Bud6 localizes to secretory vesicles and is transported on actin cables to the bud neck. (A) Colocalization of Bud6-mCherry and GFP-Sec4 in live yeast cells. Bar, 5 µm. Merge highlights two puncta used in line scan analysis below. (B) Pearson’s correlation analysis of Bud6-mCherry signal with GFP in strains containing GFP tags on different membrane compartments (n = 50 cells per strain). Error bars represent SEM. Statistical significance calculated by one-way ANOVA (****, P ≤ 0.0001). (C) Line-scan analysis shows overlap between Bud6-mCherry and GFP-Sec4 puncta, corresponding to puncta highlighted in A. (D) Time-lapse dual-color live imaging of Bud6-mCherry and GFP-Sec4. Arrow highlights the movement of a vesicle. (E) Kymograph showing Bud6-mCherry and GFP-Sec4 comigration. (F) Total Bud6-mCherry fluorescence level in cells of variable bud size from an asynchronous population (n = 100 cells). (G) Representative time-lapse images of Bnr1-GFP and Hof1-GFP strains after α-factor arrest/release. Bar, 3 µm. (H) Quantification of Bnr1-GFP and Hof1-GFP levels at the bud neck for each strain, from time lapse imaging in G (n = 5 cells per strain).