Fig. 9. Cortactin phosphorylation affects podosome dynamics. (A) RAW-derived osteoclasts were transfected with GFP-actin and dsRED (control), with GFP-actin and wild-type cortactin (corWT), or with GFP-actin and a cortactin phosphorylation mutant (cor3YF). Temporal ratio images, based on time-lapse microscopy of GFP-actin, are shown. Notice the decrease in stable podosomes in cells overexpressing either WT or mutant cortactin (corWT, cor3YF). (B) The calculated average (±s.d.) life span of cluster podosomes was 169±107 seconds in control cells, 128±73 seconds in cells overexpressing WT cortactin, and 84±54 seconds in cells overexpressing cortactin 3YF. For control cells, a total of 159 podosomes from four cells in three independent experiments were analyzed; for cells expressing WT cortactin, seven cells from three experiments, with a total of 238 podosomes were examined; for cortactin 3YF, nine cells from four experiments, with a total of 402 podosomes, were examined. Statistical differences were calculated using the Student's t-test (control vs. corWT cells: P<5 x10^-4; control vs. cor3YF, and corWT vs. cor3YF cells: P<5 x10^-13). (C) RAW cells co-expressing GFP-actin and Src251 were transfected with dsRED (control); dsRED and WT cortactin (corWT); or dsRED and cor3YF. Temporal ratio images based on time-lapse microscopy of GFP-actin are shown. Notice the increase in podosome dynamics without the development of rings or SZL structures, in cells overexpressing corWT and cor3YF. Bars, 10 µm.