Vesicles tether to the cortex for ∼18 s in wild-type cells, which lengthens and is more variable in a constitutively active Rab mutant. (A) Still frames of a typical GFP-Sec4 vesicle tracking experiment in a wild-type cell. An inverted monochrome maximum projection is shown for clarity. Arrows point to GFP-Sec4 puncta tethered to the membrane. Bar, 2 µm. See also Video 1 and Fig. S1. (B) Kymograph of GFP-Sec4 vesicle puncta shown by the arrowheads in A. (C) Column scatter plot of the time from tethering to fusion (in seconds) in wild-type cells for the whole population (n = 22), bud tip (n = 13), and bud sides (n = 9). ns, not significant. Lines show mean and 95% confidence intervals. (D) Kymograph of GFP-Sec4-Q79L vesicle puncta. See also Video 2. (E) Column scatter plot of the time from vesicle tethering to fusion (in seconds) in the indicated strains. ***, P < 0.0001. Lines show mean and 95% confidence intervals for wild type (n = 22) and GFP-Sec4-Q79L (n = 15). (F) Movement (nm/s) of puncta tethered to the cortex for the strains indicated at 21°C. Wild type (n = 22), GFP-Sec4-Q79L (n = 15), sec6-4 (n = 16), sec1-1 (n = 15), myo2-13 (n = 15), myo2-3DR (n = 14), and myo2-RAKA (n = 16) are shown. *, P < 0.05. Error bars indicate standard deviation. (G) Aborted tethering events per second for the strains indicated at 21°C. Wild type (n = 22), GFP-Sec4-Q79L (n = 15), sec6-4 (n = 16), sec1-1 (n = 15), myo2-13 (n = 15), myo2-3DR (n = 14), and myo2-RAKA (n = 16) are shown. ***, P < 0.0001. Error bars indicate standard deviation.