Figure 1.
Src is tyrosine phosphorylated by KGFR ligand stimulation.
A) HaCaT cells were serum starved and treated with KGF or FGF10 at 37°C for different time points. Immunoprecipitation with anti-Src antibodies and immunoblot with anti-phosphotyrosine antibody show that KGF treatment induces a rapid phosphorylation of Src, already evident after 10 minutes of stimulation and rapidly decreasing after 30 minutes. In FGF10-treated cells Src phosphorylation is detectable after 30 minutes of treatment, and gradually decreases, although still present up to 2 hours. The equal loading was assessed using anti-Src antibodies. The intensity of the bands was evaluated by densitometric analysis; the values from a representative experiment were normalized, expressed as fold increase with respect to the control value and reported as graph. B) HaCaT cells were pre-treated or not with the Src-specific inhibitor SU6656 and stimulated with KGF or FGF10 for 10 and 30 minutes, respectively. Immunoprecipitation with anti-Src, anti-cortactin or anti-Bek antibodies and immunoblot with anti-phosphotyrosine antibody show that the treatment with SU6656 blocks the tyrosine phosphorylation induced by KGFR ligands of Src and cortactin, but not of KGFR. The equal loading was performed using anti-src, anti-cortactin or anti-Bek antibodies.
Figure 2.
Src activity is responsible for KGFR polarization at the leading edge of migrating cells.
HaCaT cells transiently transfected with the human KGFR (HaCaT KGFR) were treated with KGF or FGF10 and then immunostained at 4°C with an anti-Bek polyclonal antibodies, which recognize the extracellular portion of the receptor, and do not compete with the ligands for binding. Cells were then fixed, permeabilized, and stained with TRITC-conjugated phalloidin to visualize the actin cytoskeleton organization. Nuclei were stained with DAPI. Images are obtained, as described in Materials and Methods, by serial optical sectioning and 3D reconstruction of a selection of three out of the total number of sections: the selected sections are all central and crossing the nucleus visualized by DAPI. Results show that, in peripheral untreated HaCaT KGFR cells, the KGFR signal appears homogeneously distributed on the entire plasma membrane and the actin is not organized in typical migratory structures. After either KGF or FGF10 treatment, the KGFR signal appears polarized at the leading edge of migrating cells, where actin cytoskeleton is mainly organized in filopodia (arrows), but also in ruffles and small lamellipodia (arrowheads). In the presence of the Src inhibitor SU6656, the KGFR signal remains uniformly distributed on the plasma membrane, also upon ligand stimulation, and the actin is organized in few thin filopodia distributed over the entire cell surface. Bars: 10 µm.
Figure 3.
The KGFR polarization is dependent on receptor endocytosis.
HaCaT KGFR cells were incubated at 4°C with the anti-Bek polyclonal antibodies to selectively stain the plasma membrane receptors and exclusively follow them during endocytosis, and then stimulated with KGF or with FGF10 for 5, 10 and 30 minutes at 37°C. The plasma membrane was decorated with the plasma membrane marker WGA-FITC. KGFR staining appears continuous and uniformly distributed on the cell surface of untreated cells, discontinuous on the plasma membrane and in some intracellular dots underlying the cell surface upon 5 minutes of KGF stimulation, and polarized at both the plasma membrane and in intracellular dots at the leading edge of migrating cells upon 10 and 30 minutes of KGF stimulation. After FGF10 stimulation the polarization appears delayed and evident only upon 30 minutes of treatment. The staining of the marker WGA appears uniformly distributed along the plasma membrane at all time points. Bars: 10 µm.
Figure 4.
Src activity is required for KGFR and cortactin colocalization.
A) HaCaT KGFR cells, treated or not with the growth factors, were incubated at 4°C with the anti-Bek polyclonal antibodies before cell fixation, as above, to selectively stain the plasma membrane KGFR, or with an anti-Bek monoclonal antibody after cell fixation and permeabilization, to visualize simultaneously the intracellular and plasma membrane KGFRs. Double immunofluorescence analysis, using anti-cortactin monoclonal antibody or anti-cortactin polyclonal antibodies, shows that in untreated cells the KGFR signal is localized along the entire surface of the cell plasma membrane, as well as in intracellular dots, and the cells do not show migratory features. The cortactin staining appears mainly localized on small intracellular dots dispersed throughout the cytoplasm, and only partially overlapping with KGFR positive dots. After treatment with either KGF or FGF10, cortactin and KGFR colocalization is significantly increased, evident not only in intracellular endocytic dots (arrows), but also at the level of the plasma membrane, where cortactin is translocated (arrowheads). Upon ligand treatment HaCaT KGFR cells located on the periphery of the colonies show a typical migratory phenotype, with a clearly defined leading edge, where the intracellular yellow dots stained for both cortactin and KGFR appear to be concentrated. Treatment with SU6656 reduces the colocalization between KGFR and cortactin, at a level comparable to that observed in untreated cells, and abolishes the migratory features. Images shown were obtained by 3D reconstruction of a selection of three out of the total number of the serial optical sections as reported in figure 2. Bars: 10 µm. B) Quantitative analysis of the percentage of colocalization of KFGR and cortactin was performed by serial optical sectioning and 3D reconstruction, as reported in Materials and Methods. Results are expressed as mean values +/- SE (standard errors): the percentage of colocalization was calculated analyzing a minimum of 50 cells for each treatment randomly taken from three independent experiments. Student's T test was performed and significance levels have been defined. *p<0,001 vs the corresponding untreated cells; **p<0,001 vs the corresponding untreated cells; ***p<0,001 vs the corresponding cells –SU6656; ****p<0,001 vs the corresponding cells –SU6656. C) Double immunofluorescence analysis, using anti-cortactin polyclonal antibodies and anti-early endosome antigene 1 (EEA1) monoclonal antibody, shows that the colocalization between cortactin and EEA1, already visible in untreated HaCaT cells, is increased upon KGF or FGF10 treatment and appears mostly relocalized in dots at the leading edge of migrating cells (arrows). The treatment with SU6656 significantly reduces the cortactin/EEA1 colocalization, as well as the migratory phenotype, upon either KGF or FGF10 stimulation. Images shown were obtained by 3D reconstruction of a selection of three out of the total number of the serial optical sections as reported in figure 2. Bars: 10 µm. D) Quantitative analysis of the percentage of colocalization of cortactin with EEA1 was performed by serial optical sectioning and 3D reconstruction as above. Results are expressed as mean values +/- SE; the percentage of colocalization was calculated analyzing a minimum of 50 cells for each treatment randomly taken from three independent experiments. Student's T test was performed and significance levels have been defined. *p<0,005 vs the corresponding untreated cells; **p<0,005 vs the corresponding untreated cells; ***p<0,001 vs the corresponding cells –SU6656; ****p<0,005 vs the corresponding cells –SU6656.
Figure 5.
Clathrin depletion affects KGFR polarization and cortatin recruitment to the endosomes.
A) HaCaT cells were coinjected with KGFR cDNA and CHC siRNA, to simultaneously obtain KGFR overexpression and CHC depletion, or with KGFR cDNA and an unrelated siRNA, as a control. After injection cells were serum starved, incubated at 4°C with the anti-Bek polyclonal antibodies, and treated with KGF or FGF10, as above. Upon KGF and FGF10 stimulation, in cells microinjected with CHC siRNA the receptor remains uniformly distributed on the plasma membrane and does not appear on intracellular dots, while the cortactin is dispersed throughout the cytoplasm and appears to be translocated just below the plasma membrane. In cells microinjected with the control siRNA, the KGFR is internalized and it colocalizes with cortactin in intracellular dots polarized at the leading edge of migrating cells. Bar: 10 µm. B) Quantitative analysis of percentage of HaCaT KGFR cells showing internalized KGFR was performed by counting 50 cells that overexpress KGFR for each condition, randomly taken from 10 microscopic fields in three different experiments, and values are expressed as the mean value ± standard errors (SE). C) HaCaT cells were coinjected with CHC siRNA and rabbit IgG to identify the microinjected cells. The immunofluorescence analysis was performed using anti-clathrin antibody: the punctate staining corresponding to clathrin-positive structures is reduced in microinjected cells compared to the surrounding uninjected cells. Bar: 10 µm.
Figure 6.
Src signaling is required for KGFR endocytosis.
A) HaCaT KGFR cells were incubated at 4°C with the anti-Bek polyclonal antibodies, to selectively stain the plasma membrane receptors, and then treated with KGF or FGF10 to induce receptor internalization from the plasma membrane. Double immunofluorescence analysis, using anti-cortactin monoclonal antibody, shows that in untreated cells the KGFR signal appears uniformly distributed on the cell surface, while the cortactin signal is evident in dots dispersed throughout the cytoplasm, which correspond to sorting endosomes. Virtually no colocalization is observed between the two proteins. After KGF or FGF10 stimulation, HaCaT KGFR cells show a migratory phenotype and the internalized KGFR appear in endocytic dots polarized at the leading edge of migrating cells, in which the receptor significantly colocalizes with cortactin (arrows). Treatment with SU6656 is able to block the ligand-induced KGFR internalization, and consequently its colocalization with cortactin in endocytic dots: the receptor staining is uniformly distributed on the plasma membrane, while the cortactin labeling remains dispersed throughout the cytosol, as observed in untreated cells. Images shown were obtained by 3D reconstruction of a selection of three out of the total number of the serial optical sections, as reported in figure 2. Bars: 10 µm. B) Quantitative analysis of percentage of HaCaT KGFR cells showing internalized KGFR was performed by counting 100 cells that overexpress KGFR for each condition, randomly taken from 10 microscopic fields in three different experiments, and values are expressed as the mean value ± standard errors (SE). C) Quantitative analysis of the percentage of colocalization of KGFR with cortactin was performed as described above. The percentage of colocalization was calculated analyzing a minimum of 50 cells for each treatment randomly taken from three independent experiments. Results are expressed as mean values +/- SE. Student's T test was performed and significance levels have been defined. Student's T test was performed and significance level has been defined as above. *p<0,005 vs the corresponding untreated cells; **p<0,005 vs the corresponding untreated cells; ***p<0,001 vs the corresponding cells –SU6656; ****p<0,005 vs the corresponding cells –SU6656.
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.
Figure 8.
KGFR expression and polarization are involved in cell motility.
A) HaCaT KGFR and HaCaT cells were seeded on coverslip and grown until confluence. A cell-free area was introduced in a monolayer of cells using a steril tip and then cells were immediately fixed (T0) or allowed to migrate from the edge of the scratch for 20 h at 37°C in the presence or not of KGF or FGF10. The cell-free area, evident in samples at time 0 (T0) from the scratch, is only partially repopulated in untreated cells. KGF stimulation induces a more intense cell migration if compared to FGF10 in HaCaT cells; KGFR overexpression induces a significant increase of cell migration upon both growth factors stimulation. Bar: 80 µm. Immunofluorescence analysis using anti-Bek antibodies in HaCaT KGFR cells shows some cells that clearly overexpresses KGFR. The receptor staining is uniformly distributed on the plasma membrane in unstimulated cells or in cells fixed at time 0 (T0) from the scratch, but is polarized at the leading edge of migrating cells that repopulate the scratch area upon KGF of FGF10 stimulation. Bar: 10 µm. B) Cells migration was quantified measuring the mean gap distance between the edges of the scratch area as reported in Materials and Methods. Student’s T test was performed and significance level has been defined as above. * NS vs the corresponding untransfected cells; **p< 0,0001 vs the corresponding untransfected cells; ***p<0,0001 vs the corresponding untransfected cells.