Exocyst rSec8-TagRFP is highly dynamic on cell protrusions and accumulates in clusters at the leading edge during cell migration. (A) Images of rSec8-TagRFP dynamics at cell protrusions by TIRFM/FRAP (see Video 4). Images of rSec8-TagRFP in stable Sec8-replaced HeLa cells in which a ROI is photobleached at time 0 s; kymographs of the ROI are shown beneath. Arrowheads show the appearance and disappearance of rSec8-TagRFP tracks. (B) ROI signal corrected for photobleaching (n = 5; error bars = SEM) fits to a single exponential (black line) and was used to calculate the half-time of recovery (τ1/2), mobile fraction (Fm), and immobile fraction (Fi). (C) rSec8-TagRFP dynamics at the leading edge of migrating cells by dual color TIRFM. (left) Images of a stable EA hy926 cell (Sec8KD/rSec8-TagRFP) transfected with TfRc-pH show the cell position at time 0 (green) and at 30 min (yellow). A gallery of the ROI is on the right. rSec8-TagRFP accumulated where the cell subsequently moved (see Video 5).
