VAMP2 is detected preferentially in fusion events (puffs) delivering MOR to the surface. (A) Schematic of a VAMP2⁺ puff in SpH-cargo and VAMP2-pHuji co-expressing cells. When a vesicle carrying both SpH-cargo and VAMP2-pHuji fuses to the plasma membrane, the fluorescence intensity increases in both the SpH channel and the pHuji channel. (B) Montage of a SpH-MOR puff colocalizing with VAMP2-pHuji (SpH-MOR in green and VAMP2-pHuji in magenta) from SpH-MOR and VAMP2-pHuji co-expressing cells 5 min after DAMGO treatment at 37°C. Scale bar = 2 µm. (C) Representative time-course traces of mean SpH-MOR (cyan) and the corresponding mean VAMP2-pHuji fluorescence (magenta) from a VAMP2⁺ puff of the SpH-MOR expressing cells. (D) Representative time-course traces of mean SpH-B2AR (red) and the corresponding mean VAMP2-pHuji fluorescence (magenta) from a VAMP2-negative puff of the SpH-B2AR expressing cells. (E) Quantification of VAMP enrichment in the population of puffs for each SpH-cargo. VAMP2 or VAMP4 enrichment of individual puff event was calculated as the fold change of VAMP-pHuji fluorescence at the peak of the event over baseline SD, as described in the text (median ± 95% confidence interval [CI]; TfR-SpH: 2 cells, puff = 85; SpH-B2AR: 3 cells, puff = 85; SpH-MOR-VAMP2: 3 cells, puff = 59; SpH-MOR-VAMP4: 4 cells, puff = 44; all conditions from two independent experiments). SpH-MOR puffs showed significantly more enrichment of VAMP2 compared with other cargos, but showed no VAMP4 enrichment (one-way ANOVA, post-hoc Kruskal–Wallis test: TfR vs. B2AR: P > 0.9999; TfR vs. MOR: P < 0.0001; B2AR vs. MOR: P < 0.0001; MOR-VAMP2 vs. MOR-VAMP4: P < 0.0001). (F) The fraction of VAMP2⁺ puffs (% population) as defined by different folds of VAMP2 enrichment over baseline SD (cutoff) shows a consistent enrichment of VAMP2 preferentially in MOR puffs across all cutoffs. (G) Kernel density estimation of the pooled population of all puffs to estimate subclasses. The best fit predicted three subclasses based on VAMP2 enrichment. Three Gaussian mixture models of the actual distribution (solid line) and predicted subclasses (dashed lines) are shown. (H) Fraction of puffs in each predicted subclass shows distinct population composition of different SpH-cargos and a preferential enrichment of VAMP2⁺ subclasses in SpH-MOR puffs.