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3, right column), confirming the SNARE dependence of the assay and the lack of free exchange of the GM1 lipid. This transfer of red fluorescence to the unstained v-SNARE population does not occur when mock-transfected cells are used instead of v-cells ( Fig. 3, arrows), which is indicative of incomplete fusion. Transfer of GM1 alone to a v-cell gives the striking result of a red-bordered cell with no fluorescent content ( Fig. Furthermore, in this assay, all the fluorescent markers (green CMFDA as soluble content marker, CFP-nls as nuclear marker, and red Alexa 594-ctxβ as membrane marker) are initially limited to the t-SNARE cell to clearly identify which fluorophore is able to diffuse to v-SNARE cells. 1 B) due to diffusional restriction of the bulky red fluorescent protein through these fusion pores. This modification would allow us to detect small nonenlarging fusion pores that might be missed with the original flipped SNARE fusion assay ( Fig. 1 C) in which a smaller fluorescent probe (5-chloromethylfluorescein diacetate ) was used as a content marker. To further characterize the novel incomplete fusion phenotypes, we developed the hemifusion assay ( Fig. Incomplete fusion phenotype corresponds to hemifusion events 2, control) or by expressing Syntaxin 1 alone (without SNAP-25 not depicted).
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No fusion subtype was observed if SNARE pairing is disrupted by either titrating free t-SNARE with the cytoplasmic domain of the v-SNARE ( Fig. All three observed fusion outcomes are SNARE dependent. We observe one such v-cell for about every 20 contacting v- and t-cell pairs. These kiss-and-run–like cells have apparently experienced transient mixing of the lipid bilayers to become GM1 positive without significant contents mixing before physically separating. We also observed a reversible version of the incomplete fusion i.e., v-cells containing lipid mixing markers that are no longer in contact with a t-cell ( Fig. Z-section analysis confirmed the absence of a t-cell–derived cyan-nucleus in these incompletely fused cells (unpublished data). Lipid transfer without content mixing (i.e., incomplete fusion) was observed in 14 ± 3% ( Fig. 2, arrowheads), which is consistent with our earlier observations in COS cells ( Hu et al., 2003). 1 B) and incubated together for 6 h, complete fusion occurs in 23 ± 3% (mean ± SD) of the cells in contact ( Fig. In stable cell lines expressing flipped SNAREs and the appropriate fluorescent markers (as depicted in the original flipped SNARE fusion assay Fig. By including various fluorescently labeled content markers, two assays were developed in which different potential membrane fusion outcomes could be distinguished according to different patterns of color mixing ( Fig. When MEF-3T3 cells expressing flipped t-SNAREs (GM1 positive) were mixed with CHO cells expressing flipped v-SNAREs (GM1 negative), membrane lipid mixing can be observed as the development of ctxβ-dependent fluorescence on the previously GM1-negative v-SNARE cells. The cell-specific localization of GM1 was detected through binding of fluorescently labeled cholera toxin β-subunit (FITC-ctxβ or Alexa 594-ctxβ ).
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To monitor lipid mixing with the cell–cell fusion assays, we took advantage of the fact that the GM1 ganglioside is absent on the surface of CHO cells due to the lack of a key enzyme in the pathway of ganglioside biosynthesis ( Rosales Fritz et al., 1997) but is present on the surface of MEF-3T3 cells ( Fig. The cell–cell fusion assays we present here use various combinations of soluble and lipidic probes to simultaneously monitor content and lipid mixing between cells.