Ankyrin-G palmitoylation and βII-spectrin binding to phosphoinositide lipids drive lateral membrane assembly

Ankyrin-G and βII-spectrin colocalize at sites of cell–cell contact in columnar epithelial cells and promote lateral membrane assembly. This study identifies two critical inputs from lipids that together provide a rationale for how ankyrin-G and βII-spectrin selectively localize to Madin-Darby canine kidney (MDCK) cell lateral membranes. We identify aspartate-histidine-histidine-cysteine 5/8 (DHHC5/8) as ankyrin-G palmitoyltransferases required for ankyrin-G lateral membrane localization and for assembly of lateral membranes. We also find that βII-spectrin functions as a coincidence detector that requires recognition of both ankyrin-G and phosphoinositide lipids for its lateral membrane localization. DHHC5/8 and βII-spectrin colocalize with ankyrin-G in micrometer-scale subdomains within the lateral membrane that are likely sites for palmitoylation of ankyrin-G. Loss of either DHHC5/8 or ankyrin-G–βII-spectrin interaction or βII-spectrin–phosphoinositide recognition through its pleckstrin homology domain all result in failure to build the lateral membrane. In summary, we identify a functional network connecting palmitoyltransferases DHHC5/8 with ankyrin-G, ankyrin-G with βII-spectrin, and βII-spectrin with phosphoinositides that is required for the columnar morphology of MDCK epithelial cells.     

Ankyrin-G and beta2-spectrin collaborate in biogenesis of lateral membrane of human bronchial epithelial cells

Ankyrins are a family of adapter proteins required for localization of membrane proteins to diverse specialized membrane domains including axon initial segments, specialized sites at the transverse tubule/sarcoplasmic reticulum in cardiomyocytes, and lateral membrane domains of epithelial cells. Little is currently known regarding the molecular basis for specific roles of different ankyrin isoforms. In this study, we systematically generated alanine mutants of clusters of charged residues in the spectrin-binding domains of both ankyrin-B and -G. The corresponding mutants were evaluated for activity in either restoration of abnormal localization of the inositol trisphosphate receptor in the sarcoplasmic reticulum in mutant mouse cardiomyocytes deficient in ankyrin-B or in prevention of loss of lateral membrane in human bronchial epithelial cells depleted of ankyrin-G by small interfering RNA. Interestingly, ankyrin-B and -G share two homologous sites that result in loss of function in both systems, suggesting that common molecular interactions underlie diverse roles of these isoforms. Ankyrins G and B also exhibit differences; mutations affecting spectrin binding had no effect on ankyrin-B function but did abolish activity of ankyrin-G in restoring lateral membrane biogenesis. Depletion of beta(2)-spectrin by small interfering RNA phenocopied depletion of ankyrin-G and resulted in a failure to form new lateral membrane in interphase and mitotic cells. These results demonstrate that ankyrin-G and beta(2)-spectrin are functional partners in biogenesis of the lateral membrane of epithelial cells.     

Lateral membrane biogenesis in human bronchial epithelial cells requires 190-kDa ankyrin-G

Ankyrin-G polypeptides are required for restriction of voltage-gated sodium channels, L1 cell adhesion molecules, and beta IV spectrin to axon initial segments and are believed to couple the Na/K-ATPase to the spectrin-actin network at the lateral membrane in epithelial cells. We report here that depletion of 190-kDa ankyrin-G in human bronchial epithelial cells by small interfering RNA results in nearly complete loss of lateral plasma membrane in interphase cells, and also blocks de novo lateral membrane biogenesis following mitosis. Loss of the lateral membrane domain is accompanied by an expansion of apical and basal plasma membranes and preservation of apical-basal polarity. Expression of rat 190-kDa ankyrin-G, which is resistant to human small interfering RNA, prevents loss of the lateral membrane following depletion of human 190-kDa ankyrin-G. Human 220-kDa ankyrin-B, a closely related ankyrin isoform, is incapable of preserving the lateral membrane following 190-kDa ankyrin-G depletion. Moreover, analysis of rat 190-kDa ankyrin G/ankyrin B chimeras shows that all three domains of 190-kDa ankyrin-G are required for preservation of the lateral membrane. These results demonstrate that 190-kDa ankyrin-G plays a pleiotropic role in assembly of lateral membranes of bronchial epithelial cells.     

Involvement of the annexin II-S100A10 complex in the formation of E-cadherin-based adherens junctions in Madin-Darby canine kidney cells

E-cadherin and nectins are major cell-cell adhesion molecules at adherens junctions (AJs) in epithelial cells. When Madin-Darby canine kidney (MDCK) cells stably expressing nectin-1 (nectin-1-MDCK cells) are cultured at normal Ca(2+), E-cadherin and nectin-1 are concentrated at the cell-cell contact sites. When these cells are cultured at low Ca(2+), E-cadherin disappears from the cell-cell contact sites, but nectin-1 persists there. When these cells are re-cultured at normal Ca(2+), E-cadherin is recruited to the nectin-based cell-cell contact sites. We found here that this recruitment was dependent on protein synthesis, because a protein synthesis inhibitor, cycloheximide, prevented the accumulation of E-cadherin. When nectin-1-MDCK cells, precultured at low Ca(2+) in the presence of a proteasome inhibitor, ALLN (N-acetyl-Leu-Leu-norleucinal), were re-cultured at normal Ca(2+), E-cadherin was recruited to the nectin-based cell-cell contact sites but the level of E-cadherin was reduced. Similar results were obtained when wild-type MDCK cells were used instead of nectin-1-MDCK cells. These results suggest that degradation of one or more protein factors and de novo synthesis of the same or different protein factor(s) are needed for the formation of the E-cadherin-based AJs. We biochemically identified the annexin II-S100A10 complex as such a candidate. Depletion of plasma membrane cholesterol, which abolished the localization of the annexin II-S100A10 complex at the plasma membrane, inhibited the re-concentration of E-cadherin at the nectin-based cell-cell contact sites in the Ca(2+) switch experiment. Knockdown of annexin II by RNA interference also inhibited the re-concentration of E-cadherin. These results indicate that the annexin II-S100A10 complex is involved in the formation of the E-cadherin-based AJs in MDCK cells.     

Identification of a membrane-cytoskeletal complex containing the cell adhesion molecule uvomorulin (E-cadherin), ankyrin, and fodrin in Madin- Darby canine kidney epithelial cells

Cell-cell contact is an important determinant in the formation of functionally distinct plasma membrane domains during the development of epithelial cell polarity. In cultures of Madin-Darby canine kidney (MDCK) epithelial cells, cell-cell contact induces the assembly and accumulation of the Na+,K+-ATPase and elements of the membrane-cytoskeleton (ankyrin and fodrin) at the regions of cell-cell contact. Epithelial cell-cell contact appears to be regulated by the cell adhesion molecule uvomorulin (E-cadherin) which also becomes localized at the lateral plasma membrane of polarized cells. We have sought to determine whether the colocalization of these proteins reflects direct molecular interactions which may play roles in coordinating cell-cell contact and the assembly of the basal-lateral domain of the plasma membrane. Recently, we identified a complex of proteins containing the Na+,K+-ATPase, ankyrin, and fodrin in extracts of whole MDCK cells (Nelson, W.J., and R. W. Hammerton. 1989. J. Cell Biol. 108:893-902). We have now examined cell extracts for protein complexes containing the cell adhesion molecule uvomorulin. Proteins were solubilized from whole MDCK cells and fractionated in sucrose gradients. The sedimentation profile of solubilized uvomorulin is well separated from the majority of cell surface proteins, suggesting that uvomorulin occurs in a protein complex. A distinct portion of uvomorulin (30%) cosediments with ankyrin and fodrin (approximately 10.5S). Further fractionation of cosedimenting proteins in nondenaturing polyacrylamide gels reveals a discrete band of proteins that binds antibodies specific for uvomorulin, Na+,K+-ATPase, ankyrin, and fodrin. Significantly, ankyrin and fodrin, but not Na+K+-ATPase, coimmunoprecipitate in a complex with uvomorulin using uvomorulin antibodies. This result indicates that separate complexes exist containing ankyrin and fodrin with either uvomorulin or Na+,K+-ATPase. These results are discussed in the context of the possible roles of uvomorulin-induced cell-cell contact in the assembly of the membrane-cytoskeleton and associated membrane proteins (e.g., Na+,K+-ATPase) at the contact zone and in the development of cell polarity.     

Cysteine 70 of Ankyrin-G Is S-Palmitoylated and Is Required for Function of Ankyrin-G in Membrane Domain Assembly

Ankyrin-G (AnkG) coordinates protein composition of diverse membrane domains, including epithelial lateral membranes and neuronal axon initial segments. However, how AnkG itself localizes to these membrane domains is not understood. We report that AnkG remains on the plasma membrane in Madin-Darby canine kidney (MDCK) cells grown in low calcium, although these cells lack apical-basal polarity and exhibit loss of plasma membrane association of AnkG partners, E-cadherin and β₂-spectrin. We subsequently demonstrate using mutagenesis and mass spectrometry that AnkG is S-palmitoylated exclusively at Cys-70, which is located in a loop of the first ankyrin repeat and is conserved in the vertebrate ankyrin family. Moreover, C70A mutation abolishes membrane association of 190-kDa AnkG in MDCK cells grown in low calcium. C70A 190-kDa AnkG fails to restore biogenesis of epithelial lateral membranes in MDCK cells depleted of endogenous AnkG. In addition, C70A 270-kDa AnkG fails to cluster at the axon initial segment of AnkG-depleted cultured hippocampal neurons and fails to recruit neurofascin as well as voltage-gated sodium channels. These effects of C70A mutation combined with evidence for its S-palmitoylation are consistent with a requirement of palmitoylation for targeting and function of AnkG in membrane domain biogenesis at epithelial lateral membranes and neuronal axon initial segments.     

Positive role of IQGAP1, an effector of Rac1, in actin-meshwork formation at sites of cell-cell contact

The small guanosine triphosphatase Rac1 is activated by E-cadherin-mediated cell-cell adhesion and is required for the accumulation of actin filaments, E-cadherin, and β-catenin at sites of cell-cell contact. However, the modes of activation and action of Rac1 remain to be clarified. We here found that suppression of IQGAP1, an actin-binding protein and an effector of Rac1, by small interfering RNA apparently reduced the accumulation of actin filaments, E-cadherin, and β-catenin at sites of cell-cell contact in Madin-Darby canine kidney II epithelial cells under the conditions in which knockdown of Rac1 reduced them. Knockdown of Rac1 did not affect the localization of these junctional components in cells expressing a constitutively active IQGAP1 mutant defective in Rac1/Cdc42 binding. Knockdown of either Rac1 or IQGAP1 accelerated the 12-O-tetradecanoylphorbol-13-acetate-induced cell-cell dissociation. The basal Rac1 activity, which was maintained by E-cadherin-mediated cell-cell adhesion, was inhibited in the IQGAP1-knocked down cells, whereas the Rac1 activity was increased in the cells overexpressing IQGAP1. Together, these results indicate that Rac1 enhances the accumulation of actin filaments, E-cadherin, and β-catenin by acting on IQGAP1 and suggest that there exists a positive feedback loop comprised of “E-cadherin-mediated cell-cell adhesion→Rac1 activation→actin-meshwork formation by IQGAP1→increasing E-cadherin-mediated cell-cell adhesion.”     

Viral determinants of polarized assembly for the murine leukemia virus

Retrovirus transmission via direct cell-cell contact is more efficient than diffusion through the extracellular milieu. This is believed to be due to the ability of viruses to efficiently coordinate several steps of the retroviral life cycle at cell-cell contact sites (D. C. Johnson et al., J. Virol. 76:1-8, 2002; D. M. Phillips, AIDS 8:719-731, 1994; Q. Sattenau, Nat. Rev. Microbiol. 6:815-826, 2008). Using the murine leukemia virus (MLV) as a model retrovirus, we have previously shown that interaction between viral envelope (Env) and receptor directs viral assembly to cell-cell contact sites to promote efficient viral spreading (J. Jin et al., PLoS Biol. 7:e1000163, 2009). In addressing the underlying mechanism, we observed that Env cytoplasmic tail directs this contact-induced polarized assembly. We present here the viral determinants in the Env cytoplasmic tail and Gag that are important in this process. A tyrosine residue within the cytoplasmic tail of Env was identified, which directs polarized assembly. MLV matrix-mediated membrane targeting is required for Gag recruitment to sites of cell-cell contact. Our results suggest that MLV polarized assembly is mediated by a direct or indirect interaction between both domains, thereby coupling Gag recruitment and virus assembly to Env accumulation at the cell-cell interface. In contrast, HIV Gag that assembles outside of cell-cell interfaces can subsequently be drawn into contact zones mediated by MLV Env and receptor, a finding that is consistent with the previously observed lateral movement of HIV into the virological synapse (W. Hubner et al., Science 323:1743-1747, 2009; D. Rudnicka et al., J. Virol. 83:6234-6246, 2009). As such, we observed two distinct modes of virus cell-to-cell transmission that involve either polarized or nonpolarized assembly, but both result in virus transmission.     

Differential behavior of E-cadherin and occludin in their colocalization with ZO-1 during the establishment of epithelial cell polarity

At the initial stage of cell-cell contact of epithelial cells, primordial spot-like junctions are formed at the tips of thin cellular protrusions radiating from adjacent cells, where E-cadherin and ZO-1 are precisely coconcentrated (Yonemura et al., 1995, J. Cell Sci. 108:127-142). In fully polarized epithelial cells, E-cadherin and ZO-1 are completely sorted into belt-like adherens junctions (AJ) and tight junctions (TJ), respectively. Here we examined the behavior of occludin, an integral membrane protein consisting of TJ, during the establishment of epithelial cell polarity. Using confocal immunofluorescence microscopy, we quantitatively compared the spatial relationship of occludin/ZO-1 with that of E-cadherin/ZO-1 during epithelial cellular polarization by replating or wounding cultured mouse epithelial cells (MTD1-A). At the initial stage of cell-cell contact, E-cadherin and ZO-1 appeared to be simultaneously recruited to the primordial form of spot-like junctions at the tips of cellular processes which showed no concentration of occludin. Then, as cellular polarization proceeded, occludin was gradually accumulated at the ZO-1-positive spot-like junctions to form belt-like TJ, and in a complementary manner E-cadherin was sorted out from the ZO-1-positive spot-like junctions to form belt-like AJ. The molecular mechanism of TJ/AJ formation during epithelial cellular polarization is discussed with special reference to the roles of ZO-1.     

Localized zones of Rho and Rac activities drive initiation and expansion of epithelial cell-cell adhesion

Spatiotemporal coordination of cell-cell adhesion involving lamellipodial interactions, cadherin engagement, and the lateral expansion of the contact is poorly understood. Using high-resolution live-cell imaging, biosensors, and small molecule inhibitors, we investigate how Rac1 and RhoA regulate actin dynamics during de novo contact formation between pairs of epithelial cells. Active Rac1, the Arp2/3 complex, and lamellipodia are initially localized to de novo contacts but rapidly diminish as E-cadherin accumulates; further rounds of activation and down-regulation of Rac1 and Arp2/3 occur at the contacting membrane periphery, and this cycle repeats as a restricted membrane zone that moves outward with the expanding contact. The cortical bundle of actin filaments dissolves beneath the expanding contacts, leaving actin bundles at the contact edges. RhoA and actomyosin contractility are activated at the contact edges and are required to drive expansion and completion of cell-cell adhesion. We show that zones of Rac1 and lamellipodia activity and of RhoA and actomyosin contractility are restricted to the periphery of contacting membranes and together drive initiation, expansion, and completion of cell-cell adhesion.     

A molecular mechanism directly linking E-cadherin adhesion to initiation of epithelial cell surface polarity

Mechanisms involved in maintaining plasma membrane domains in fully polarized epithelial cells are known, but when and how directed protein sorting and trafficking occur to initiate cell surface polarity are not. We tested whether establishment of the basolateral membrane domain and E-cadherin-mediated epithelial cell-cell adhesion are mechanistically linked. We show that the basolateral membrane aquaporin (AQP)-3, but not the equivalent apical membrane AQP5, is delivered in post-Golgi structures directly to forming cell-cell contacts where it co-accumulates precisely with E-cadherin. Functional disruption of individual components of a putative lateral targeting patch (e.g., microtubules, the exocyst, and soluble N-ethylmaleimide-sensitive factor attachment protein receptors) did not inhibit cell-cell adhesion or colocalization of the other components with E-cadherin, but each blocked AQP3 delivery to forming cell-cell contacts. Thus, components of the lateral targeting patch localize independently of each other to cell-cell contacts but collectively function as a holocomplex to specify basolateral vesicle delivery to nascent cell-cell contacts and immediately initiate cell surface polarity.     

Quantitative analysis of cadherin-catenin-actin reorganization during development of cell-cell adhesion

Epithelial cell-cell adhesion requires interactions between opposing extracellular domains of E-cadherin, and among the cytoplasmic domain of E-cadherin, catenins, and actin cytoskeleton. Little is known about how the cadherin-catenin-actin complex is assembled upon cell-cell contact, or how these complexes initiate and strengthen adhesion. We have used time-lapse differential interference contrast (DIC) imaging to observe the development of cell-cell contacts, and quantitative retrospective immunocytochemistry to measure recruitment of proteins to those contacts. We show that E-cadherin, alpha-catenin, and beta- catenin, but not plakoglobin, coassemble into Triton X-100 insoluble (TX-insoluble) structures at cell-cell contacts with kinetics similar to those for strengthening of E-cadherin-mediated cell adhesion (Angres, B., A. Barth, and W.J. Nelson. 1996. J. Cell Biol. 134:549- 557). TX-insoluble E-cadherin, alpha-catenin, and beta-catenin colocalize along cell-cell contacts in spatially discrete micro-domains which we designate "puncta," and the relative amounts of each protein in each punctum increase proportionally. As the length of the contact increases, the number of puncta increases proportionally along the contact and each punctum is associated with a bundle of actin filaments. These results indicate that localized clustering of E- cadherin/catenin complexes into puncta and their association with actin is involved in initiating cell contacts. Subsequently, the spatial ordering of additional puncta along the contact may be involved in zippering membranes together, resulting in rapid strengthening of adhesion.     

IQGAP1 and calmodulin modulate E-cadherin function

Ca(2+)-dependent cell-cell adhesion is mediated by the cadherin family of transmembrane proteins. Adhesion is achieved by homophilic interaction of the extracellular domains of cadherins on adjacent cells, with the cytoplasmic regions serving to couple the complex to the cytoskeleton. IQGAP1, a novel RasGAP-related protein that interacts with the cytoskeleton, binds to actin, members of the Rho family, and E-cadherin. Calmodulin binds to IQGAP1 and regulates its association with Cdc42 and actin. Here we demonstrate competition between calmodulin and E-cadherin for binding to IQGAP1 both in vitro and in a normal cellular milieu. Immunocytochemical analysis in MCF-7 (E-cadherin positive) and MDA-MB-231 (E-cadherin negative) epithelial cells revealed that E-cadherin is required for accumulation of IQGAP1 at cell-cell junctions. The cell-permeable calmodulin antagonist CGS9343B significantly increased IQGAP1 at areas of MCF-7 cell-cell contact, with a concomitant decrease in the amount of E-cadherin at cell-cell junctions. Analysis of E-cadherin function revealed that CGS9343B significantly decreased homophilic E-cadherin adhesion. On the basis of these data, we propose that disruption of the binding of calmodulin to IQGAP1 enhances the association of IQGAP1 with components of the cadherin-catenin complex at cell-cell junctions, resulting in impaired E-cadherin function.     

Immediate-early signaling induced by E-cadherin engagement and adhesion

Epithelial cell-cell interactions require localized adhesive interactions between E-cadherin on opposing membranes and the activation of downstream signaling pathways that affect membrane and actin dynamics. However, it is not known whether E-cadherin engagement and activation of these signaling pathways are locally coordinated or whether signaling is sustained or locally down-regulated like other receptor-mediated pathways. To obtain high spatiotemporal resolution of immediate-early signaling events upon E-cadherin engagement, we used laser tweezers to place beads coated with functional E-cadherin extracellular domain on cells. We show that cellular E-cadherin accumulated rapidly around beads, reaching a sustained plateau level in 1-3 min. Phosphoinositides and Rac1 co-accumulated with E-cadherin, reached peak levels with E-cadherin, but then rapidly dispersed. Both E-cadherin and Rac1 accumulated independently of Rac1 GTP binding/hydrolysis, but these activities were required for Rac1 dispersal. E-cadherin accumulation was dependent on membrane dynamics and actin polymerization, but actin did not stably co-accumulate with E-cadherin; mathematical modeling showed that diffusion-mediated trapping could account for the initial E-cadherin accumulation. We propose that initial E-cadherin accumulation requires active membrane dynamics and involves diffusion-mediated trapping at contact sites; to propagate further contacts, phosphatidylinositol 3-kinase and Rac1 are transiently activated by E-cadherin engagement and initiate a new round of membrane dynamics, but they are subsequently suppressed at that site to allow maintenance of weak E-cadherin mediated adhesion.     

N-glycosylation affects the adhesive function of E-Cadherin through modifying the composition of adherens junctions (AJs) in human breast carcinoma cell line MDA-MB-435

E-cadherin mediates calcium-dependent cell-cell adhesion between epithelial cells. The ectodomain of human E-cadherin contains four potential N-glycosylation sites at Asn residues 554, 566, 618, and 633. In this study, the role of N-glycosylation in E-cadherin-mediated cell-cell adhesion was investigated by site-directed mutagenesis. In MDA-MB-435 cells, all four potential N-glycosylation sites of human E-cadherin were N-glycosylated. Removal of N-glycan at Asn-633 dramatically affected E-cadherin stability. In contrast, mutant E-cadherin lacking the other three N-glycans showed similar protein stability in comparison with wild-type E-cadherin. Moreover, N-glycans at Asn-554 and Asn-566 were found to affect E-cadherin-mediated calcium-dependent cell-cell adhesion, and removal of either of the two N-glycans caused a significant decrease in calcium-dependent cell-cell adhesion accompanied with elevated cell migration. Analysis of the composition of adherens junctions (AJs) revealed that removal of N-glycans on E-cadherin resulted in elevated tyrosine phosphorylation level of beta-catenin and reduced beta- and alpha-catenins at AJs. These findings demonstrate that N-glycosylation may affect the adhesive function of E-cadherin through modifying the composition of AJs.     

Rap1 regulates the formation of E-cadherin-based cell-cell contacts

In epithelial tissues, cells are linked to their neighbors through specialized cell-cell adhesion proteins. E-cadherin is one of the most important membrane proteins for the establishment of intimate cell-cell contacts, but the molecular mechanism by which it is recruited to contact sites is largely unknown. We report here that the cytoplasmic domain of E-cadherin interacts with C3G, a guanine nucleotide exchange factor for Rap1. In epithelial cell cultures, ligation of the extracellular domain of E-cadherin enhances Rap1 activity, which in turn is necessary for the proper targeting of E-cadherin molecules to maturing cell-cell contacts. Furthermore, our data suggest that Cdc42 functions downstream of Rap1 in this process. We conclude that Rap1 plays a vital role in the establishment of E-cadherin-based cell-cell adhesion.     

Adducin promotes micrometer-scale organization of beta2-spectrin in lateral membranes of bronchial epithelial cells

Adducin promotes assembly of spectrin-actin complexes, and is a target for regulation by calmodulin, protein kinase C, and rho kinase. We demonstrate here that adducin is required to stabilize preformed lateral membranes of human bronchial epithelial (HBE) cells through interaction with beta2-spectrin. We use a Tet-on regulated inducible small interfering RNA (siRNA) system to deplete alpha-adducin from confluent HBE cells. Depletion of alpha-adducin resulted in increased detergent solubility of spectrin after normal membrane biogenesis during mitosis. Conversely, depletion of beta2-spectrin resulted in loss of adducin from the lateral membrane. siRNA-resistant alpha-adducin prevented loss of lateral membrane, but only if alpha-adducin retained the MARCKS domain that mediates spectrin-actin interactions. Phospho-mimetic versions of adducin with S/D substitutions at protein kinase C phosphorylation sites in the MARCKS domain were not active in rescue. We find that adducin modulates long-range organization of the lateral membrane based on several criteria. First, the lateral membrane of adducin-depleted cells exhibited reduced height, increased curvature, and expansion into the basal surface. Moreover, E-cadherin-GFP, which normally is restricted in lateral mobility, rapidly diffuses over distances up to 10 mum. We conclude that adducin acting through spectrin provides a novel mechanism to regulate global properties of the lateral membrane of bronchial epithelial cells.     

Dynamics of cell adhesion and motility in living cells is altered by a single amino acid change in E-cadherin fused to enhanced green fluorescent protein

E-Cadherin regulates epithelial cell adhesion and is critical for the maintenance of tissue integrity. In sporadic diffuse-type gastric carcinoma, mutations of the E-cadherin gene are frequently observed that predominantly affect putative calcium binding motifs located in the linker region between the second and third extracellular domains. A single amino acid change (D370A) as found in a gastric carcinoma patient reduces cell adhesion and up-regulates cell motility. To study the effect of this mutation on the dynamics of cell adhesion and motility in living cells, enhanced green fluorescent protein (EGFP) was C-terminally fused to E-cadherin. The resulting mutant E-cadherin-EGFP fusion protein with a point mutation in exon 8 (p8-EcadEGFP) and a wild-type E-cadherin-EGFP fusion construct (wt-EcadEGFP) were expressed in human MDA-MB-435S cells. Fluorescent images were acquired by time-lapse laser scanning microscopy and E-cadherin was visualized during contact formation and in moving cells. Spatial and temporal localization of p8- and wt-EcadEGFP differed significantly. While wt-EcadEGFP was mainly localized at lateral membranes of contacting cells and formed E-cadherin puncta and plaques, p8-EcadEGFP-expressing cells frequently formed transient cell-cell contacts. During random cell migration, p8-EcadEGFP was found in lamellipodia. In contrast, wt-EcadEGFP localized at lateral cell-cell contact sites in low or non-motile cells. Inhibition of the epidermal growth factor (EGF) receptor, which plays a major role in lamellipodia formation and cell migration, reduced the motility of p8-EcadEGFP-expressing cells and caused lateral membrane staining of p8-EcadEGFP. Conversely, EGF induced cell motility and caused formation of lamellipodia that were E-cadherin positive. In conclusion, our data show that mutant E-cadherin significantly alters the dynamics of cell adhesion and motility in living cells and interferes with the formation of stable cell-cell contacts.     

A Single Divergent Exon Inhibits Ankyrin-B Association with the Plasma Membrane

Vertebrate ankyrin-B and ankyrin-G exhibit divergent subcellular localization and function despite their high sequence and structural similarity and common origin from a single ancestral gene at the onset of chordate evolution. Previous studies of ankyrin family diversity have focused on the C-terminal regulatory domain. Here, we identify an ankyrin-B-specific linker peptide connecting the ankyrin repeat domain to the ZU5₂-UPA module that inhibits binding of ankyrin-B to membrane protein partners E-cadherin and neurofascin 186 and prevents association of ankyrin-B with epithelial lateral membranes as well as neuronal plasma membranes. The residues of the ankyrin-B linker required for autoinhibition are encoded by a small exon that is highly divergent between ankyrin family members but conserved in the ankyrin-B lineage. We show that the ankyrin-B linker suppresses activity of the ANK repeat domain through an intramolecular interaction, likely with a groove on the surface of the ANK repeat solenoid, thereby regulating the affinities between ankyrin-B and its binding partners. These results provide a simple evolutionary explanation for how ankyrin-B and ankyrin-G have acquired striking differences in their plasma membrane association while maintaining overall high levels of sequence similarity.