**5. Conclusion**

240 Crosstalk and Integration of Membrane Trafficking Pathways

The number of SNARE complexes that cooperate to mediate vesicle fusion is under active investigation. To investigate the cooperativity of VAMPs in membrane fusion, we determine the dependence of cell fusion activity on cell surface expression level of VAMP1. We choose VAMP1 in this experiment because it has high membrane fusion activity (Fig. 6). In t-cells, the cell surface expression levels of syntaxin1 and SNAP-25 are kept constant. v-Cells are transfected with increasing concentrations of the flipped VAMP1 plasmid. At each concentration, we measure the cell surface expression level of VAMP1 proteins using flow cytometry, and determine cell fusion activity of VAMP1 with syntaxin1/SNAP-25 using the enzymatic fusion assay. Cell fusion activity is then plotted as a function of the mean fluorescence intensity of VAMP1 staining (Fig. 7A). The correlation is best fit with a polynomial regression. The hyperbolic instead of sigmoidal correlation (Fig. 7A) suggests

**VAMP1 fluorescence**

**log (VAMP1 fluorescence)**

Fig. 7. Dependence of cell fusion activity on cell surface density of VAMP1. (A) v-cells expressing increasing amount of VAMP1 at the cell surface are combined with t-cells expressing syntaxin1/SNAP-25. Cell fusion activities are quantified and correlated with the mean fluorescence intensity of VAMP1 staining. (B) Log-log plot of cell fusion activity vs.

**4.3 Cooperativity of VAMP proteins in the cell fusion reaction** 

that there is no cooperativity of VAMP1 proteins in driving cell fusion.

**β-Galactosidase activity (OD420)**

**B**

**log (β-Galactosidase activity)**

mean fluorescence intensity of VAMP1 staining.

**A**

To examine v-/t-SNARE interactions quantitatively, we developed an enzymatic cell fusion assay that utilizes activated expression of β-galactosidase and spectrometric measurement. Using this assay, we show that VAMPs 1, 2, 3, 4, 7 and 8 mediate membrane fusion efficiently with plasma membrane t-SNAREs syntaxin1/SNAP-25 and syntaxin4/SNAP-25, whereas VAMP5 does not drive fusion with the t-SNAREs. By expressing VAMPs 1, 3, 4, 7 and 8 at the same level, we further compare their membrane fusion activities. VAMPs 1 and 3 exhibit comparable and the highest fusion activities, whereas VAMPs 4, 7 and 8 have 30 - 50% lower fusion activities. Collectively, these data indicate that VAMPs have differential membrane fusion activities, and imply that with the exception of VAMP5, VAMPs are essentially redundant in mediating membrane fusion with plasma membrane t-SNAREs. Furthermore, no cooperativity of VAMP1 proteins is observed in the cell fusion reaction, suggesting that concerted action of multiple SNARE complexes is not required to fuse cellular membranes.

## **6. Acknowledgement**

We thank Adrienne Bushau and David Humphrey for critically reading the manuscript. This work was supported by startup funds from the University of Louisville and CA135123 from the National Institutes of Health (to C.H.).

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