Fig 2: 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 3: 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 4: Mammalian exocyst complex comprises two subcomplexes that can localize to the PM independently. a Quantitative full-scan LC–MS/MS analysis of endogenous EXO70-GFP pulldowns from intact or Sec8-depleted cells. Inputs for the samples were calibrated and normalized using MRM–MS analysis on the same samples. The schematic shows the experimental design in which EXO70-GFP was captured using GFP-Trap beads and SEC8 was depleted by shRNA. b Heatmap summarizing relative binding of SEC6, SEC8, EXO70, and EXO84 to SEC3-GFP in SEC10-depleted cells compared to control shRNA-treated cells, as assessed by western blot. Also see Figure S2H-I. c TIRFM images of EXO70-GFP and SEC3-GFP in control or SEC10 shRNA-treated cells. Scale bars 20 µm. d Quantification of relative fluorescence intensities in C. e Quantification of fluorescence intensities from TIRFM images of SEC5-GFP cells treated with control or SEC10 shRNA. f TIRFM images of double knock-in NMuMG cells, showing localizations of EXO70-GFP and SEC5-Sc at the PM in control, SEC8 shRNA, and SEC3 shRNA-treated cells. Scale bars = 20 µm. g Quantification of relative fluorescence intensities in D. Center lines show the median; box limits indicate 25th and 75th percentiles; whiskers extend 1.5X the IQR from the 25th and 75th percentiles. Experiments were repeated at least three times with similar results

Fig 5: Dual-color FCCS of exocyst subunits in the membrane and cytosol. a Schematic of dual-color cross-correlation spectroscopy (FCCS). Top diagram shows cross-correlation between co-diffusing molecules. Bottom shows molecules that do not co-localize. b SEC5-Halo and EXO70-GFP FCS measurements in the cytosol and the PM. EXO70-GFP and Akt-Halo fluorescence cross-correlation was used as a negative control. HaloTag was labeled using JF646 Halo ligand (200 nM for 1.5 h). c SEC5-Halo and SEC8-GFP, or SEC8-GFP and Akt-Halo FCS measurements in the cytosol or the PM. d SEC5-Halo and SEC3-GFP, or SEC3-GFP and Akt-Halo FCS measurements in the cytosol or the PM. e Statistics of the fraction of GFP-tagged exocyst subunit bound in the cytosol. SEC8-GFP+SEC5-Halo: 92 ± 3.86%, SEC3-GFP + SEC5-Halo: 67 ± 3.84%, and EXO70-GFP+SEC5-Halo: 64 ± 3.46% (mean ± SEM). f Fractional coincidence between SEC5-Halo and SEC8-GFP or SEC5-Halo and EXO70-GFP was analyzed using TIRFM. Calculations were corrected for HaloTag labeling efficiency and expression levels of SEC8-GFP and EXO70-GFP with respect to SEC5-GFP. Center line denotes median. SEC5-Halo bound to SEC8-GFP was 85 ± 3.3% (mean ± SEM). SEC8-GFP bound to SEC5-Halo was 85 ± 5.1%. SEC5-Halo bound to EXO70-GFP was 60 ± 3.2% and EXO70-GFP bound to SEC5-Halo was 69.8 ± 2.9%. Object detection parameters were set between 0.30 and 0.35 µm and contrast was adjusted to faithfully detect objects of interest. White circles show the medians; box limits indicate the 25th and 75th percentiles as determined by R software; whiskers extend 1.5 times the interquartile range from the 25th and 75th percentiles; polygons represent density estimates of data and extend to extreme values. p values were calculated using Kruskal–Wallis test followed by Dunn’s multiple comparison test. The hydrodynamic radius (RH) for the slow diffusing fraction was median 466 nm (95% CI: 355.2–606.8), and that of the fast diffusing component, 14.82 nm (95% CI: 13.22–17.27). FCS measurements were fitted (lines) to the model described in Methods (Eqs 2–4). Curves shown are representative, taken from a single cell. Statistics are average from > 25 measurements for each condition from two experiments and > 5 cells. p-values were calculated by one-way ANOVA followed by Tukey’s post hoc test