SNAREs can promote complete fusion and hemifusion as alternative outcomes

Abstract

Using a cell fusion assay, we show here that in addition to complete fusion SNAREs also promote hemifusion as an alternative outcome. Approximately 65% of events resulted in full fusion, and the remaining 35% in hemifusion; of those, approximately two thirds were permanent and approximately one third were reversible. We predict that this relatively close balance among outcomes could be tipped by binding of regulatory proteins to the SNAREs, allowing for dynamic physiological regulation between full fusion and reversible kiss-and-run-like events.

Figure 1.

Schemes of constructs used and fusion outcomes obtained with the cell–cell fusion assays. (A) The domain structure of GPI-anchored SNAREs. The preprolactin signal sequence (SS) was fused to the NH₂ terminus of VAMP2 and the Syntaxin H3 domain. The transmembrane domains (TMD) of VAMP2 and Syntaxin were replaced with the GPI-anchoring sequence of decay-accelerating factor. A Myc tag (red) was engineered between the NH₂ terminus of the Syntaxin H3 domain and the signal sequence. Two additional GPI-anchored SNAREs (GPI-VAMP_2-84 and GPI-Syntaxin_186-256), in which amino acids 85–92 in VAMP2 and amino acids 257–265 in Syntaxin H3 were deleted, were generated to bring the coiled-coil domains closer to the membrane anchors. (B) Cell fusion assay design monitoring lipid mixing and content mixing. The cytoplasm of CHO cells (GM1 negative) that expressed flipped VAMP2 or GPI-VAMP2 was labeled with RFP-nes. The nuclei of MEF-3T3 cells (GM1 positive) that express flipped t-SNAREs or GPI-anchored t-SNAREs were labeled with CFP-nls. The t-cells were harvested with an EDTA buffer, and overlaid on the v-cells. Cells were fixed after 6 h at 37°C. GM1 was stained with FITC-cholera toxin β-subunit (green). Complete fusion resulted in cells containing red cytoplasm, cyan nuclei, and green cell surface staining. In hemifused cells, GM1 transferred from t-cells to the contacting CHO v-cells in the absence of the mixing of the cytoplasmic markers. In the event of reversible hemifusion, after GM1 is transferred from t-cells to v-cells, the v- and t-cells detached from each other. In the no fusion cells, all the markers remained within the original cells. (C) Cell fusion assay designed to detect small fusion pores using CMFDA. MEF-3T3 cells (GM1 positive) that express CFP-nls and either flipped t-SNAREs or GPI-anchored t-SNAREs were preloaded with soluble dye CMFDA, detached, and mixed with CHO v-cells (GM1 negative) expressing flipped VAMP2 or GPI-VAMP2. GM1 lipid, originally present only in the t-cell plasma membrane, was labeled with Alexa⁵⁹⁴-cholera toxin β-subunit (red). In completely fused cells, all three markers (CFP-nls, CMFDA, and GM1) are transferred from the t-cells to the v-cells, evidenced as a cell with multiple cyan nucleus, green cytoplasm, and red plasma membrane staining. In hemifused cells, only the lipid probe GM1 is transferred to the v-cells, showing a v-cell red-labeled plasma membrane without green cytoplasm and cyan nuclei staining. Reversible hemifusion resulted in v-cells that were positive for GM1 staining but not in contact with any t-cells.

Figure 2.

Complete fusion, incomplete fusion, and reversible incomplete fusion by flipped SNAREs. As described in Fig. 1 B, CHO stable v-cells expressing flipped VAMP2 and RFP-nes were detached and overlaid on MEF-3T3 cells expressing flipped Syntaxin, flipped SNAP-25, and CFP-nls. The cells were fixed after 6 h at 37°C. GM1 was stained with FITC-cholera toxin β-subunit (green). Arrowheads indicate fused cells, arrows indicate incomplete fused cells, and asterisks indicate reversible incomplete fusion. (Control) The cytoplasmic domain of VAMP2 (20 μM) completely inhibited complete fusion, incomplete fusion, and reversible incomplete fusion mediated by flipped SNAREs. Bars, 10 μm.

Figure 3.

Hemifusion mediated by flipped SNAREs. As described in Fig. 1 C, MEF-3T3 stable cells expressing flipped Syntaxin, flipped SNAP-25, and CFP-nls were preloaded with CMFDA as a fluid phase marker, detached, and then seeded on a coverslip that already contained CHO stable cells expressing flipped VAMP2. Cells were fixed after 6 h at 37°C and stained with Alexa⁵⁹⁴-cholera toxin β-subunit (red). Arrowheads indicate fused cells and arrows indicate hemifused cells. (top) A fused cell in contact with a hemifused one. (control) No fusion or hemifusion was observed using CHO cells that were mock transfected. Bars, 10 μm.

Figure 4.

Time course of complete fusion, hemifusion, and reversible hemifusion mediated by flipped SNAREs. CHO stable v-cells were mixed with MEF-3T3 stable t-cells. At different time points, the percentage of v- and t-cells in contact that underwent complete fusion or hemifusion was determined using the assay described in Fig. 1 B. Images in 50 random fields were used for calculation of each time point. Reversible hemifusion events were calculated as a fraction of the number of v-cells in contact with t-cells. Values are mean ± SD of three independent experiments. Dashed line is the mean value from 6 to 48 h showing that the hemifusion curve reached a plateau.

Figure 5.

Cell surface expression of GPI-anchored SNAREs. (A) Soluble t-SNAREs bind to GPI-VAMP2. COS cells were transfected with an IRES plasmid that encodes GPI-VAMP_2-92 and enhanced GFP (EGFP). The cells were incubated with a soluble t-SNARE complex (5 μM) for 1 h at 37°C. Surface-bound t-SNARE was detected with an antibody to Syntaxin (red, merged with EGFP fluorescence). (right) When phosphatidylinositol-specific phospholipase C (PI-PLC) was included in the binding assay, little t-SNARE binding was observed. (B) Expression of GPI-anchored t-SNAREs on the cell surface. COS cells cotransfected with GPI-Syntaxin_186-265 and flipped SNAP-25 were stained with antibody to c-myc (Syntaxin, green) and antibody to SNAP-25 (red). Top, unpermeabilized cells; bottom, permeabilized cells. (C) Release of GPI-anchored t-SNAREs from the cell surface by PI-PLC. COS cells expressing Syntaxin_186-265 and flipped SNAP-25 were incubated with PI-PLC (100 mU/ml), and then stained with antibody to c-myc (Syntaxin, green) and antibody to SNAP-25 (red). Bar, 10 μm.

Figure 6.

GPI-anchored SNAREs promote hemifusion. (A) MEF-3T3 cells expressing GPI-Syntaxin and flipped SNAP-25 were overlaid on CHO cells expressing GPI-VAMP2. First column, hemifusion detected with the assay described in Fig. 1 B. (third column) Hemifusion detected with the assay described in Fig. 1C. (fourth column) No hemifusion was observed when GPI-VAMP2 was not expressed in the CHO cells. Arrows point to hemifused cells. (B) The percentage of v- and t-cells in contact that underwent complete fusion or hemifusion in the presence of different combinations of flipped or GPI anchored SNAREs. Hemifusion was also detected in cell fusion mediated by flipped SNAREs. Vc-Peptide (60 μM) produced a slight increase in hemifusion mediated by GPI-anchored SNAREs, whereas the cytoplasmic domain of VAMP2 (CD-VAMP2; 20 μM) inhibited it. Filled bars, fused cells; open bars, hemifused cells. Values are mean ± SD of four independent experiments; n = 200 cells. Bars, 10 μm.

Figure 7.

Hemifusion does not involve the mixing of the inner monolayers of the membranes or of soluble content. (A) CHO cells transiently transfected with GPI-VAMP2 and EGFP-f were detached and incubated with the MEF 3T3 t-cells (top). GM1 was detected with Alexa⁵⁹⁴-ctxβ (red). No transfer of EGFP-f was observed in the hemifused cells. CHO v-cells that were transiently transfected with GPI-VAMP2 and CFP-nls were preloaded with the cytoplasmic dye Calcein AM (green), and then detached and incubated with MEF-3T3 t-cells that expressed CFP-nls, GPI-Syntaxin, and flipped SNAP-25 (bottom). The cells were fixed after 6 h and stained with Alexa⁵⁹⁴-ctxβ (red). Arrows indicate hemifused cells. Transfer of GM1, but not Calcein AM, was observed in the hemifused cells. Bars, 10 μm. (B) Summary of results using different types of probes. 5-Chloromethylfluorescein diacetate (CMFDA), Calcein AM, and 4-chloromethyl-6,8-difluoro7-hydroxycoumarin (Blue CMF₂HC) are small soluble dyes; EFGP-f, EYFP bearing a palmitoylation sequence (EYFP-pal), and HA epitope fused to a farnesylation sequence (HA-f) labeled the inner leaflet of the plasma membrane; GPI-anchored EYFP (GPI-EYFP), GPI-anchored epitope AU1 (GPI-AU1), and ctxβ labeled the outer monolayer of the plasma membrane. Filled bars, percentage of hemifused cells showing probe transfer mediated by flipped SNAREs; open bars, percentage of hemifused cells showing probe transfer mediated by GPI-anchored SNAREs. Only GPI-AU1 and GM1 were transferred in the hemifused cells. Values are mean ± SD of two independent experiments; n = 100 cells.

Figure 8.

Hemifused cells do not show electrically active connecting fusion pores. Electrical diagram (A) and equivalent circuit (B) of hemifused cells in the whole cell configuration. R _a is the access resistance, R _m1 and R _m2 are the resistances of cells 1 and 2, respectively, C _m1 and C _m2 are the membrane capacitances of cells 1 and 2, respectively, and R _p is the resistance of a theoretical pore connecting the two cells. (C) Cell capacitances ± SD averaged over 100 s of recording. A 20-Hz sampling frequency (see Materials and methods) was used. Measurements of single CHO cells (n = 3) as well as hemifused cells patched from the CHO cell (n = 3) and cell capacitance of completely fused cells are shown (n = 3). Hemifused cells appear to have capacitances similar to single cells, whereas fused cells have membrane capacitances equal to the sum of one CHO and 3T3 cell. (D) A representative recording of cell resistance (n = 4) taken over 100 s. A 20-kHz sampling frequency was used. Pore opening would be observed as a downward current step.

Figure 9.

Alternative outcomes of SNARE-mediated membrane fusion. Formation of the SNARE complex leads to the formation of a transitional pore. The thermodynamic properties of the transitional pore drive membrane fusion to complete fusion, hemifusion, reversible hemifusion, or kiss and run.