In this paper, we have identified and characterized a novel plasma membrane–associated, F-actin–binding protein (supervillin/p205), which is present in bovine neutrophils and in various transformed and nontransformed cell lines. We have shown previously that this protein fractionates with crude neutrophil membranes and binds directly and specifically to the sides of α-actin filaments in blot overlay assays (66
). We show here that supervillin is a tightly bound peripheral protein (Fig. ) that can associate with the plasma membranes of both neutrophils (Fig. ) and MDBK cells (Figs. –). We further show that supervillin can be isolated from neutrophil plasma membranes as part of a high molecular weight complex with endogenous actin (Fig. ) and that the interaction with actin persists after immunoprecipitation and high stringency washing (Fig. ). Although many tissue culture cell lines contain both supervillin and another F-actin–binding protein of similar size (Fig. B
), all of our results to date suggest that supervillin is the only 205-kD F-actin–binding protein in bovine neutrophils. The presence of supervillin in numerous cell lines (Fig. ) suggests that this protein is one of a small, but growing group of membrane skeleton proteins known to bind actin at the peripheries of many cells (73
We also show here that the structure of supervillin is novel (Figs. , , and ). Not only is this protein currently unrepresented in the protein databases, but it is unique in that it contains both a strong homology to cytosolic actin-binding proteins in the villin/gelsolin family (Fig. ) and four nuclear localization signals (Fig. ). The demonstrated tight binding of supervillin to actin filaments (Figs. and ) is reflected by the prediction from the primary sequence that this protein may contain as many as three binding sites for F-actin (Fig. B
). Based upon the extent of the sequence similarities and the known properties of the homologous sequences in villin and gelsolin, supervillin may also bundle actin filaments. Interestingly, amino acids required for filament severing in gelsolin (Lys-150 through Gln-160; see reference 41
) and villin (Arg-137; see reference 23
) are not conserved in supervillin (Fig. B
), suggesting that, like protovillin (43
), supervillin probably lacks this activity. At least one of the F-actin–binding sites in supervillin is insensitive to the presence of free calcium ions since F-actin binding on blot overlays is similar in the presence of either 1 mM EGTA or 0.1 mM CaCl2
(data not shown). However, the definitive determination of the nature of the interaction(s) between supervillin, actin filaments, and calcium ions awaits the identification of a source from which biochemically significant amounts of native supervillin are readily obtainable.
The colocalization of supervillin with E-cadherin at sites of initial (Fig. ) and established (Fig. ) cell–cell contact and its internalization with E-cadherin and actin during EGTA-mediated cell dissociation (Fig. ) suggest that supervillin may be involved in the formation and/or stabilization of actin filament bundles at adherens junctions. Such an activity would be analogous to that documented for villin in the microvilli of highly organized brush borders. Villin both nucleates microvillar assembly in transfected cells and cross-links actin filaments in the mature microvillar core (34
). A second precedent may be the actin bundling protein, p30a, which stabilizes filaments against depolymerization (89
) and apparently potentiates their association with intercellular junctional membranes, even though p30a does not itself bind tightly to the membrane (30
). An even more intriguing paradigm may be α-actinin, an actin-bundling protein that also appears to facilitate the attachment of stress fibers to integrins at focal contacts (64
). We hypothesize that supervillin plays an analogous role in actin filament bundling and/or attachment to the membrane at adherens junctions.
Because the COOH terminus of supervillin apparently constitutes the actin-binding domain of the molecule, an attractive hypothetical function for the NH2
terminus is the targeting of supervillin to appropriate intracellular compartment(s). The localization of supervillin at regions of lateral cell–cell contact (Figs. , ) is quite distinct from the observed concentration of villin in apical microvilli (11
). Also, no supervillin is observed in association with the actin filament meshwork at the basal surfaces of MDBK cells. Thus, either the unique supervillin NH2
terminus or one of the supervillin-specific, “linker” sequences interspersed between the villin/gelsolin homology regions must contain a sequence responsible for targeting to some component of adherens junctions. The punctate cytoplasmic distribution observed in nonadherent cells implies that this target may be membrane associated.
Another intracellular destination for supervillin might be the nucleus. The nuclear localization predicted from the presence of NH2
-terminal targeting signals is supported by the observation that nuclei of low density cells label with an antibody against a supervillin peptide (Fig. ). Although nuclear localization artifacts are common in fixed cells (57
), no significant nuclear staining is observed in EGTA-treated cells (Fig. ), suggesting that the localization observed in Fig. is not a consequence of our fixation conditions or a fortuitous cross-reaction with a similar epitope in a nuclear protein.
A role for supervillin outside the adherens junction is also suggested by its comparatively high abundance in bovine neutrophils (Figs. and A
) and HeLa cells (Fig. B
). These cells are not adherent and either lack (neutrophils) or are grossly deficient (HeLa cells) in classical cadherins detectable by antibodies against the highly conserved cadherin cytoplasmic domain (data not shown). While these cell types might contain a divergent cadherin with an immunologically distinct cytoplasmic domain (76
), it is also possible that supervillin plays different roles in nonadherent and adherent cells. Such a multiplicity of functions is supported by the changing intracellular localization of supervillin as a function of the growth and adhesive state of MDBK cells (Figs. and ).
Assuming that subsequent analyses with additional antibodies and with epitope-tagged supervillin confirm its multiple intracellular localizations, this protein is an excellent candidate for a signaling molecule that transduces information to the nucleus from the membrane skeleton at sites of cell–cell adhesion. Precedents include β-catenin and the Drosophila melanogaster
armadillo protein, both of which bind cadherin, potentiate cell–cell adhesion, and function in the Wnt-1/Wingless signal transduction pathway (39
). When present at high levels, both β-catenin (6
) and armadillo protein (81
) can functionally interact with the LEF-1/Tcf family of transcription factors, an interaction that provides one explanation for the apparent involvement of β-catenin in tumor progression (50
). Thus, supervillin may be one of a small group of candidate proteins, which also includes the focal contact proteins zyxin and cCRP (70
), that could function as relatively direct signaling molecules between the nucleus and the actin cytoskeleton at sites of cell adhesion.
In conclusion, we have shown that supervillin is a novel F-actin–binding protein that cofractionates with endogenous actin, binds peripherally but tightly to neutrophil plasma membranes, and conditionally localizes with E-cadherin at sites of intercellular adhesion. Future work will be directed towards elucidating supervillin function and regulation in adherent vs. nonadherent cells and determining the role of this protein in the modulation of adhesion, motility, and adhesion-mediated signal transduction.