α-Catenin-mediated cadherin clustering couples cadherin and actin dynamics

Abstract

The function of the actin-binding domain of α-catenin, αABD, including its possible role in the direct anchorage of the cadherin-catenin complex to the actin cytoskeleton, has remained uncertain. We identified two point mutations on the αABD surface that interfere with αABD binding to actin and used them to probe the role of α-catenin-actin interactions in adherens junctions. We found that the junctions directly bound to actin via αABD were more dynamic than the junctions bound to actin indirectly through vinculin and that recombinant αABD interacted with cortical actin but not with actin bundles. This interaction resulted in the formation of numerous short-lived cortex-bound αABD clusters. Our data suggest that αABD clustering drives the continuous assembly of transient, actin-associated cadherin-catenin clusters whose disassembly is maintained by actin depolymerization. It appears then that such actin-dependent αABD clustering is a unique molecular mechanism mediating both integrity and reassembly of the cell-cell adhesive interface formed through weak cis- and trans-intercadherin interactions.

Figure 1.

Characterization of the actin-uncoupled αABD mutants. (a) Structure of αABD in the context of α-catenin and in isolation in both open and closed conformations (PDB ID 4IGG, chain A and B, respectively). αABD is colored in orange, whereas the other domains of α-catenin (αH, M1, M2, and M3) are in gray. The residues that decrease αABD–actin binding in vitro upon mutation to alanine (Fig. 1 c) are shown as spheres and colored according to conservation score estimated via the ConSurf algorithm (see legend). The conserved W859 is in stick representation. The structurally resolved parts of the C-terminal extension of αABD are colored in blue. I792A is exposed on the surface of αABD in isolation but is buried in the interface between αABD and M1 of full-length α-catenin (pink stars). (b) SDS-PAGE showing the results of actin cosedimentation assays with GST-α(671–883) and its deletion mutant GST-α(671–864), each at 3 µM. Pellet (P) and supernatant (S) fractions are shown. Note that the GST-α(671–883) mutant (one asterisk) cosedimented with the actin filaments, whereas its deletion mutant, GST-α(671–864), marked by two asterisks, remains in the supernatant. Bar shows position of ovalbumin (45 kD). (c) In vitro actin binding assays of αABD mutants. The K842-K883 region of αABD (top line) was divided into triplets, and binding of each triple alanine mutant (blue bars) was plotted as the quantity of mutant protein in the pellet relative to total protein (pellet + supernatant). Based on these data, several point mutations were selected (black bars). For comparison, the binding of control proteins, such as GST, GST-α(671–883), and GST-α(671–864), as well as the point mutant GST-α(671–883)-I792A, is also shown. (d) Cosedimentation assays with GST-α(671–883) and its K866A point mutant at constant F-actin concentration (1 µM) while varying the amount of GST-tagged proteins (ligand [Lig]) from 1.5 to 24 µM. (e) Actin binding curves of GST-α(671–883), GST-α(671–864), GST-α(671–883)-K866A, GST-α(671–883)-K842A, and GST-α(671–883)-I792A. Binding affinities were approximated only for two recombinant proteins that showed evidence of saturation at higher ligand concentrations: K_d(GST-α(671–883)) = 1 µM and K_d(GST-α(671–883)-K842A) = ∼30 µM.

Figure 2.

Direct αABD binding to actin drives junction formation. (a–c) Immunofluorescence microscopy of cadherin-deficient A431D cells expressing the following chimera molecules: (a) EcΔ-Dn-α(280–906); (b) αABD point mutants (mt) of the EcΔ-Dn-α(280–906) chimera (K842A, K866A, or I792A); and (c) vinculin-uncoupled EcΔ-Dn-α(280–906)-ΔVin mutant. Schematic representation of the chimeras is given atop of the microscopy images. Each chimera includes extracellular, transmembrane, and a 17-aa-long cytoplasmic region of E-cadherin lacking all known cytoplasmic protein binding sites (EcΔ). Dn denotes the fluorescent protein Dendra2. α-(280–906) denotes a region of α-catenin, which includes the M1–M3 domains and the C-terminal actin binding domain (αABD). The mutated domains are in yellow. The dash line boxed regions are magnified on the right or at the bottom. The cells were stained for Dendra2 to reveal chimera (Dn) as well as costained with actin (Dn+Act), vinculin (Dn+Vin), or actin and vinculin together. Expression of EcΔ-Dn-α(280–906)-ΔVin mutant results in formation of actin-enriched junctions devoid of vinculin though actin structures, which are colocalized with chimera are no longer organized into bundles. Numbers above the scale bars indicate micrometers.

Figure 3.

Polymorphism of AJs in α-catenin–expressing 468 cells. (a) Immunofluorescence staining of the parental α-catenin–deficient 468 cells for E-cadherin (Ec) and actin filaments (Act). The boxed regions are magnified on the right. Note that E-cadherin molecules can assemble only into tiny clusters. (b) Dn-αCat–expressing 468 cells triple stained for Dendra2 (Dn), actin (Act), and vinculin (Vin). The arrows and the asterisk point to the basolateral and apicolateral junctions, respectively, which are positive for all three markers. The α-catenin–negative focal contacts are indicated by arrowheads. (c) Dn-αCat–expressing 468 cells triple stained for Dendra2 (Dn), actin (Act), and a TJ protein cingulin (Cin). Apical (left) and basal (right) focus planes are shown. Note that the apicolateral AJs associate with TJs and with a fine actin staining (apical focus plane). The lateral cell membranes are enriched with numerous lateral spot-like junctions, which did not show clear association with actin structures. The base of the lateral membranes formed basolateral AJs associated with the radial actin bundles (basal focus plane). Schematic representation of the Dn-αCat is given on the top of image. Dn denotes the GFP Dendra2. αH denotes the head domain of α-catenin implicated in binding to β-catenin and homodimerization. M1–M3 and αABD denote middle domains and αABD of α-catenin, respectively. Bars, 10 µm.

Figure 4.

Comparison of junctions formed by Dn-αCat or its mutants in 468 cells. The cells were double stained for occludin to reveal TJs (top row) and for Dendra2 to reveal transgene products (bottom row). The maps of Dn-αCat proteins are shown as in Fig. 3. The mutated domains are in yellow. Dn-αCat can interact with actin both directly through αABD and indirectly through vinculin, whereas its Dn-αCat-ΔVin and Dn-αCat(1–505) mutants can only associate with actin specifically through αABD or vinculin, respectively. Both modes of interactions are inactivated in the double mutant Dn-αCat-ΔVin+I792A. Note that cells expressing intact α-catenin were the only ones to produce fully closed rings of TJs. All the mutants showed comparable levels of expression (Fig. S4 a). Bars, 40 µm.

Figure 5.

AJs interact with actin filaments through αABD. (a–c) Immunofluorescence microscopy of 468 cells expressing the α-catenin mutant, which interacts with actin specifically through αABD, Dn-αCat-ΔVin (a and b), or the same mutant with additional K866A point mutation (c). Cells were double stained for Dendra2 (Dn) and actin (Act; a) or Dn and vinculin (Vin; b). The boxed regions are magnified on the right. (a) The apical and basal focus planes are shown. Many apicolateral AJs of these cells were associated with the radial actin bundles. The cells were completely unable to produce basolateral AJs. (b) Vinculin is recruited only into the focal contacts. (c) 468 cells expressing Dn-αCat-ΔVin-K866A chimera have a phenotype similar to that of the parental cells (Fig. 3 a). Bars, 10 µm.

Figure 6.

AJs interacting with actin through vinculin produce static junctions. (a and b) Immunofluorescence microscopy of 468 cells expressing the α-catenin deletion mutant, Dn-αCat(1–505). Cells were double stained for Dendra2 (Dn) and actin (Act; a) or Dn and vinculin (Vin; b). (a) Apicolateral AJs are associated with fine actin structures (apical focus plane). The basolateral junctions were also present but not associated with actin bundles (arrows, basal focus plane). (b) Apicolateral and lateral junctions both recruit vinculin. Bars, 10 µm. (c) Dendra photoconversion assay of the apicolateral AJs in 468 cells expressing Dn-αCat, Dn-αCat-ΔVin, or Dn-αCat(1–505). The intensity of the red fluorescence in the photoconverted spots decreases over time. The error bars represent SEs (n = 20).

Figure 7.

Selective interaction of αABD with the actin cytoskeleton. (a–d) Spatial localization of Dn-αABD (Dn, stained for Dendra2) and actin filaments (Act): (a) in A431 cells; (b) in A431 cells treated with a high dose of Latrunculin A for 10 min; (c) in A431 cells treated with a low dose of Latrunculin A for 10 min; and (d) in A431 cells treated with Blebbistatin for 15 min. The Dn-αABD chimera includes Dendra2 (Dn) and the 671–906 region of α-catenin (αABD). (e) Spatial localization of the Dn-αABD point mutants—K842A, I792A, and K866A—in A431 cells. (f) Confocal microscopy of A431 cells expressing Dn-αABD and its point mutants—K842A and K866A in horizontal and vertical cross sections (indicated by the yellow lines). (g) wt A431 cells were permeabilized with 0.025% saponin for 3 min and then incubated for another 5 min with His-mCherry–tagged αABD (αABD) or its inactive I792A version (αABD-I792A) and stained for actin. The boxed regions are magnified on the insets. Note that αABD preferentially decorates the cortex but shows very weak binding to the actin bundles. The I792A mutant shows only nonspecific binding. Arrows point to bundles (a) and cortical clumps (c). Bars: (main images) 10 µm; (magnified images) 2.5 µm.

Figure 8.

SMLM of αABD clusters. A431 cells expressing Dn-αABD (αABD), its point mutant Dn-αABD-I792A (I792A), or Dn-αABD after a 10-min treatment with Latrunculin A (LnA). The corresponding heat maps of molecular cluster densities are shown at the bottom with the heat bar given in the right corner. The boxed regions are magnified in the insets.

Figure 9.

Dynamic properties of αABD clusters. (a) Dendra photoconversion assay of A431 cells expressing Dn-αABD (αABD) or its K842A or K866A point mutants. The images of green (−2) and red (−1) fluorescence were taken 2 and 1 s before photoconversion, respectively. The encircled areas (d = 2.5 µm) were converted from green to red fluorescence at time 0. After photoconversion, the red fluorescence was imaged in a stream mode with an image acquisition time of 1 s. Selected frames taken 1, 6, or 11 s after photoconversion (+1, +6, and +11) are shown. (b) Red fluorescence intensity over time in the photoconverted spots of A431 cells expressing Dn-αABD or its K842A point mutant. The curves were plotted based on experiments shown in Fig. 8 a (repeated 15 times) in unaffected A431 cells (control), in the cells with cytoskeleton stabilized by ATP depletion (ATP depletion), and in the cells with actin dynamics arrested by a triple-drug cocktail (JYL [Jasplakinolide, Latrunculin B, and Y27632]). Error bars indicate SEs. (c) FSM of A431 cells expressing Dn-αABD or its I792A point mutant. The encircled areas of the cells (d = ∼4 µm) were photoconverted to image the adjacent area (yellow boxes) for 40 s in a stream mode with 200-ms acquisition time (Videos 1 and 2, respectively). Frames taken 20 s after photoconversion (+20) are shown in the images on the right. (d) Spatial localization of αABD speckles plotted based on Video 1. The color of a given speckle corresponds to the moment of its appearance (the time bar is given at the bottom). (e) Lifetime distribution of αABD speckles. (f) Displacement of speckles (in nanometers) during their entire lifetime. Error bars in f and e indicate SDs. (g) The photobleaching curves of the individual speckles. A.U., arbitrary unit. Bars, 10 µm.