**3.1 Expression of flipped SNAREs at the cell surface**

By fusing the pre-prolactin signal sequence, which specifies translocation across the endoplasmic reticulum, to N-termini of the neuronal SNAREs VAMP2, syntaxin1 and SNAP-25, the cell fusion assay was originally developed in Dr. James Rothman's lab (Hu et al., 2003). The engineered SNAREs are called 'flipped' SNAREs because the orientation of their SNARE motifs against cellular membranes is flipped. A Myc tag is inserted between the signal sequence and N-termini of SNAREs to detect flipped SNARE proteins (Fig. 3A). When COS-7 cells are transfected with flipped SNARE constructs, flipped SNARE proteins are expressed at the cell surface (Fig. 3B). To express plasma membrane t-SNAREs, flipped syntaxins 1 or 4 are cotransfected with flipped SNAP-25. SNAP-25 does not contain a transmembrane domain. After expression, flipped SNAP-25 proteins are anchored to the cell surface by assembling with flipped syntaxin proteins.

Flow cytometry is used to measure the expression levels of SNARE proteins at the cell surface (Figs. 3C and D). When flipped VAMPs 1, 3, 4, 5, 7 and 8, and syntaxins 1 and 4 plasmids are transfected at the same concentration, cell surface expression of VAMPs 5 and 8 is higher than VAMPs 1, 3, 4 and 7, and cell surface expression of syntaxin4 is higher than syntaxin1 (Hasan et al., 2010). To express the v- and t-SNAREs at the same level, we optimized the concentration of each flipped SNARE plasmid used in transfection (concentrations see Section 4.2), so that VAMPs 1, 3, 4, 5, 7 and 8, and syntaxins 1 and 4 are expressed at the same level at the cell surface, respectively (Fig. 3D).

#### **3.2 Microscopic cell fusion assay**

Multiple readout systems have been developed to detect fusion of the cells that express flipped v-SNAREs (v-cells) and the cells that express flipped t-SNARE proteins (t-cells) (Hasan et al., 2010; Hu et al., 2003; Hu et al., 2007). In the microscopic assay shown in Fig. 4A, flipped v-SNARE constructs are cotransfected with a plasmid that encodes the green fluorescent protein EGFP. In t-cells, the flipped t-SNARE constructs are cotransfected with a plasmid that encodes the red fluorescent protein DsRed2. Fusion of the v- and t-cells results in fused cells that contain both EGFP and DsRed2. In the merged channel, the cytoplasm of the fused cells is yellow (arrows, Fig. 4A).

#### **3.3 Enzymatic cell fusion assay**

The microscopic cell fusion assay becomes less efficient when used to analyze multiple v-/t-SNARE combinations quantitatively. To develop a quantitative cell fusion assay, we take advantage of the strong transcriptional activation by binding of the tetracycline-controlled transactivator (tTA) to the tetracycline-response element (TRE) (Gossen and Bujard, 1992). Two plasmids in CLONTECH's Tet-Off gene expression system are used (Hasan et al., 2010). The first plasmid pTet-Off encodes the transcriptional activator tTA, and the second

Randhawa et al., 2000; Veale et al., 2010). Therefore, multiple VAMPs are co-expressed in mammalian cells. However, it is not clear if the seven VAMPs have differential membrane fusion activities. Using a cell fusion assay, we compare the membrane fusion activities of

By fusing the pre-prolactin signal sequence, which specifies translocation across the endoplasmic reticulum, to N-termini of the neuronal SNAREs VAMP2, syntaxin1 and SNAP-25, the cell fusion assay was originally developed in Dr. James Rothman's lab (Hu et al., 2003). The engineered SNAREs are called 'flipped' SNAREs because the orientation of their SNARE motifs against cellular membranes is flipped. A Myc tag is inserted between the signal sequence and N-termini of SNAREs to detect flipped SNARE proteins (Fig. 3A). When COS-7 cells are transfected with flipped SNARE constructs, flipped SNARE proteins are expressed at the cell surface (Fig. 3B). To express plasma membrane t-SNAREs, flipped syntaxins 1 or 4 are cotransfected with flipped SNAP-25. SNAP-25 does not contain a transmembrane domain. After expression, flipped SNAP-25 proteins are anchored to the cell

Flow cytometry is used to measure the expression levels of SNARE proteins at the cell surface (Figs. 3C and D). When flipped VAMPs 1, 3, 4, 5, 7 and 8, and syntaxins 1 and 4 plasmids are transfected at the same concentration, cell surface expression of VAMPs 5 and 8 is higher than VAMPs 1, 3, 4 and 7, and cell surface expression of syntaxin4 is higher than syntaxin1 (Hasan et al., 2010). To express the v- and t-SNAREs at the same level, we optimized the concentration of each flipped SNARE plasmid used in transfection (concentrations see Section 4.2), so that VAMPs 1, 3, 4, 5, 7 and 8, and syntaxins 1 and 4 are

Multiple readout systems have been developed to detect fusion of the cells that express flipped v-SNAREs (v-cells) and the cells that express flipped t-SNARE proteins (t-cells) (Hasan et al., 2010; Hu et al., 2003; Hu et al., 2007). In the microscopic assay shown in Fig. 4A, flipped v-SNARE constructs are cotransfected with a plasmid that encodes the green fluorescent protein EGFP. In t-cells, the flipped t-SNARE constructs are cotransfected with a plasmid that encodes the red fluorescent protein DsRed2. Fusion of the v- and t-cells results in fused cells that contain both EGFP and DsRed2. In the merged channel, the cytoplasm of

The microscopic cell fusion assay becomes less efficient when used to analyze multiple v-/t-SNARE combinations quantitatively. To develop a quantitative cell fusion assay, we take advantage of the strong transcriptional activation by binding of the tetracycline-controlled transactivator (tTA) to the tetracycline-response element (TRE) (Gossen and Bujard, 1992). Two plasmids in CLONTECH's Tet-Off gene expression system are used (Hasan et al., 2010). The first plasmid pTet-Off encodes the transcriptional activator tTA, and the second

VAMPs.

**3. Cell fusion assays** 

**3.1 Expression of flipped SNAREs at the cell surface** 

surface by assembling with flipped syntaxin proteins.

**3.2 Microscopic cell fusion assay** 

the fused cells is yellow (arrows, Fig. 4A).

**3.3 Enzymatic cell fusion assay** 

expressed at the same level at the cell surface, respectively (Fig. 3D).

Fig. 3. Expression of flipped SNAREs at the cell surface. (A) Domain structure of flipped SNAREs. (B) Twenty-four hours after transfection with empty vector pcDNA3.1(+) or flipped VAMP5 plasmid, unpermeabilized COS-7 cells are stained with an anti-Myc antibody. Representative confocal images are shown. Scale bar, 50 m. (C and D) Twentyfour hours after transfection with empty vector or flipped SNARE plasmids, unpermeabilized cells are stained with the anti-Myc antibody and analyzed by flow cytometry. (C) Representative FACS profiles of the cells transfected with empty vector or flipped VAMP1. (D) To express VAMPs and syntaxins at the same level at the cell surface, flipped SNARE plasmids are transfected at titrated concentrations. The mean fluorescence intensity of staining is obtained using CellQuest Pro software.

Analysis of SNARE-Mediated Exocytosis Using a Cell Fusion Assay 235

plasmid pBI-G encodes the *LacZ* gene under control of the tetracycline-response element (TRE-*LacZ*). 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*, resulting in the expression of -galactosidase. We hypothesize that if tTA is located in v-cells and TRE-*LacZ* is located in t-cells, -galactosidase will not be expressed. Fusion of the v- and t-cells would

The neuronal SNAREs are used to test feasibility of the assay. VAMP2 is coexpressed with tTA in v-cells, and syntaxin1 and SNAP-25 are coexpressed with TRE-*LacZ* in t-cells. When the v- and t-cells are combined, robust -galactosidase expression is indeed detected (Fig. 4B). However, when either VAMP2 or SNAP-25 is not expressed, only baseline galactosidase activity is detected, indicating that cell fusion and expression of -

COS-7 cells were obtained from the American Type Culture Collection, and cultured in Dulbecco Modified Eagle's Medium (DMEM) supplemented with 4.5 g/l glucose and 10% fetal bovine serum (FBS). The anti-Myc monoclonal antibody 9E10, developed by Dr. Bishop, was obtained from the Developmental Studies Hybridoma Bank maintained by the University of Iowa. pEGFP-N3, pDsRed2-N1, pTet-Off and pBI-G were obtained from CLONTECH. Plasmid transfection is done with Lipofectamine according to the

Expression levels of SNAREs at the cell surface are measured using immunostaining and flow cytometry as we previously reported (Hasan and Hu, 2010). Briefly, COS-7 cells are seeded in 6-well plates. Twenty-four hours after transfection with the flipped SNARE, pTet-Off and pBI-G plasmids, cells are fixed with 1% paraformaldehyde in PBS++ (PBS supplemented with 0.1 g/l CaCl2 and 0.1 g/l MgCl2). After labeling with the anti-Myc monoclonal antibody 9E10 and FITC-conjugated secondary antibodies, the cells are scraped off the plates with a cell scraper. 15,000 cells are analyzed using a FACSCalibur flow cytometer (BD Biosciences). The mean fluorescence intensity of each sample is obtained

The day before transfection, 1.2 × 106 COS-7 cells are seeded in each 100-mm cell culture dish, and 5 × 104 COS-7 cells are seeded on sterile 12-mm glass coverslips contained in 24 well plates. For v-cells, 5 g of flipped v-SNARE and pEGFP-N3 are cotransfected into the cells grown in each 100-mm culture dish. For t-cells, 0.25 g each of flipped syntaxin, SNAP-25 and pDsRed2-N1 are cotransfected into the cells seeded in the 24-well plates. Twentyfour hours after transfection, the v-cells are detached from culture dishes with EDTA (Enzyme-free Cell Dissociation Buffer (Invitrogen)). Detached cells are counted with a hemocytometer and resuspended in HEPES-buffered DMEM supplemented with 10% FBS,

result in the binding of tTA to TRE and transcriptional activation of *LacZ* (Fig. 4B).

galactosidase rely on interactions of v- and t-SNAREs.

**3.4 Experimental procedures 3.4.1 Cell culture and reagents** 

manufacturer's instructions (Invitrogen).

**3.4.2 FACS analysis** 

using CellQuest Pro software.

**3.4.3 Microscopic cell fusion assay** 

Fig. 4. Cell fusion assays. (A) Microscopic cell fusion assay. Cells that express flipped v-SNAREs (v-cells) are labeled by the green fluorescent protein EGFP, whereas cells that express flipped t-SNAREs (t-cells) are labeled by the red fluorescent protein DsRed2. Fusion of v- and t-cells results in fused cells (arrows) whose cytoplasm is yellow under fluorescence microscope. Scale bar, 50 m. (B) Enzymatic cell fusion assay. The tetracycline-controlled transactivator (tTA) is expressed in v-cells, and a reporter plasmid that encodes galactosidase under control of the tetracycline-response element (TRE-LacZ) is transfected into t-cells. Fusion of the v- and t-cells leads to the binding of tTA to TRE and the expression of -galactosidase, which is measured using a colorimetric method by absorbance at 420 nm. Only baseline -galactosidase activity is detected when either flipped VAMP2 or SNAP-25 is not expressed.

plasmid pBI-G encodes the *LacZ* gene under control of the tetracycline-response element (TRE-*LacZ*). 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*, resulting in the expression of -galactosidase. We hypothesize that if tTA is located in v-cells and TRE-*LacZ* is located in t-cells, -galactosidase will not be expressed. Fusion of the v- and t-cells would result in the binding of tTA to TRE and transcriptional activation of *LacZ* (Fig. 4B).

The neuronal SNAREs are used to test feasibility of the assay. VAMP2 is coexpressed with tTA in v-cells, and syntaxin1 and SNAP-25 are coexpressed with TRE-*LacZ* in t-cells. When the v- and t-cells are combined, robust -galactosidase expression is indeed detected (Fig. 4B). However, when either VAMP2 or SNAP-25 is not expressed, only baseline galactosidase activity is detected, indicating that cell fusion and expression of galactosidase rely on interactions of v- and t-SNAREs.

#### **3.4 Experimental procedures**

234 Crosstalk and Integration of Membrane Trafficking Pathways

**GFP RFP Fusion**

**No Fusion Cell Fusion**

**A v-cell t-cell**

**0.0 0.1 0.2 0.3 0.4 0.5 0.6 0.7 0.8 0.9**

**VAMP2 + + - Syntaxin1A + + + SNAP-25 + + -**

transactivator (tTA) is expressed in v-cells, and a reporter plasmid that encodes -

Fig. 4. Cell fusion assays. (A) Microscopic cell fusion assay. Cells that express flipped v-SNAREs (v-cells) are labeled by the green fluorescent protein EGFP, whereas cells that express flipped t-SNAREs (t-cells) are labeled by the red fluorescent protein DsRed2. Fusion of v- and t-cells results in fused cells (arrows) whose cytoplasm is yellow under fluorescence microscope. Scale bar, 50 m. (B) Enzymatic cell fusion assay. The tetracycline-controlled

galactosidase under control of the tetracycline-response element (TRE-LacZ) is transfected into t-cells. Fusion of the v- and t-cells leads to the binding of tTA to TRE and the expression of -galactosidase, which is measured using a colorimetric method by absorbance at 420 nm. Only baseline -galactosidase activity is detected when either flipped VAMP2 or SNAP-25 is

**Fusion**

**-Galactosidase activity (OD420)**

**v-cell t-cell**

**B**

not expressed.

#### **3.4.1 Cell culture and reagents**

COS-7 cells were obtained from the American Type Culture Collection, and cultured in Dulbecco Modified Eagle's Medium (DMEM) supplemented with 4.5 g/l glucose and 10% fetal bovine serum (FBS). The anti-Myc monoclonal antibody 9E10, developed by Dr. Bishop, was obtained from the Developmental Studies Hybridoma Bank maintained by the University of Iowa. pEGFP-N3, pDsRed2-N1, pTet-Off and pBI-G were obtained from CLONTECH. Plasmid transfection is done with Lipofectamine according to the manufacturer's instructions (Invitrogen).

#### **3.4.2 FACS analysis**

Expression levels of SNAREs at the cell surface are measured using immunostaining and flow cytometry as we previously reported (Hasan and Hu, 2010). Briefly, COS-7 cells are seeded in 6-well plates. Twenty-four hours after transfection with the flipped SNARE, pTet-Off and pBI-G plasmids, cells are fixed with 1% paraformaldehyde in PBS++ (PBS supplemented with 0.1 g/l CaCl2 and 0.1 g/l MgCl2). After labeling with the anti-Myc monoclonal antibody 9E10 and FITC-conjugated secondary antibodies, the cells are scraped off the plates with a cell scraper. 15,000 cells are analyzed using a FACSCalibur flow cytometer (BD Biosciences). The mean fluorescence intensity of each sample is obtained using CellQuest Pro software.

#### **3.4.3 Microscopic cell fusion assay**

The day before transfection, 1.2 × 106 COS-7 cells are seeded in each 100-mm cell culture dish, and 5 × 104 COS-7 cells are seeded on sterile 12-mm glass coverslips contained in 24 well plates. For v-cells, 5 g of flipped v-SNARE and pEGFP-N3 are cotransfected into the cells grown in each 100-mm culture dish. For t-cells, 0.25 g each of flipped syntaxin, SNAP-25 and pDsRed2-N1 are cotransfected into the cells seeded in the 24-well plates. Twentyfour hours after transfection, the v-cells are detached from culture dishes with EDTA (Enzyme-free Cell Dissociation Buffer (Invitrogen)). Detached cells are counted with a hemocytometer and resuspended in HEPES-buffered DMEM supplemented with 10% FBS,

Analysis of SNARE-Mediated Exocytosis Using a Cell Fusion Assay 237

six VAMPs drive fusion to a similar degree. With syntaxin4/SNAP-25, VAMP8 fuses less efficiently than VAMPs 1, 2, 3 and 4 (31% lower fusion activity and *P* = 0.046 vs. VAMP1, Fig. 5B). In contrast, when VAMP5 is combined with the t-SNAREs, only baseline βgalactosidase activity is detected (Figs. 5A and B), suggesting that VAMP5 does not drive membrane fusion with the t-SNAREs. The stronger fusion activities of syntaxin4/SNAP-25 than syntaxin1/SNAP-25 (compare Figs. 5A and B) are likely caused by higher cell surface expression of syntaxin4/SNAP-25 than syntaxin1/SNAP-25 and higher fusion activity of syntaxin4 than syntaxin1 (Hasan et al., 2010). Together, the data shown in Fig. 5 indicate that VAMPs 1, 2, 3, 4, 7 and 8, but not VAMP5, drive membrane fusion when partnering with

**t-SNARE Syntaxin4/SNAP-25**

**4 5**

**VAMP 1 2 3 78 -**

**t-SNARE Syntaxin1/SNAP-25**

**VAMP 1 2 3 4 5 78 -**

**\* <sup>β</sup>-Galactosidase**

Fig. 5. Cell fusion by VAMPs and plasma membrane t-SNAREs. Twenty-four hours after combining v-cells that express VAMPs 1, 2, 3, 4, 5, 7 or 8 and t-cells that express (A)

syntaxin1/SNAP-25 or (B) syntaxin4/SNAP-25, cell fusion is quantified using the enzymatic cell fusion assay. Control cells (-VAMP) are transfected with the empty vector. The flipped SNARE plasmids are transfected at the same concentration. Error bars represent standard

plasma membrane t-SNAREs.

**A**

**β-Galactosidase activity (OD420)**

> **0.0 0.2 0.4 0.6 0.8 1.0 1.2 1.4 1.6 1.8**

deviation of three independent experiments. \* *P* < 0.05 vs. VAMP1.

**0.0 0.1 0.2 0.3 0.4 0.5 0.6 0.7 0.8 0.9 1.0**

**activity (OD420)**

**B**

6.7 g/ml tunicamycin and 0.67 mM DTT. v-Cells (1.2 × 105) are added to each coverslip already containing the t-cells. After 24 hours at 37C in 5% CO2, the cells are gently washed once with PBS++, then fixed with 4% paraformaldehyde. Confocal images are collected on an Olympus laser scanning confocal microscope. The 488 nm argon laser line is used to excite EGFP and the 543 nm HeNe laser line is used to excite DsRed2. To prevent crosscontamination between EGFP and DsRed2, each channel is imaged sequentially before merging the images.
