MT1-MMP actin binding is both necessary and sufficient to induce matrix degradation. (A–C) 25 µM final concentration of WT (A and C) cytoplasmic MT1-MMP tail peptide or LLY/AAA (B and C) cytoplasmic MT1-MMP tail peptide were tested for their ability to bind to increasing amounts of F-actin in an in vitro binding assay. Actin was allowed to polymerize for 1 h. After centrifugation, 10% of total F-actin pellets (p) and supernatants (s) were run on a 4–12% NuPAGE gel using MES running buffer. (D) Quantification of Coomassie gel densitometry of peptide bands. n = 5. (E) Relative steady-state fluorescence anisotropy was measured between IAEDANS-labeled filamentous actin (2 µM) and increasing concentration of MT1 peptide constructs: MT1_tail (closed squares), MT1tail_L1Y/A (open circles), and MT1tail_scrambled (open triangles). The binding curve was fitted with the equation given in the Materials and methods section (“Steady-state fluorescence anisotropy experiments”), and the Kd value was calculated (see Results). (F) MDA-MB-231 cells were transfected with MT1-MMP-GFP alone or together with Lifeact-TagRFP and invaded in Matrigel in CIA. Cells were imaged using a spinning disc FLIM system. Fluorescence lifetime was measured in the pseudopod area and quantified from 30 cells in n = 3. Bar, 10 µm. (G) Immunofluorescence images of mCherry, mCherry-MT1-MMP, mCherry-MT1-MMPΔCT, and mCherry-MT1-MMPEZ-ABD expressing MDA-MB-231 MT-MMP knockdown cells (red) on Alexa Fluor 488–conjugated gelatin (green). The percentage of cells showing matrix degradation was quantified after 3 h incubation. At least 30 cells of each expressing construct were imaged for quantification from three independent experiments. Bar, 10 µm.
