Fig. 6
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
