Figure 5
Figure 5. Podosome rosette–associated ECM degradation is a protease-dependent process altered in Hck−/− BMDMs. (A) Podosome rosettes exhibit ECM degradation activity. BMDMs were seeded on FITC-coupled gelatin-coated coverslips. After 24 hours, cells were fixed and stained for F-actin and microscopically examined. Large degradation area is formed underneath large podosome rosettes (i). In contrast, small degradation area is observed underneath small podosome rosettes (ii). Measurements of fluorescent intensities along the white dashed line depicted in subpanels i and ii in the merged image shows increase of intensities of F-actin (red line) correlating with loss of fluorescent intensity of the gelatin (green line). (B) Podosome rosette is a site for LysoTracker accumulation. (C) MT1-MMP and MMP2 localize at sites of gelatin-FITC degradation. BMDMs were subjected to cell surface labeling with MT1-MMP and MMP2 antibodies. (D-E) BMDMs were seeded on FITC-coupled gelatin-coated coverslips and incubated overnight in the presence or absence of a protease inhibitor cocktail, then fixed and stained for F-actin and nuclei and microscopically examined for quantification. (D top 2 panels) wt BMDMs form large podosome rosettes associated with large areas of gelatin-FITC degradation (arrowheads). Hck−/− BMDMs form small podosome rosettes and degrade small gelatin areas (arrowheads). (Bottom 2 panels) The same experiments performed in the presence of protease inhibitors (PI), which significantly block matrix degradation (scale bar represents 10 μm; magnification ×40). (E) Quantification of FITC-coupled gelatin degradation by BMDMs in the presence and in the absence of protease inhibitors. The percentage of degradation corresponds to the number of pixels of degradation for 100 pixels of cell surface. Results are expressed as mean ± SEM (n = 8) of FITC-gelatin degradation areas. (F) wt and Hck−/− BMDMs express similar levels of MMP2, MMP9, and MT1-MMP. Immunoblot analysis performed on BMDM cell lysates for MMP9 (92 kDa), MMP2 (72 kDa), MT1-MMP (64 kDa), and actin (46 kDa). (G) Hck−/− BMDMs are not defective in MMP9 and MMP2 activity and release. Gelatin zymograph of BMDM cell lysates or conditioned media collected from cells adhering on fibronectin.

Podosome rosette–associated ECM degradation is a protease-dependent process altered in Hck−/− BMDMs. (A) Podosome rosettes exhibit ECM degradation activity. BMDMs were seeded on FITC-coupled gelatin-coated coverslips. After 24 hours, cells were fixed and stained for F-actin and microscopically examined. Large degradation area is formed underneath large podosome rosettes (i). In contrast, small degradation area is observed underneath small podosome rosettes (ii). Measurements of fluorescent intensities along the white dashed line depicted in subpanels i and ii in the merged image shows increase of intensities of F-actin (red line) correlating with loss of fluorescent intensity of the gelatin (green line). (B) Podosome rosette is a site for LysoTracker accumulation. (C) MT1-MMP and MMP2 localize at sites of gelatin-FITC degradation. BMDMs were subjected to cell surface labeling with MT1-MMP and MMP2 antibodies. (D-E) BMDMs were seeded on FITC-coupled gelatin-coated coverslips and incubated overnight in the presence or absence of a protease inhibitor cocktail, then fixed and stained for F-actin and nuclei and microscopically examined for quantification. (D top 2 panels) wt BMDMs form large podosome rosettes associated with large areas of gelatin-FITC degradation (arrowheads). Hck−/− BMDMs form small podosome rosettes and degrade small gelatin areas (arrowheads). (Bottom 2 panels) The same experiments performed in the presence of protease inhibitors (PI), which significantly block matrix degradation (scale bar represents 10 μm; magnification ×40). (E) Quantification of FITC-coupled gelatin degradation by BMDMs in the presence and in the absence of protease inhibitors. The percentage of degradation corresponds to the number of pixels of degradation for 100 pixels of cell surface. Results are expressed as mean ± SEM (n = 8) of FITC-gelatin degradation areas. (F) wt and Hck−/− BMDMs express similar levels of MMP2, MMP9, and MT1-MMP. Immunoblot analysis performed on BMDM cell lysates for MMP9 (92 kDa), MMP2 (72 kDa), MT1-MMP (64 kDa), and actin (46 kDa). (G) Hck−/− BMDMs are not defective in MMP9 and MMP2 activity and release. Gelatin zymograph of BMDM cell lysates or conditioned media collected from cells adhering on fibronectin.

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