Fig 2: Establishment and validation of tagged exocyst subunit cell lines. a Schematic showing a region of subunit gene targeted by sgRNA and tags to be inserted C-terminally, in-frame with the coding region. b Strategy to isolate CRISPR/Cas9-mediated sfGFP-tagged clones of exocyst subunits in NMuMG cells. c Western blots with subunit specific antibodies show successful incorporation of sfGFP in both alleles (EXO70, SEC5, and SEC8), or in one allele (SEC3 and SEC6). A shRNA specific to SEC3 was used to confirm the identity of multiple bands in the blot. d Confocal images of live wild-type NMuMG cells and endogenously tagged SEC3-GFP, SEC5-GFP, SEC6-GFP, SEC8-GFP, and EXO70-GFP cell lines. Scale bar = 10 µm. e Protein–protein interaction heat map for endogenous SEC3-GFP, SEC5-GFP, and EXO70-GFP pulldown using GFP-Trap nanobodies and MS. Yellow indicates baits, blue the protein identified with high confidence, and gray denotes preys that were not identified. The experiments were repeated three times for EXO70-GFP and twice for SEC3-GFP and SEC5-GFP. All experiments were also partially confirmed by IP–WB in at least three independent experiments, with similar results. f Western blot analysis to assess the relative abundance of exocyst subunits fused to sfGFP, using anti-GFP antibodies (SEC3 and SEC6 quantifications corrected for heterozygosity). GAPDH was the loading control. Y axis shows the ratio of GFP/GAPDH. Quantification shows pooled data from three experiments

Fig 3: Assessment of fractional binding using single-molecule approach. a Diagram showing experimental workflow. b Cleared cell lysates from the indicated double knock-in cells were spread between quartz slides and glass coverslips for TIRF imaging. The GFP and Halo + JF585 fluorophores were excited simultaneously with 488-nm and 561-nm lasers at 12.5 Hz for 40 s. Scale bar = 1 µm. c Number of photobleaching steps detected for SEC5-Halo and SEC8-GFP in experiments shown in panel b. d Quantification of the fraction of subunit A bound to (–> ) subunit B from experiments shown in panel b. Numbers of particles analyzed for each pair are shown in Supplementary Fig. 4c. e Fraction of SEC5-Halo and SEC8-GFP bound to each other in SEC3 or SEC6 shRNA-treated cells compared to control. Center line denotes mean. f Western blot analysis of SEC3 and SEC6 shRNA knockdown efficiencies in the experiment shown in e. Error bars denote ± SD. Statistical significance was computed by one-way (d) or two-way (e) ANOVA, followed by Tukey’s multiple comparison tests. Experiments were repeated three times and data were pooled

Fig 4: Exocyst molecule counting at vesicle fusion sites. a Stepwise photobleaching of CD86 fused to one or two sfGFP molecules used to determine intensities of known numbers of GFP molecules. b Standard curve for counting protein molecules, from intensities measured on CD86 fused to one, two, or three sfGFP molecules. Error bars = ±s.d.; pink shaded region denotes 95% confidence band. c Fluorescence landscape of SEC8-GFP shows a typical resolvable distribution of molecules within an ROI from which intensity measurements were taken. d Intensity (y) was measured from the peak and converted to the number of molecules using the regression equation determined in b. Numbers for SEC3 and SEC6 were corrected for heterozygosity. Mean ± SD = 7.6 ± 3.6 (SEC3-GFP), 9.8 ± 3.5 (SEC5-GFP), 9.8 ± 3.4 (SEC6-GFP), 9.35 ± 3.2 (SEC8-GFP), and 9.19 ± 2.8 (EXO70-GFP). Cohen’s d value for the difference between SEC3-GFP and SEC5-GFP is –0.208. Centers indicate median, box limits indicate 25th and 75th percentiles, and whiskers extend 1.5x IQR from 25th to 75th percentiles. n = 96, 50, 51, 50, 58. Coefficient of variation (CV) = 47.9, 35.2, 39.2%, 34.3, and 30.7% in the order indicated in the graph. CV between each subunit was 7.9%. Experiments were repeated at least three times with similar results. P-values were computed using one-way ANOVA test followed by Scheffe’s post hoc test

Fig 5: Sec3 departs from the exocyst complex prior to vesicle fusion. a Schematic representation of the data. b NMuMG cells were transduced with mApple-Rab11 and VAMP2-pHluorin lentivirus and imaged by TIRFM. Kymographs show arrival and departure of mApple-Rab11 and vesicle fusion, highlighted by VAMP2-pHluorin flashes. Speed = 5 Hz. Scale bar = 5 s. c Distribution of mApple-Rab11 arrival times prior to vesicle fusions. The inset shows box and whisker plot; center line = −14.6 s (median; 95% CI: −16.6 to −12.5). Error bars = Tukey’s range. d Intensity trace over time for VAMP2-pHluorin and TfR-pHuji. The peak intensities denote flashes resulting from vesicle fusion. Speed = 5 Hz. e Effects of exocyst subunits depletion on vesicle fusion activities in NMuMG cells assessed using VAMP2-pHluorin. n = 32, 32, 18, 22, 21, 22. P-values compared to control. f Kymographs showing an itinerary of SEC3-GFP, SEC5-GFP, SEC6-GFP, and EXO70-GFP arrivals/departures with respect to the vesicle fusion marker TfR-pHuji. Square images show snapshots in the green and red channels at the time of vesicle fusions. Scale bar = 5 s. g, h Quantification of exocyst subunit arrival and departure times at and from the vesicle fusion site shown in E. Data points shown as dots. g Median arrival times in seconds: −11.7 (95% CI: −12.9 to −9.1; EXO70), −11.6 (95% CI: −14.6 to −10.2; SEC3), −10.6 (95% CI: −14.0 to −8.2; SEC5), and −10.3 (95% CI: −12.3 to −8.8; SEC6). n = 146, 132, 108, and 132 objects in the order data shown. h Median departure times in seconds: 1.4 (95% CI: 1.2–1.5; EXO70), −0.4 (95% CI: −0.98 to −0.69; SEC3), 1.3 (95% CI: 1.21.6; SEC5), and 1.3 (95% CI: 1.21.7; SEC6). n = 145, 117, 108, and 132 particles from 33, 19, 29, and 26 cells in the order data are shown. Center lines = medians; hinges extend from 25th to 75th percentiles. Statistical significance measured by Kruskal–Wallis test followed by Dunn’s post hoc analysis. Experiments were repeated three times with similar results