Dynamics of the exocyst rSec8-TagRFP trafficking on vesicles. (A) HeLa cells were knocked down for Sec8, cotransfected with Vamp2-GFP/rSec8-TagRFP, and imaged by TIRFM at 2 Hz. Maximum projections (5 min) were generated (Video 2; arrows show colocalization). (B) Kymograph of box in A shows the appearance and disappearance of vesicles (arrowheads). (C) Track length measurements (∼3,000) were made to determine the median duration (∼7.5 s) of rSec8-TagRFP (histogram; inset shows cumulative probability graph). (D) rSec8-TagRFP colocalization analysis with post-Golgi cargos (VSVG-GFP and NPY-GFP) or recycling endosomes markers (GFP-Rab11 and Vamp2-GFP). n = 4 cells per cargo; error bars = SD; *, P = 0.001, t test. (E) Two galleries of a dual labeled vesicle show the appearance (open arrowhead) and disappearance (closed arrowhead) of vesicles at the PM. Asterisks show when the Vamp2-GFP intensity increases.