Vesicle tethering does not occur at the restrictive temperature in sec6-4 cells, whereas vesicles remain tethered in sec1-1 cells. (A) Kymograph of GFP-Sec4 vesicle puncta shown in a sec6-4 strain at the permissive temperature. (B) Kymograph of GFP-Sec4 vesicle puncta shown in the sec1-1 strain at the permissive temperature. (C) Column scatter plot of the time from tethering to fusion (in seconds) in wild-type (n = 22), sec6-4 (n = 16), and sec1-1 (n = 15) cells at 21°C. *, P < 0.05; ns, not significant. Lines show mean and 95% confidence intervals. (D) Schematic diagram for creating kymographs along the entire bud cortex over time. (E) Kymograph of GFP-Sec4 along the entire bud cortex in an sec6-4 strain at the permissive and restrictive temperature (5 min shift). (F) Kymograph of GFP-Sec4 along the entire bud cortex in an sec1-1 strain at the permissive and restrictive temperatures (5 min shift).