Figure 5.
Figure 5. Rac3 targets GIT1 to regulate MT1-MMP–mediated matrix degradation at invadopodia. (A) Images of control (top) or Rac3 siRNA (bottom)–treated MTLn3 cells plated on 405 nm fluorescent gelatin for 16 h. Cells were stained with anti–MT1-MMP antibody for 30 min before fixation. Quantification of the intensity of MT1-MMP at invadopodia in control or Rac3 siRNA–treated cells. n ≥ 20 in ≥8 cells; three independent experiments. (B) MTLn3 cells with control (top) or GIT1 siRNA (bottom) and plated on 405 nm fluorescent gelatin overnight. White arrows indicate invadopodia. (B, left) Normalized mean degradation area per field. n ≥ 10 fields for each condition; three independent experiments. (B, right) Mean number of invadopodia per cell. n ≥ 50 invadopodia from ≥25 cells for each condition; three independent experiments. (B, bottom) Western blots of cell lysates of control and GIT1 siRNA–treated MTLn3 cells blotted for GIT1 and β-actin with quantification. (C) Expression of GFP-GIT1 rescues the matrix degradation defect when GIT1 is depleted. n ≥ 36 invadopodia fields and ≥21 cells for each condition; three independent experiments. (D) Representative MTLn3 cells expressing EGFP-GIT1 and plated on 405 nm fluorescent gelatin for 16 h. (D, top) Ringlike localization of EGFP-GIT1. (D, bottom) Core localization of EGFP-GIT1 at invadopodia. (E) The percentage of invadopodia with EGFP-GIT1 in MTLn3 cells after starvation and EGF stimulation for indicated times. Significance is shown relative to the relevant bar at 0 min. n ≥ 50 from ≥15 cells per condition; three independent experiments. (F) Differential localization times of EGFP-GIT1 at the core or ring of invadopodia in MTLn3 cells. n = 7 invadopodia from ≥5 different cells pooled from three independent experiments. (G) The percentage of GIT1-positive (total, core, or ring localization) invadopodia in MTLn3 cells treated with control (black bars) or Rac3 siRNA (gray bars) plated on gelatin for 16 h. (H) The percentage of GIT1-positive (total, core, or ring localization) invadopodia in MTLn3 cells transfected with WT (black bars) or Rac3 T35S/Y40C effector binding mutant (gray bars) plated on gelatin for 16 h. (I) Normalized mean degradation area per field for cells overexpressing EGFP-GIT1 WT (black bars) or EGFP-GIT1-R39K (Arf-GAP deficient mutant). n ≥ 10 fields for each condition; three independent experiments. (J) Percentage of invadopodia with cytoplasmic (internal) MT1-MMP or surface (external) MT1-MMP in cells treated with the indicated siRNA. Significance is shown relative to the relevant control bar. *, P ≤ 0.05; **, P ≤ 0.01; ***, P ≤ 0.001. All error bars are SEM.

Rac3 targets GIT1 to regulate MT1-MMP–mediated matrix degradation at invadopodia. (A) Images of control (top) or Rac3 siRNA (bottom)–treated MTLn3 cells plated on 405 nm fluorescent gelatin for 16 h. Cells were stained with anti–MT1-MMP antibody for 30 min before fixation. Quantification of the intensity of MT1-MMP at invadopodia in control or Rac3 siRNA–treated cells. n ≥ 20 in ≥8 cells; three independent experiments. (B) MTLn3 cells with control (top) or GIT1 siRNA (bottom) and plated on 405 nm fluorescent gelatin overnight. White arrows indicate invadopodia. (B, left) Normalized mean degradation area per field. n ≥ 10 fields for each condition; three independent experiments. (B, right) Mean number of invadopodia per cell. n ≥ 50 invadopodia from ≥25 cells for each condition; three independent experiments. (B, bottom) Western blots of cell lysates of control and GIT1 siRNA–treated MTLn3 cells blotted for GIT1 and β-actin with quantification. (C) Expression of GFP-GIT1 rescues the matrix degradation defect when GIT1 is depleted. n ≥ 36 invadopodia fields and ≥21 cells for each condition; three independent experiments. (D) Representative MTLn3 cells expressing EGFP-GIT1 and plated on 405 nm fluorescent gelatin for 16 h. (D, top) Ringlike localization of EGFP-GIT1. (D, bottom) Core localization of EGFP-GIT1 at invadopodia. (E) The percentage of invadopodia with EGFP-GIT1 in MTLn3 cells after starvation and EGF stimulation for indicated times. Significance is shown relative to the relevant bar at 0 min. n ≥ 50 from ≥15 cells per condition; three independent experiments. (F) Differential localization times of EGFP-GIT1 at the core or ring of invadopodia in MTLn3 cells. n = 7 invadopodia from ≥5 different cells pooled from three independent experiments. (G) The percentage of GIT1-positive (total, core, or ring localization) invadopodia in MTLn3 cells treated with control (black bars) or Rac3 siRNA (gray bars) plated on gelatin for 16 h. (H) The percentage of GIT1-positive (total, core, or ring localization) invadopodia in MTLn3 cells transfected with WT (black bars) or Rac3 T35S/Y40C effector binding mutant (gray bars) plated on gelatin for 16 h. (I) Normalized mean degradation area per field for cells overexpressing EGFP-GIT1 WT (black bars) or EGFP-GIT1-R39K (Arf-GAP deficient mutant). n ≥ 10 fields for each condition; three independent experiments. (J) Percentage of invadopodia with cytoplasmic (internal) MT1-MMP or surface (external) MT1-MMP in cells treated with the indicated siRNA. Significance is shown relative to the relevant control bar. *, P ≤ 0.05; **, P ≤ 0.01; ***, P ≤ 0.001. All error bars are SEM.

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