invadopodia (Fig. 6), suggesting a FA-specific mechanism. Finally, the formation of this trimeric complex is regulated by Src, and is essential for tumor cell invasion in vitro. FAK as an essential adaptor for the recruitment of MT1-MMP to focal adhesions Two of the interesting central concepts addressed in this study focus on the mechanics of MT1-MMP recruitment to FAs and the functional equilibrium of matrix degradation occurring at FAs and invadopodia during cancer invasion. Although degradation predominates at invadopodia in most tumor cells, FA-based degradation does occur and is not from mechanical tearing of the ECM, as inhibiting MMP activity with the drug BB94 abolished FA-centric degradation. This is further sup- ported by the fact that MT1-MMP–deficient cells (PANC-1) or HT-1080 cells with MT1 knockdown failed to degrade ECM at FAs. These data suggest that matrix degradation at FAs, like that at invadopodia, is a proteolytic event, and the essential role of a membrane-type MMP (MT1-MMP) is consistent with the confined degradation pattern at FAs. Key evidence supporting MT1-MMP’s function at FAs is an interaction with FAK. Although FAs and invadopodia share a substantial number of components, FAK is generally believed to be FA specific. Accordingly, we hypothesized that MT1-MMP may be selectively recruited to FAs by FAK. Further analysis revealed that FAK–MT1 interaction is mediated by the PRR domain of FAK and the cytoplasmic tail of MT1-MMP (Figs. 2 and 3). Functionally, FAK knockdown in HT-1080 cells resulted in a reduction of FA-mediated degradation, and consistent with a previous report (Chan et al., 2009) invadopodia-type degradation was not affected (Fig. 6 b), again supporting FAK’s distinct roles between these two structures. However, this defect could not be rescued by reexpression of WT FAK. We believe this is due to the fact that FAK overexpression promotes FA turnover (Ezratty et al., 2005; Mitra et al., 2005) and thus reduces the time allotted for matrix degradation to occur. This concept is supported by a subsequent finding that FAK Y397F, a mutant known to inhibit FA turnover (Hamadi et al., 2005), exhibited a significant rescue of FA-based degradation (Fig. 2), and importantly, no rescue was observed by FAK Y397F carrying the two PXXP mutations (P2/3A) that reduce MT1 binding. Moreover, cells expressing a MT1-MMP mutant that cannot interact with FAK (MT1CP) were defective in degrading ECM at FAs (Fig. 3). These data strongly suggest that the FAK–MT1 interaction is important for FA-based degradation, but is dispensable for degradation by invadopodia. As a direct interaction between FAK and MT1 was not detected, we had searched for an adaptor protein that could act as a functional linker. Based on previous reports (Harte et al., 1996; Gingras et al., 2008) we pursued, and sub- sequently found, that the FAK–MT1 connection is largely medi- ated by p130Cas (Fig. 4). A FAK–p130Cas–MT1-MMP complex regulates FA-based tumor cell invasion An interesting component of this study is the Src-dependent regulation of protease–adaptor interactions. Consistent with the findings of others (Gingras et al., 2008; Gonzalo et al., 2010), we observed that phosphorylation of a specific tyrosine (Tyr 573) on the cytoplasmic tail of MT1 by Src facilitates interactions with p130Cas (Fig. 5) and FAK (Fig. S4). It is well known that Src signaling promotes ECM degradation (Yeatman, 2004; Courtneidge et al., 2005; Kelley et al., 2010), yet it appears that Src regulates FAs and invadopodia through distinct mechanisms. For example, Src-mediated phosphory- lation of cortactin, AMAP1, paxillin, and p190 RhoGAP (Stylli et al., 2008) are essential for invadopodia-mediated degrada- tion, presumably by regulating the assembly of this organelle. In contrast, Src phosphorylation of MT1-MMP showed no obvious effects on FA structure but did enhance MT1-MMP’s recruitment to FAs (Fig. 5, I and j). This is both significant and consistent with our biochemical observations that Src facilitates MT1 interactions with FAK–p130Cas (Fig. 5 and Fig. S4). Moreover, FAK or p130Cas knockdown (Figs. 2 and 4) significantly compromised degradation at FAs, but not at invadopodia (Fig. 6, b and c). From these contrasting findings we believe that matrix degradations at these two structures are related yet distinct. Despite the fact that FAs and invadopodia share many structural components, the relative contributions of these two organelles to cell migration and invasion remain unclear. One interesting study (Oikawa et al., 2008) has suggested that FAs could be transformed into podosomes, an invadopodia homo- logue, in a Src-dependent manner. As many signaling mole- cules, including Src, are elevated or activated in cancer cells, it is attractive to predict that FAs could acquire some invadopodia- like properties during cancer progression, while largely main- taining its FA morphology and function. The Boyden chamber–based invasion assay (Fig. 7) suggests that FA-centric degradation is relevant for cancer invasion. As demonstrated in Figs. 2–4, disruption of the FAK–p130Cas–MT1 complex significantly impaired ECM degradation at FAs, as well as invasion through Matrigel (Fig. 7). Admittedly, due to the mul- tifunctional nature of these proteins, we cannot rule out the possibility that the mutant FAK, p130Cas, and MT1-MMP pro- teins affect invasion through other mechanisms. Finally, it is attractive to speculate that FA-based degra- dation may play a central role during cancer invasion in vivo, in which cells acquire a compact spindle-like morphology (Cukierman et al., 2001, 2002) while FAs become smaller and predominate at the pseudopodia–ECM contact at the leading edge (Friedl and Wolf, 2009; Wolf and Friedl, 2009; Kubow and Horwitz, 2011). From this organization it is likely that in situ FAs act to macerate, degrade, and reorganize matrix at the front edge and allow the cell body to migrate through. Although the biology of FAs in situ remains elusive and a topic of debate (Fraley et al., 2010; Kubow and Horwitz, 2011), it has been reported that the leading edge localization of FAK–p130Cas is important for invasion (Hsia et al., 2003), and proteolytic activity has been observed at the pseudopodia– ECM contacts (Wolf and Friedl, 2009). Moreover, specific targeting of an enzymatic defective chimeric MT1 to FAs sig- nificantly hinders cancer invasion (Takino et al., 2007). All of these related findings suggest that FA-mediated ECM degradation Selective recruitment of MT1-MMP to focal adhesions • Wang and McNiven 383