Fig. 3. Microinjected cells move during TIR-FM recording of exocytic events. (A,B) Monolayers of NRK fibroblasts were wounded. Cells at the edge of the wound were microinjected with cDNA encoding LDLR-GFP, incubated for 1 hour at 37°C and then shifted to 20°C for 2 hours prior to image acquisition. Wound-edge cells expressing LDLR-GFP were identified at low magnification by epifluorescence (black outlines). The cells were then imaged under transmitted light at the same magnification to localize the wound edge at the beginning of the experiment (green outline). At the end of the experiment, 90 minutes later, the same field was imaged again at low magnification (red outline). Cells at the edge of the wound advanced significantly (10-20 µm) during that time. All injected cells advanced with the wound edge. (C) High magnification contrast images were taken of the injected cell marked by the asterisk in (B). The outlines of the cell and the nuclei were traced just before the first TIR-FM recording (0 min, green) and after the last TIR-FM recording (30 min, red). (D) The cell shown in (C) was imaged by TIR-FM in eight intervals (each 500 frames, 5 frames per second). The complete (yellow crosses) and partial (cyan stars) fusion events were mapped. The fusion map was overlayed on top of an epifluorescence image taken at the beginning of the TIR-FM recording. Notice that the Golgi complex is oriented towards the direction of migration (white arrows). Scale bars, 50 µm (A,B), 10 µm (C,D).