PFKL forms dynamic particles in MTLn3 rat breast cancer cells. (A) TIRF image of PFKL-EGFP expressing MTLn3 rat mammary adenocarcinoma cell from a time-lapse sequence acquired at 10 frames per second and 1-s (10 frames) and 5-s (50 frames) rolling averages highlighting docked PFKL-EGFP particles. (B) Kymographs showing docked PFKL-EGFP particles in the yellow box indicated in A. Black arrowheads correspond with the individual time points shown on the left. The plot shows fluorescence intensity of the two example particles highlighted by colored arrowheads as a function of time. (C) Docked PFKL-EGFP lifetime distribution from control cells (n = 306 particles from 10 cells). Histogram bin width was set according to the Freedman–Diaconis rule. The red line is a log-logistic fit of the lifetime distribution. The inset is a box plot of the mean particle docking time per cell for control cells or cells treated with 2-deoxyglucose (DOG; n = 298 particles from 10 cells) for 23 h. A t test was used to determine statistical significance. (D) Kymograph of the area in the red box indicated in A showing PFKL-EGFP particles docking at the similar location over the course of the video. (E) Single exposure and 5-s rolling averages for MTLn3 cells expressing EGFP or various EGFP-PFK1 constructs as labeled. The frame rate of time-lapse sequences was: EGFP, 2.6 frames per second; PFKP-EGFP, 1.5 frames per second; PFKL-F638R-EGFP, 2.8 frames per second; CatP/RegL-EGFP, 2.9 frames per second. Higher-magnification images of the areas of the red boxes in the CatP/RegL-EGFP cells highlight a docked particle. Data are representative of three independent cell preparations.