope. The images are processed with the Adobe Photoshop software.</li>
</ol>
3. FACS Analysis of Cell Surface SNARE Expression
<ol>
 <li>The day before transfection, 2&times;105 COS-7 cells are seeded in each well of 6-well plates.</li>
 <li>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.</li>
 <li>The cells are incubated with the anti-Myc monoclonal antibody 9E10 for 60 min.</li>
 <li>After three washes with PBS++, the cells are labeled with FITC-conjugated secondary antibodies (1:200 dilution) for 45 min.</li>
 <li>After three washes with PBS++, the cells are scraped off the plates with a cell scraper.</li>
 <li>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.</li>
</ol>
4. Enzymatic Cell Fusion Assay
<ol>
 <li>The day before transfection, 1.2&times;106 COS-7 cells are seeded in each 100-mm tissue culture dish, and 2&times;105 COS-7 cells are seeded in each well of 6-well plates.</li>
 <li>For v-cells, 0.5 - 5 &mu;g each of flipped VAMP plasmid is cotransfected with 5 &mu;g 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 &mu;g/ml) is included in cell culture medium during transfection.</li>
 <li>For t-cells in each well of the 6-well plates, 1 &mu;g of flipped SNAP-25 plasmid and pBI-G are cotransfected with 0.5 &mu;g of flipped syntaxin1 plasmid or 0.05 &mu;g of flipped syntaxin4 plasmid.</li>
 <li>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 &mu;g/ml tunicamycin and 0.67 mM DTT.</li>
 <li>4.8&times;105 resuspended v-cells are added to each well already containing the t-cells.</li>
 <li>After 6, 12 or 24 hr rat 37 &deg;C in 5% CO2, the expression of &beta;-galactosidase is measured using the &beta;-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&times; Buffer. As a blank control, the Reporter Lysis Buffer is mixed with the Assay 2&times; Buffer.</li>
 <li>After 90 min, the colorimetric reaction is stopped by adding 1 M sodium carbonate.</li>
 <li>Absorbance at 420 nm is measured using a HITACHI 100-40 spectrophotometer.</li>
</ol>

<ol><li>Rothman, J. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Mechanisms+of+intracellular+protein+transport%5BTitle%5D)+AND+%22Nature%22%5BJournal%5D)">Mechanisms of intracellular protein transport.</a> Nature. 372, 55-63 (1994).</li>
<li>Jahn, R., Lang, T., Sudhof, T. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Membrane+fusion%5BTitle%5D)+AND+%22Cell%22%5BJournal%5D)">Membrane fusion.</a> Cell. 112, 519-533 (2003).</li>
<li>Bonifacino, J. S., Glick, B. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((The+mechanisms+of+vesicle+budding+and+fusion%5BTitle%5D)+AND+%22Cell%22%5BJournal%5D)">The mechanisms of vesicle budding and fusion.</a> Cell. 116, 153-166 (2004).</li>
<li>Jahn, R., Scheller, R. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((SNAREs--engines+for+membrane+fusion%5BTitle%5D)+AND+%22Nature%22%5BJournal%5D)">SNAREs--engines for membrane fusion.</a> Nature. 7, 631-643 (2006).</li>
<li>Weber, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((SNAREpins%3A+minimal+machinery+for+membrane+fusion%5BTitle%5D)+AND+%22Cell%22%5BJournal%5D)">SNAREpins: minimal machinery for membrane fusion.</a> Cell. 92, 759-772 (1998).</li>
<li>Hu, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Fusion+of+cells+by+flipped+SNAREs%5BTitle%5D)+AND+%22Science+%28New+York%2C+N.Y%29%22%5BJournal%5D)">Fusion of cells by flipped SNAREs.</a> Science (New York, N.Y). 300, 1745-1749 (2003).</li>
<li>Hu, C., Hardee, D., Minnear, F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Membrane+fusion+by+VAMP3+and+plasma+membrane+t-SNAREs%5BTitle%5D)+AND+%22Experimental+cell+research%22%5BJournal%5D)">Membrane fusion by VAMP3 and plasma membrane t-SNAREs.</a> Experimental cell research. 313, 3198-3209 (2007).</li>
<li>Hasan, N., Corbin, D., Hu, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Fusogenic+Pairings+of+Vesicle-Associated+Membrane+Proteins+%28VAMPs%29+and+Plasma+Membrane+t-SNAREs+-+VAMP5+as+the+Exception%5BTitle%5D)+AND+%22PloS+one%22%5BJournal%5D)">Fusogenic Pairings of Vesicle-Associated Membrane Proteins (VAMPs) and Plasma Membrane t-SNAREs - VAMP5 as the Exception.</a> PloS one. 5, e14238(2010).</li>
<li>Gossen, M., Bujard, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Tight+control+of+gene+expression+in+mammalian+cells+by+tetracycline-responsive+promoters%5BTitle%5D)+AND+%22Proceedings+of+the+National+Academy+of+Sciences+of+the+United+States+of+America%22%5BJournal%5D)">Tight control of gene expression in mammalian cells by tetracycline-responsive promoters.</a> Proceedings of the National Academy of Sciences of the United States of America. 89, 5547-5551 (1992).</li>
<li>Hanson, P. I., Heuser, J. E., Jahn, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Neurotransmitter+release+-+four+years+of+SNARE+complexes%5BTitle%5D)+AND+%22Current+opinion+in+neurobiology%22%5BJournal%5D)">Neurotransmitter release - four years of SNARE complexes.</a> Current opinion in neurobiology. 7, 310-315 (1997).</li>
<li>McMahon, H. T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Cellubrevin+is+a+ubiquitous+tetanus-toxin+substrate+homologous+to+a+putative+synaptic+vesicle+fusion+protein.+%5Bsee+comments%5BTitle%5D)+AND+%22Nature%22%5BJournal%5D)">Cellubrevin is a ubiquitous tetanus-toxin substrate homologous to a putative synaptic vesicle fusion protein. [see comments.</a> Nature. 364, 346-349 (1993).</li>
<li>Steegmaier, M., Klumperman, J., Foletti, D. L., Yoo, J. S., Scheller, R. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Vesicle-associated+membrane+protein+4+is+implicated+in+trans-Golgi+network+vesicle+trafficking%5BTitle%5D)+AND+%22Molecular+biology+of+the+cell%22%5BJournal%5D)">Vesicle-associated membrane protein 4 is implicated in trans-Golgi network vesicle trafficking.</a> Molecular biology of the cell. 10, 1957-1972 (1999).</li>
<li>Zeng, Q. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((A+novel+synaptobrevin%2FVAMP+homologous+protein+%28VAMP5%29+is+increased+during+in+vitro+myogenesis+and+present+in+the+plasma+membrane%5BTitle%5D)+AND+%22Molecular+biology+of+the+cell%22%5BJournal%5D)">A novel synaptobrevin/VAMP homologous protein (VAMP5) is increased during in vitro myogenesis and present in the plasma membrane.</a> Molecular biology of the cell. 9, 2423-2437 (1998).</li>
<li>Galli, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((A+novel+tetanus+neurotoxin-insensitive+vesicle-associated+membrane+protein+in+SNARE+complexes+of+the+apical+plasma+membrane+of+epithelial+cells%5BTitle%5D)+AND+%22Molecular+biology+of+the+cell%22%5BJournal%5D)">A novel tetanus neurotoxin-insensitive vesicle-associated membrane protein in SNARE complexes of the apical plasma membrane of epithelial cells.</a> Molecular biology of the cell. 9, 1437-1448 (1998).</li>
<li>Wong, S. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Endobrevin%2C+a+novel+synaptobrevin%2FVAMP-like+protein+preferentially+associated+with+the+early+endosome%5BTitle%5D)+AND+%22Molecular+biology+of+the+cell%22%5BJournal%5D)">Endobrevin, a novel synaptobrevin/VAMP-like protein preferentially associated with the early endosome.</a> Molecular biology of the cell. 9, 1549-1563 (1998).</li>
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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><tbody><tr><td>DMEM</td><td>Invitrogen</td><td>12800-017</td><td></td></tr><tr><td>COS-7 cells</td><td>ATCC</td><td>CRL-1651</td><td></td></tr><tr><td>tunicamycin</td><td>Sigma-Aldrich</td><td>T7765-5MG</td><td></td></tr><tr><td>pTet-Off</td><td>Clontech</td><td>K1620-A</td><td></td></tr><tr><td>pBI-G</td><td>Clontech</td><td>6150-1</td><td></td></tr><tr><td>lipofectamine</td><td>Invitrogen</td><td>18324-012</td><td></td></tr><tr><td>Enzyme-free cell dissociation solution</td><td>Invitrogen</td><td>13151-014</td><td></td></tr><tr><td>Rabbit anti-TET Repressor polyclonal antibody</td><td>Millipore</td><td>AB3541</td><td></td></tr><tr><td>FITC-conjugated donkey anti-mouse IgG (H+L)</td><td>Jackson ImmunoResearch</td><td>715-095-150</td><td></td></tr><tr><td>Laser scanning confocal microscope</td><td>Olympus</td><td>FV1000</td><td></td></tr><tr><td>FACSCalibur flow cytometer</td><td>BD Biosciences</td><td></td><td></td></tr><tr><td>&amp;#946;-Galactosidase Enzyme Assay System with Reporter Lysis Buffer</td><td>Promega</td><td>E2000</td><td></td></tr><tr><td>Model 100-40 UV-VIS spectrophotometer</td><td>Hitachi</td><td>C740843</td><td></td></tr></tbody></table>

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

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...

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 ...

The overall goal of this procedure is to quantify snare mediated cell fusion events by activated expression of beta galacto. This is accomplished by first expressing v and t snare proteins ectopically at the surface of cost seven cells. The next step of the procedure is to combine the cells expressing V snare proteins with those expressing T snare proteins.This results in snare mediated cell fusion and the activated expression of beta galacto. Ultimately, the cell fusion induced beta galacto activity is measured using a spectrophotometer. The main advantage of this technique or the existing methods like the microscopic SAL region assay, is that it can analyze multiple snare combinations.Quantitatively, Though this method may be applied to understand the mechanism and regulation of snare mediated membrane fusion. It may also be applied to have throughput studies. Demonstrating the procedure will be Doc Na Hassan, a postdoc and David Humphrey, a technician from my lab The day before the transfection load.24 well plates with 12 millimeter glass cover slips prepare two wells for each combination of cells expressing unique plasmids in the experiment on those cover slips. Seed 30, 000 cost seven cells cultured in DMEM supplemented with 4.5 grams per liter glucose and 10%FBS to transfect. The V cells add to each, well a quarter microgram of the TTA plasmid and one set of plasmids expression.Flipped proteins of interest in this case flipped vamps to transfect the T cells add a quarter microgram of the plasmid encoding TRE lacks Z, and the complementary set of plasmids encoding the flipped proteins of interest. In this case, snap 25 syntaxin one and syntaxin four. Now transfect the cost seven with lipectomy according to the manufacturer's instructions and culture, the cells for 24 hours, 24 hours after the transfection fix the cells in supplemented para formaldehyde for 10 minutes after the fixation, rinse the cells three times with PBS plus plus, then block the cells with supplemented FBS for 30 minutes.After the block, incubate the cells with the anti mic monoclonal antibody. Nine E 10 for 30 minutes after four washes with PBS plus plus incubate the cells for an hour with fitzy conjugated secondary antibodies at a dilution of one to 500. After four more washes with PBS plus plus mount the labeled cells in commercially available anti fade reagent.The cells are now ready for confocal microscopy the day before transfection seed 1.2 million costs seven cells in each 100 millimeter tissue culture dish and 200, 000 costs seven cells in each well of six well plates for each plasmid combination to be analyzed. Prepare one, well culture the cells for 24 hours. The next day add the plasmids for the transfection for V cells transfect a plasmid expressing the flipped protein of interest and five micrograms of p tet off to the cells in each 100 millimeter culture.Dish cot transfect control cells in a 100 millimeter culture dish with an empty vector and P te off at the same relative concentrations as the V cells. After all the plasmids have been added to the V cells and control cells, add tcom mycin to those wells to prevent the N glycosylation of vamps. In each well of the six well plates cot transfect the T cells with one microgram of PBIG with the complementary plasmids expressing proteins of interest.Now transfect the cost seven cells with lip according to the manufacturer's instructions. Then culture the cells for 24 hours, 24 hours after the transfection, detach the V cells from their culture dishes with enzyme free cell dissociation buffer. Start by washing the cells once with 20 milliliters of PBS.Then add five milliliters of enzyme free dissociation buffer. After a 10 minute incubation at room temperature, the cells will dislodge by sharply tapping the plate check that they have detached under microscope. If less than half the cells have detached, wait another five minutes and tap the plate again.Continue the incubation in detachment buffer until more than half of the cells have detached. Then count the V cells with a hemo cytometer. Then resus the V cells at a concentration of approximately 480, 000 cells per milliliter in supplemented heis buffer DMEM.Now add one milliliter of resuspended V cells to each well containing the T cells and return the plates to the incubator. After six, 12, or 24 hours of co culturing the cells, wash them with PBS and add lysis buffer. Next, scrape the cells from the wells and transfer them to tubes for centrifuging.After centrifuging, transfer the supernatant to a new tube and measure the expression of beta galacto using a commercially available assay according to the manufacturer's instructions during that protocol, be sure to stop the cholera metric reaction after 90 minutes by adding one molar sodium carbonate to score the results. Measure absorbance at 420 nanometers on a spectrophotometer ter. When CO seven cells were transfected with flipped snare plasmids flipped snare proteins were expressed at the cell surface.Flow cytometry was used to measure the expression levels of snare proteins at the cell surface. In order to compare their membrane fusion capacities, vamps needed to be expressed at the same level. Therefore, the concentration of each flipped snare plasmid used in the transfection was titrated and optimized for transfection.Under such conditions, vamps 1, 3, 4, 5, 7, and eight and syntaxin one and four were expressed at the same level at the cell surface respectively, the neuronal snares that mediate synaptic exocytosis were used to test the feasibility of the assay. Indeed, when the V cells expressing vamp two and T cells expressing SYNTAXIN one snap 25 were combined, robust beta galacto expression was detected. However, when either vamp two or SNAP 25 were not expressed, only baseline beta galacto sase activity was detected, indicating that cell fusion and the expression of beta galacto sase rely on interactions of v and t snares overall SYNTAXIN one, snap 25, vamp one, vamp three, and vamp eight had comparable and the highest fusion activities.In contrast, baseline beta GAL activity suggests the protein combination does not drive membrane fusion such as the case between vamp five and SYNTAXIN one. Snap 25. While performing the enzymatic fusion assay, it is important to remember to add the unica mycin to prevent the an glycosylation of esna proteins and add DTT to prevent the disulfide bond formation among esna proteins.After watching this video, you should have a good understanding of how to perform the cell fusion assay.

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

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Learn to quantify SNARE-mediated membrane fusion using an enzymatic cell fusion assay with β-galactosidase reporter. This method enables quantitative analysis of multiple SNARE combinations in COS-7 cells.

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

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