Cadherin controls nectin recruitment into adherens junctions by remodeling the actin cytoskeleton

Abstract

The mechanism that coordinates activities of different adhesion receptors is poorly understood. We investigated this mechanism by focusing on the nectin-2 and E-cadherin adherens junction receptors. We found that, cadherin was not required for the basic process of nectin junction formation because nectin-2 formed junctions in cadherin-deficient A431D cells. Formation of nectin-2 junctions in these cells, however, became regulated by cadherin as soon as E-cadherin was re-expressed. E-cadherin recruited nectin-2 into adherens junctions, where both proteins formed distinct but tightly associated clusters. Live-cell imaging showed that the appearance of E-cadherin clusters often preceded that of nectin-2 clusters at sites of junction assembly. Inactivation of E-cadherin clustering by different strategies concomitantly suppressed the formation of nectin clusters. Furthermore, cadherin significantly increased the stability of nectin clusters, thereby making them resistant to the BC-12 antibody, which targets the nectin-2 adhesion interface. By testing different E-cadherin-α-catenin chimeras, we showed that the recruitment of nectin into chimera junctions is mediated by the actin-binding domain of α-catenin. Our data suggests that E-cadherin regulates assembly of nectin junctions through α-catenin-induced remodeling of the actin cytoskeleton around the cadherin clusters.

Keywords: Actin; Adhesion; Cadherin; Nectin; α-catenin.

Fig. 1.

Nectin junctions depend on cadherin. (A) Representative image of anti-nectin-2-stained A431D cells. (B) The left column shows A431D cells double-stained for nectin-2 (Nt) and afadin (Af). Nectin forms apicolateral rings of wavy linear junctions that are associated with afadin. The set of images to the right shows A431D cells double-stained for nectin-2 (Nt) and α-catenin (αC). (C) Structured-illumination microscopy of nectin junctions in A431D cells stained for nectin-2. The apical cell region is shown. (D) A431D cells stably expressing EcadDn stained for nectin-2 (Nt). The boxed area is zoomed in the inset and is shown in two colors: red, nectin-2 (Nt), and green, EcadDn (Ec). Note that upon E-cadherin expression, nectin is localized at the apical adherens junctions. The images at the bottom show that apical adherens junctions (Ec, green) recruit vinculin (Vin, red). (E) EcadDn-expressing A431D cells were cultured for 20 min in the presence of function-blocking anti-cadherin antibody SHE78-7 (SHE) or in low Ca²⁺ medium (Low Ca) and then double-stained using mouse anti-cadherin (Ec, green) and rabbit anti-nectin-2 (Nt, red) antibodies. Note that both treatments severely affected both nectin and cadherin junctions. Higher magnifications of the selected regions (indicated by arrows) are shown in the insets. (F) Structured-illumination microscopy of nectin junctions in EcadDn- (EcDn) and cis-EcadDn (cis-EcDn)-expressing A431D cells double stained for nectin-2 (Nt) and E-cadherin (Ec). Note that the nectin-positive junctions were perpendicular to the cell–cell contact and associated with the apical cadherin junctions. Also note that mutation of the cadherin cis interface significantly disturbed cadherin junctional localization. Scale bars: 30 µm (A,B,E), 10 µm (D), 5 µm (C,F).

Fig. 2.

Nectin-2 localization depends on cadherin adhesion properties. (A) A431D cells stably transfected to express EcadDn (Ec) or its mutants bearing point mutations inactivating either its adhesive (W2A) or cis-binding (Cis) interfaces. Cells were double-stained for cadherin (left column) and nectin-2 (Nt, right column). Higher magnifications of the selected regions (indicated by arrows) are shown in the insets. Scale bars: 30 µm. (B) High magnifications of the junctions produced in A431D cells expressing the cis-EcadDn mutant. The cells were double-stained for the mutant (Cis, green) and for nectin-2 (Nt), vinculin (Vin) or actin (Act). Green and red channels are shown separately for the boxed regions. Note that the cells contact one another through numerous actin-rich filopodia co-recruiting nectin and vinculin. Scale bars: 10 µm.

Fig. 3.

Kinetics of cadherin and nectin junction assembly. (A) A431 cells co-expressing EcadDn (Ec, green) and N2mCh (Nt, red) were imaged in 1-s intervals and with 1-s-long acquisition. Time (s) is shown below the frames. Time 0 is the moment when the cells touch one another. Note that cadherin clusters as fast as 2 s after time 0. Nectin clusters become visible only 2 s later. Merged images of the cells 2 s before (−2) and 20 s after (20) contact formation are also shown. Scale bars: 6 µm. (B) A431 cells expressing an adhesion-incompetent mutant of nectin-2, F136D-N2Dn. The mutant is randomly distributed along the cell surface. Scale bars: 30 µm. (C) Circular areas (diameter ∼2.5 µm) of F136D-N2Dn-expressing cells were photoconverted from green to red fluorescence. The green image taken 1 s before photoconvesion and the first red image taken 1 s after photoconversion are shown. The left pair of images was obtained from prefixed cells and the right pair from live cells. Note that the photoconverted red spot is much larger in live cells. Scale bar: 10 µm. (D) Graphs showing the change in intensity of the photoconverted red fluorescence over time (s) in the entire laser-irradiated area in the control conditions (Ctrl) and 10 min after Latrunculin A applications (LnA). The time needed for 50% decrease of the red fluorescence (t_1/2) is shown for each case. The photoconversion experiments were repeated ten times with cells in each condition. Results are mean±s.d.

Fig. 4.

E-cadherin reinforces the nectin junctions. Parental A431D cells (A431D) and their derivatives expressing EcadDn (EcDn) or cis-EcadDn (cis-EcDn) were incubated for 20 min with BC-12 antibody (10 µg/ml) at 37°C (37°C) and then double-stained using anti-mouse antibody (Nt), detecting localization of BC-12–nectin-2 complexes, and rabbit anti-Dendra antibody (Dn), detecting recombinant Dendra-tagged E-cadherin. The control cells (4°C) were incubated with BC-12 antibody (20 min, 10 µg/ml) on ice. Higher magnifications of the selected regions (indicated by arrows) are shown in the insets. Scale bars: 10 µm. Note that BC-12 antibody disintegrates nectin junctions in A431D cells but is unable to do so in presence of cadherin.

Fig. 5.

The actin-binding domain of α-catenin is sufficient for nectin–cadherin junction association. (A) Schematic representation of the E-cadherin–α-catenin chimeras. Each chimera includes a different α-catenin region fused with the EcΔDn module consisting of dendra2, Dn (green) and the tailless cadherin, EcΔ (brown), which lacks any known cadherin intracellular binding sites. The C-terminal portion of α-catenin (amino acids 506–906), encompassing the M3 domain and αABD, is present in the EcΔDn–α506 chimera. Two other chimeras, EcΔDn–M3 and EcΔDn–HαABD, incorporate either one of these domains. (B–D) Double immunostaining of A431D cells expressing EcΔDn (B), EcΔDn–α506 (C), EcΔDn–M3 (D), EcΔDn–HαABC (E). All cells were stained with rabbit anti-dendra2 (Dn) to detect the chimeras and mouse anti-nectin-2 (Nt), mouse anti-afadin (Af), or Alexa-Fluor-555–phalloidin (Act). Higher magnifications of the boxed regions (shown in the merged images) are shown either in the insets or in the separate rows. Some of the radial and polymorphic non-radial junctions are indicated by arrows and arrowheads, respectively. Scale bars: 20 µm (5 µm for the zoomed areas).

Fig. 6.

Replacement of the αABD with other actin-binding domains abolishes cadherin–nectin junction association. (A) Schematic representation of non-αABD cadherin chimeras tested. In all cases the cadherin module, EcΔDn, was the same as in the cadherin–α-catenin chimeras (see Fig.  5). The αABD was replaced with the actin-binding domain of utrophin (Utr) in the EcΔDn–Utr chimera or with the actin-binding peptide Lifeact (Lat) in the EcΔDn–Lat chimera. The EcΔDn–M3Utr chimera contained the α-catenin M3 domain in front of the Utr. (B) Immunoblot analysis of the stable cell lines expressing different chimeras: EcΔDn (Δ), EcΔDn-α506 (506), EcΔDn-M3 (M3), EcΔDn-HαABD (ABD), EcΔDn-Utr (Utr), EcDDn-Lat (Lat), EcΔDn-M3Utr (M3Utr), EcΔDn-LatUtr (LatUtr); and the control A431 cells expressing endogenous E-cadherin (A431). Equal amounts of total cell lysates were analyzed using anti-E-cadherin SHE78-7 (upper panel) and anti-tubulin (lower panel) antibodies. (C–E) Double immunostaining of A431D cells expressing EcΔDn-Utr (C), EcΔDn-Lat (D), and EcΔDn-M3Utr (E) using rabbit anti-dendra2 (Dn, green) antibody, to detect the chimeras, and mouse anti-nectin-2 (Nt, red) antibody, to detect nectin junctions. Higher magnifications of the selected regions (indicated by arrows in the merged images) are shown in the insets. Scale bars: 20 µm. Note that the cell–cell contacts enriched with the chimera junctions are mostly devoid of nectin junctions. If present, nectin junctions do not colocalize with the chimeras.

Fig. 7.

Hypothetical mechanism of cadherin and nectin cluster assembly. (A) Actin filaments are disorganized in the actin cortex. (B) Filaments are specifically aligned owing to the adhesive clustering of the cadherin–catenin complex. (C) The resulting cadherin-associated actin filament structure becomes the substrate for nectin–afadin clustering. Nectin–afadin clusters could reinforce the actin structure and facilitate further clustering of cadherin.