Figure 7. Cortactin depletion inhibits KGFR internalization and polarization.

A) HaCaT cells were coinjected with cortactin siRNA and KGFR cDNA, to simultaneously induce cortactin silencing and KGFR overexpression. Control cells were injected with an unrelated siRNA. After injection cells were incubated at 4°C with anti-Bek polyclonal antibodies and treated with KGF or FGF10, as described above. Quantitative double immunofluorescence analysis, using anti-cortactin monoclonal antibody, shows that in cortactin siRNA/KGFR cDNA coinjected cells, KGFR signal is evident, while the cortactin signal appears strongly decreased if compared to the surrounding uninjected cells in the same microscopic fields or to control cells injected with unrelated siRNA (compare upper panels to lower panels). Upon ligands treatment, in cortactin-depleted cells KGFR signal remains uniformly distributed on the plasma membrane, while in cells microinjected with unrelated control siRNA, expressing cortactin, the KGFR appears internalized and its colocalization with cortactin is evident, as well as their polarization at the leading edge of migrating cells (arrows). Images shown were obtained by 3D reconstruction performed as reported in figure 2. Bar: 10 µm. B) Quantitative analysis of the percentage of KGFR internalization was performed as above. Results are expressed as mean values +/- SE and Student's T test was performed and significance level has been defined as above. *p<0,005 vs the corresponding untreated cells; **p<0,0001 vs the corresponding untreated cells. C) HaCaT cells were coinjected with a mixture of cortactin siRNA and rabbit IgG, to identify the microinjected cells, and then treated with EGF-TRITC or Transferrin-Texas Red (Tf-TxRed) for 1 h at 4°C or for 20 minutes at 37°C before fixation and permeabilization. Triple immunofluorescence analysis, using anti-cortactin monoclonal antibody, shows that in cells microinjected with the cortactin siRNA very low levels of cortactin staining were detectable, Tf internalization was strongly impaired, while EGF uptake appeared unaffected, if compared to uninjected cells or to cells injected with unrelated siRNA. Bar: 10 µm.