lysis of b2adaptin-depleted cells demonstrated an increase in Src activity when compared to control cells, as measured by blotting with antiphospho Src Y418. An 
increase in FAK Y397 phosphorylation was also observed. To determine if the increase in Src activity was required for b2-adaptin regulated matrix degradation, b2-adaptin knockdown cells were plated on fluorescent 488-gelatin in the presence of vehicle or the Src inhibitor, PP2. PP2 treatment abolished the ability of b2-adaptin knockdown cells to degrade matrix. These data indicate that b2-adaptin may be involved in negatively regulating Src activity in non-transformed cells, as depletion of b2adaptin resulted in increased Src activity. This in turn promotes invadopodia formation and matrix degradation in normal MCF10A, demonstrating a key role for b2-adaptin regulation of B2-Adaptin Regulates Cell Spreading and Migration Src activity in maintaining a non-invasive phenotype in epithelial cells. Global clathrin-mediated endocytosis can be inhibited with monodansylcadaverine as shown using a TRITC-transferrin uptake assay. Interestingly, inhibition of global clathrin-mediated endocytosis failed to induce matrix degradation in either the MCF10A or U2OS cells, suggesting that the increased matrix degradation we observed following b2adaptin knockdown is specific to perturbation of AP-2-dependent processes. In addition, these data suggest that the increase in Src activity and matrix degradation is not an indirect consequence of sustained growth factor receptor signaling. Discussion In this study, the endocytic adapter protein b2-adaptin was shown to bind directly to the focal adhesion protein actopaxin. Furthermore, GST-R-7128 Actopaxin pull downs also contained a-, but not c-adaptin, confirming a selective interaction with the AP-2 complex. Beta2-adaptin also localized to focal adhesions during cell spreading and migration in an actopaxin-dependent fashion. In contrast, localization of actopaxin to coated pits was not observed, suggesting that actopaxin may be involved in mediating AP-2 specific endocytosis at sites of adhesion via its interaction with b2-adaptin. The interaction of b2-adaptin with actopaxin occurred within the N-terminal 195 region, that lacks the paxillin or ILK-binding site and when over expressed, this region of actopaxin does not localize to focal adhesions. This suggests that b2-adaptin localization to adhesions occurs indirectly via actopaxin binding to paxillin or ILK. Actopaxin is also an actin binding protein, but the functional significance of this interaction has not been evaluated. As actin plays a key role in the process of endocytosis, with a number of actin binding proteins such as Arp2/3, NWASP, cortactin and mammalian Abp1p being recruited to coated pits in mammalian cells, it is possible that actopaxin B2-Adaptin Regulates Cell Spreading and Migration is playing a specific role in regulating focal adhesion associated endocytosis via the ability to link the clathrin endocytic machinery with the actin cytoskeleton. Alternatively, actopaxin may be binding b2-adaptin, and therefore the AP-2 complex, in order to ensure that selective endocytosis of integrins and associated proteins occurs specifically at focal adhesions. A number of integrin subunits contain the internalization signal NPXY motif that is associated with clathrinmediated endocytosis and clathrin containing structures have been shown to facilitate microtubule-dependent focal adhesion disassembly a