The interaction of cadherins with cytoplasmic proteins and the actin cytoskeleton is thought to underlie many aspects of cell-cell adhesion, including clustering of cadherins, strengthening of adhesive contacts, and downstream effects on membrane and cell organization (Kobielak and Fuchs, 2004
). To understand these processes, it is essential that protein assemblies that link cadherins to the actin cytoskeleton are rigorously defined. Virtually all of the candidate components of these assemblies (see Introduction) were identified through binary interactions such as yeast two-hybrid, pull-down, or coimmunoprecipitation assays. A general assumption has been that binding of a given protein to two distinct partners means that all three proteins are in the same complex. In particular, independent binding of α-catenin to β-catenin-E-cadherin and to actin filaments has led to the assumption that α-catenin binds to both simultaneously although this quaternary complex has not been demonstrated previously. A direct test of this widely believed conclusion presented here shows, in fact, that this is not the case.
Using purified proteins in solution or membrane patches containing clustered E-cadherin, we reconstituted, in the correct order of protein-protein interactions, a ternary complex of E-cadherin-β-catenin-α-catenin and the interaction between α-catenin and actin filaments in vitro. However, we were unable to bind α-catenin simultaneously to the E-cadherin-β-catenin complex and to actin filaments, even when E-cadherin was clustered in vitro (COMP-Ecyto) or on membrane patches from cells. Similarly, we could reconstitute vinculin binding to β-catenin or α-catenin but not to the cadherin-β-catenin complex and actin filament simultaneously. α-actinin could not be reconstituted into complexes with either β-catenin or α-catenin.
An additional activity might be needed to enable simultaneous binding of α-catenin or vinculin to E-cadherin-β-catenin complex and actin filaments or to relieve the head-to-tail autoinhibition of vinculin bound to β-catenin. Neither lipids in the membrane patches nor MDCK or bovine brain cytosol provided such an activity, although we cannot exclude that such a factor was missing or inactivated in our cytosol preparations. We also tested whether specific post-translational modifications shown previously to enhance complex assembly were involved (Lilien et al., 2002
). Serine/ threonine phosphorylation by CKII had no effect on α- or β-catenin interaction or on binding of actin filaments to reconstituted cadherin-catenin complexes on membrane patches. The small GTPase Rac1 and PI3K have been suggested to be transiently activated during initial phases of cell-cell contact formation and cadherin ligation (Ehrlich et al., 2002
; Kovacs et al., 2002
; Noren et al., 2001
). However, our preparations of membrane patches would not be able to capture these transient activation states that might be important for actin-filament interactions with the cadherin-catenin complex. Alternatively, linkage of the E-cadherin-β-catenin complex to actin filaments could be mediated by other α-catenin and actin binding proteins, including ZO-1 (Itoh et al., 1997
), afadin (Pokutta et al., 2002
), spectrin (Pradhan et al., 2001
), Ajuba (Marie et al., 2003
), or formin-1 (Kobielak et al., 2004
). However, there is no direct evidence that these proteins can bind simultaneously to α-catenin and actin filaments. In this context, it is noteworthy that, when membrane extracts of adherent cells are immunoprecipitated with anti-cadherin antibody, only β- and α-catenin are coprecipitated stoichiometrically in the complex (Hinck et al., 1994
; Ozawa and Kemler, 1992
). Thus, although a number of actin binding proteins are reported to colocalize with cadherins and/or interact with α-catenin, it is unclear whether any of them represents a significant structural component of the cadherin-catenin complex in cells.
If the core cadherin-catenin complex does not bind to actin filaments directly, we would expect that interactions between this complex and underlying the actin cytoskeleton in cells might be very dynamic rather than being relatively static as has been assumed. E-cadherin, β-catenin, and α-catenin had essentially identical mobile fractions and recovery rates, consistent with an integrated complex of these proteins on the membrane. In contrast, actin, vinculin, and the Arp2/3 complex were highly mobile and had recovery rates completely different from those of the cadherin-catenin complex. These data are also inconsistent with a static linkage of the cadherin-catenin complex, either directly or indirectly, to the actin cytoskeleton and support our biochemical studies that the cadherin-catenin complex does not bind to actin filaments.
That a stable linkage does not exist between membrane-anchored cadherin cell-adhesion molecules and the underlying cytoskeleton may be surprising. However, adhesion must be a dynamic process to enable morphogenetic changes during cell and tissue development (Takeichi, 1995
). The interaction of clustered cadherin extracellular domains on opposing cells may provide the necessary adhesive force as long as the underlying actin cytoskeleton is correctly organized to provide the mechanical properties required for cell and tissue function. Data presented in the accompanying paper (Drees et al., 2005
) provide mechanistic evidence of why α-catenin does not bind simultaneously to the E-cadherin-β-catenin complex and actin filaments and new insights into how α-catenin may regulate actin dynamics at cell-cell contacts.