and the dynamic turnover of FAs is essential for the motility of both normal and tumor cells (Mitra et al., 2005; Gimona and Buccione, 2006). Despite distinct morphologies, FAs and invadopodia share many components including integrins, talin, paxillin, actin, cortactin, and dynamin, to name just a few (Linder and Aepfelbacher, 2003). A central component of FAs that provides both a scaffolding and signaling function is focal adhesion kinase (FAK). Although not observed at invadopodia (Bowden et al., 2006), FAK has been implicated in the regu- lation of invadopodia formation by modulating downstream signaling through Src and p130Cas (Hsia et al., 2003; Chan et al., 2009). Despite these molecular similarities, FAs are gen- erally perceived to mediate cell migration but are not involved in matrix degradation. Recently we have observed that a variety of cancer cell lines degrade matrix not only at invadopodia, but also at nu- merous peripheral sites that resemble FAs. Here we have confirmed that this peripheral ECM degradation represents bona fide FA sites, and have further revealed that this FA-type degradation is MT1-MMP dependent. Moreover, we find that this protease is targeted to FAs by a physical interaction between MT1-MMP and a FAK–p130Cas complex in response to the activation of Src. Formation of this FAK–p130Cas–MT1 complex is essential for the FA-centric ECM degradation as well as invasive migration of tumor cells through Matrigel. Results MT1-MMP mediates matrix degradation at focal adhesions Matrix degradation by a wide variety of different tumor cell lines appears to occur at linear, peripherally located FA-like sites as well as the well-characterized, centrally situated and dot- shaped invadopodia. To determine if this pattern represents the action of FAs, human fibrosarcoma cells (HT-1080) were plated on fluorescent gelatin-coated coverslips for 6 h, and stained for the FA marker vinculin. As shown in Fig. 1 a, many of the peripheral, linear degradation sites colocalized with vinculin. The enlarged images reveal that mature FAs (Fig. 1 a’) gen- erated a solid, dark pattern of degradation whereas nascent FAs (Fig. 1 a’’) appeared to be in the process of degrading matrix. A similar result was obtained from several cell lines (Fig. S1). As many actin stress fibers terminate in FAs, we colo- calized actin with a FA marker, and sites of matrix degradation as just one feature of many used to distinguish FAs from other adhesive structures (Fig. S1, c and d). It is well known that cells exert physical tension on the ECM at FA sites that can result in matrix tearing. To distinguish enzymatic activity from physical effects, HT-1080 cells were treated with 10 µM of the MMP inhibitor BB94 (Davies et al., 1993) as the cells were plated on gelatin. As shown in Fig. S2 (a–a’), overall degradation includ- ing the ones at FAs were completely inhibited, while no sig- nificant change in FA morphology was observed. From these observations we conclude that ECM degradation at FAs occurs by the proteolytic activity of MMPs. Because the FA-like degradation was sharply focused rather than diffuse in nature, we hypothesized that a membrane-type 376 JCB • VOLUME 196 • NUMBER 3 • 2012 Figure 1. MT1-MMP mediates ECM degradation at focal adhesions. (a) To test if extracellular matrix degradation occurs at focal adhesions, HT-1080 cells were plated on fluorescent gelatin (green)-coated coverslips for 6 h before fixation and staining with a vinculin antibody (red). Matrix degrada- tion occurred at vinculin-positive FA sites. Boxed regions provide a higher magnification showing the correlation between vinculin and matrix degra- dation in the gelatin matrix (dark spots in a’ and a’’). (b–c’) To define if MT1-MMP is required for the FA-centered degradation, HT-1080 cells were treated with MT1 siRNA for 72 h to knock down endogenous MT1-MMP (b), and then were plated on gelatin-coated coverslips (c and c’). MT1 knockdown cells (dashed lines) exhibit a significant reduction in matrix degradation. (d) Quantification of the percentage of cells (in c) that degraded ECM at FAs indicates that MT1 is required for this type of degra- dation. Values represent the mean of three independent experiments ± SD. (e and f) In reciprocal experiments MT1-MMP was overexpressed in a human pancreatic tumor cell line (PANC-1) that does not express endogenous MTI-MMP (e) or degrade matrix (Fig. S2). Just 2 h after plating on gelatin the transfected PANC-1 cells exhibited substantial matrix degradation at FA sites (f–f’’). Additional examples of pancreatic tumor cells degrading matrix at FAs are provided in Fig. S1. Bars, 10 µm. MMP, not a secreted form, was responsible for degradation at FAs. MT1-MMP is the most prominent member of MT-MMPs and has been shown by many others to support ECM degradation (Sato et al., 2005; Itoh, 2006; Strongin, 2006). As shown in Fig. 1, c–c’, knocking down MT1 in HT-1080 cells (dashed line) significantly impaired ECM degradation, including the