Assembly of the TgrB1-TgrC1 cell adhesion complex during Dictyostelium discoideum development.

In Dictyostelium discoideum, TgrB1 and TgrC1 are partners of a heterophilic cell-adhesion system. To investigate its assembly process, the split GFP complementation assay was used to track the oligomeric status of both proteins. The ability of TgrC1 to form cis-homodimers spontaneously was demonstrated by fluorescence complementation studies and confirmed by chemical cross-linking. In contrast, TgrB1 failed to form cis-homodimers in the absence of TgrC1. Treatment of cell aggregates with antibodies against TgrB1 or TgrC1 did not affect TgrC1 dimerization, but inhibited TgrB1 dimer formation, suggesting that TgrB1 cis-homodimerization is dependent on trans-interaction with TgrC1. When TgrB1 and TgrC1 conjugated with the complementary halves of GFP were co-expressed in cells, cis-heterodimers were not detected. However, weak FRET signals were detected in cells expressing TgrB1-RFP and TgrC1-GFP, suggesting that TgrB1 dimers and TgrC1 dimers were arranged juxtapose to each other in the adhesion complex. The results of the present study suggest that the assembly process is initiated upon trans-interaction of monomeric TgrB1 with TgrC1 homodimers on adjacent cells, which triggers the formation of TgrB1 dimers. The homodimerization of TgrB1 in turn induces the clustering of TgrB1 and TgrC1, and the coalescence of TgrB1-TgrC1 clusters results in the formation of large adhesion complexes.

The cell adhesion molecule DdCAD-1 regulates morphogenesis through differential spatiotemporal expression in Dictyostelium discoideum.

During development of Dictyostelium, multiple cell types are formed and undergo a coordinated series of morphogenetic movements guided by their adhesive properties and other cellular factors. DdCAD-1 is a unique homophilic cell adhesion molecule encoded by the cadA gene. It is synthesized in the cytoplasm and transported to the plasma membrane by contractile vacuoles. In chimeras developed on soil plates, DdCAD-1-expressing cells showed greater propensity to develop into spores than did cadA-null cells. When development was performed on non-nutrient agar, wild-type cells sorted from the cadA-null cells and moved to the anterior zone. They differentiated mostly into stalk cells and eventually died, whereas the cadA-null cells survived as spores. To assess the role of DdCAD-1 in this novel behavior of wild-type and mutant cells, cadA-null cells were rescued by the ectopic expression of DdCAD-1-GFP. Morphological studies have revealed major spatiotemporal changes in the subcellular distribution of DdCAD-1 during development. Whereas DdCAD-1 became internalized in most cells in the post-aggregation stages, it was prominent in the contact regions of anterior cells. Cell sorting was also restored in cadA(-) slugs by exogenous recombinant DdCAD-1. Remarkably, DdCAD-1 remained on the surface of anterior cells, whereas it was internalized in the posterior cells. Additionally, DdCAD-1-expressing cells migrated slower than cadA(-) cells and sorted to the anterior region of chimeric slugs. These results show that DdCAD-1 influences the sorting behavior of cells in slugs by its differential distribution on the prestalk and prespore cells.

TgrC1 mediates cell-cell adhesion by interacting with TgrB1 via mutual IPT/TIG domains during development of Dictyostelium discoideum.

Cell-cell adhesion plays crucial roles in cell differentiation and morphogenesis during development of Dictyostelium discoideum. The heterophilic adhesion protein TgrC1 (Tgr is transmembrane, IPT, IG, E-set, repeat protein) is expressed during cell aggregation, and disruption of the tgrC1 gene results in the arrest of development at the loose aggregate stage. We have used far-Western blotting coupled with MS to identify TgrB1 as the heterophilic binding partner of TgrC1. Co-immunoprecipitation and pull-down studies showed that TgrB1 and TgrC1 are capable of binding with each other in solution. TgrB1 and TgrC1 are encoded by a pair of adjacent genes which share a common promoter. Both TgrB1 and TgrC1 are type I transmembrane proteins, which contain three extracellular IPT/TIG (immunoglobulin, plexin, transcription factor-like/transcription factor immunoglobulin) domains. Antibodies raised against TgrB1 inhibit cell reassociation at the post-aggregation stage of development and block fruiting body formation. Ectopic expression of TgrB1 and TgrC1 driven by the actin15 promoter leads to heterotypic cell aggregation of vegetative cells. Using recombinant proteins that cover different portions of TgrB1 and TgrC1 in binding assays, we have mapped the cell-binding regions in these two proteins to Lys(537)-Ala(783) in TgrB1 and Ile(336)-Val(360) in TgrC1, corresponding to their respective TIG3 and TIG2 domain.

ATP-Binding Cassette Transporter B4 Anchors the Cell Adhesion Molecule DdCAD-1 to Cell Membrane in Dictyostelium discoideum.

In Dictyostelium, soluble cell adhesion molecule, DdCAD-1, regulates cell-cell interaction through an unknown anchoring protein on the plasma membrane. Far western blot analysis using different probes revealed that the potential DdCAD-1 interacting protein was between 64 and 98 kDa. To isolate and identify the anchoring protein, GST-DdCAD-1 and anchoring protein were cross-linked in vivo by chemical cross-linker and stable protein complex was isolated by co-immunoprecipitation assays. The protein cross-linked to DdCAD-1 was extracted from the gel slice and trypsinized. The peptides were subjected to analysis by mass spectrometry, which showed that the putative anchoring protein belongs to ATP-binding cassette transporter family.

Regulation of spatiotemporal expression of cell-cell adhesion molecules during development of Dictyostelium discoideum.

The social amoeba Dictyostelium discoideum is a simple but powerful model organism for the study of cell-cell adhesion molecules and their role in morphogenesis during development. Three adhesive systems have been characterized and studied in detail. The spatiotemporal expression of these adhesion proteins is stringently regulated, often coinciding with major shifts in the morphological complexity of development. At the onset of development, amoeboid cells express the Ca(2+) -dependent cell-cell adhesion molecule DdCAD-1, which initiates weak homophilic interactions between cells and assists in the recruitment of individuals into cell streams. DdCAD-1 is unique because it is synthesized as a soluble protein in the cytoplasm. It is targeted for presentation on the cell surface by an unconventional protein transport mechanism via the contractile vacuole. Concomitant with the aggregation stage is the expression of the contact sites A glycoprotein csA/gp80 and TgrC1, both of which mediate Ca(2+) /Mg(2+) -independent cell-cell adhesion. Whereas csA/gp80 is a homophilic binding protein, TgrC1 binds to a heterophilic receptor on the cell. During cell aggregation, csA/gp80 associates preferentially with lipid rafts, which facilitate the rapid assembly of adhesion complexes. TgrC1 is synthesized at low levels during aggregation and rapid accumulation occurs initially in the peripheral cells of loose mounds. The extracellular portion of TgrC1 is shed and becomes part of the extracellular matrix. Additionally, analyses of knockout mutants have revealed important biological roles played by these adhesion proteins, including size regulation, cell sorting and cell-type proportioning.

Solution structures of the adhesion molecule DdCAD-1 reveal new insights into Ca(2+)-dependent cell-cell adhesion.

DdCAD-1 is a novel Ca(2+)-dependent cell adhesion molecule that lacks a hydrophobic signal peptide and a transmembrane domain. DdCAD-1 is expressed by the social amoeba Dictyostelium discoideum at the onset of development. It is synthesized as a soluble protein and then transported to the plasma membrane by contractile vacuoles. Here we describe the novel features of the solution structures of Ca(2+)-free and Ca(2+)-bound monomeric DdCAD-1. DdCAD-1 contains two beta-sandwich domains, belonging to the betagamma-crystallin and immunoglobulin fold classes, respectively. Whereas the N-terminal domain has a major role in homophilic binding, the C-terminal domain tethers the protein to the cell membrane. From structural and mutational analyses, we propose a model for the Ca(2+)-bound DdCAD-1 dimer as a basis for understanding DdCAD-1-mediated cell-cell adhesion at the molecular level. Our results provide new insights into Ca(2+)-dependent mechanisms for cell-cell adhesion.

Cell adhesion molecule DdCAD-1 is imported into contractile vacuoles by membrane invagination in a Ca2+- and conformation-dependent manner.

The cadA gene in Dictyostelium encodes a Ca(2+)-dependent cell adhesion molecule DdCAD-1 that contains two beta-sandwich domains. DdCAD-1 is synthesized in the cytoplasm as a soluble protein and then transported by contractile vacuoles to the plasma membrane for surface presentation or secretion. DdCAD-1-green fluorescent protein (GFP) fusion protein was expressed in cadA-null cells for further investigation of this unconventional protein transport pathway. Both morphological and biochemical characterizations showed that DdCAD-1-GFP was imported into contractile vacuoles. Time-lapse microscopy of transfectants revealed the transient appearance of DdCAD-1-GFP-filled vesicular structures in the lumen of contractile vacuoles, suggesting that DdCAD-1 could be imported by invagination of contractile vacuole membrane. To assess the structural requirements in this transport process, the N-terminal and C-terminal domains of DdCAD-1 were expressed separately in cells as GFP fusion proteins. Both fusion proteins failed to enter the contractile vacuole, suggesting that the integrity of DdCAD-1 is required for import. Such a requirement was also observed in in vitro reconstitution assays using His(6)-tagged fusion proteins and purified contractile vacuoles. Import of DdCAD-1 was compromised when two of its three Ca(2+)-binding sites were mutated, indicating a role for Ca(2+) in the import process. Spectral analysis showed that mutations in the Ca(2+)-binding sites resulted in subtle conformational changes. Indeed, proteins with altered conformation failed to enter the contractile vacuole, suggesting that the import signal is somehow integrated in the three-dimensional structure of DdCAD-1.

Involvement of Src family kinases in N-cadherin phosphorylation and beta-catenin dissociation during transendothelial migration of melanoma cells.

N-cadherin is recruited to the heterotypic contact during transendothelial migration of melanoma cells in a coculture system with tumor cells seeded on top of a monolayer of endothelial cells. However, beta-catenin dissociates from N-cadherin and redistributes to the nucleus of transmigrating melanoma cells to activate gene transcription. In this report, we demonstrate that Src becomes activated at the heterotypic contact between the transmigrating melanoma cell and neighboring endothelial cells. Src activation shows close temporal correlation with tyrosine phosphorylation of N-cadherin. Expression of a dominant-negative Src in melanoma cells blocks N-cadherin phosphorylation, beta-catenin dissociation, and nuclear translocation in transmigrating cells, consistent with the involvement of Src family kinases. In in vitro binding assays, Src-mediated phosphorylation of the N-cadherin cytoplasmic domain results in a significant reduction in beta-catenin binding. Although five phospho-tyrosine residues can be identified on the N-cadherin cytoplasmic domain by mass spectrometry, site-specific mutagenesis indicates that Tyr-860 is the critical amino acid involved in beta-catenin binding. Overexpression of N-cadherin carrying the Y860F mutation inhibits the transmigration of transfected cells across the endothelium. Together, the data suggest a novel role for tyrosine phosphorylation of N-cadherin by Src family kinases in the regulation of beta-catenin association during transendothelial migration of melanoma cells.

Transendothelial migration of melanoma cells involves N-cadherin-mediated adhesion and activation of the beta-catenin signaling pathway.

Cancer metastasis is a multistep process involving many types of cell-cell interactions, but little is known about the adhesive interactions and signaling events during extravasation of cancer cells. Transendothelial migration of cancer cells was investigated using an in vitro assay, in which melanoma cells were seeded on top of a monolayer of endothelial cells. Attachment of melanoma cells on the endothelium induced a twofold increase in N-cadherin expression in melanoma cells and the redistribution of N-cadherin to the heterotypic contacts. Transendothelial migration was inhibited when N-cadherin expression was repressed by antisense RNA, indicating a key role played by N-cadherin. Whereas N-cadherin and beta-catenin colocalized in the contact regions between melanoma cells and endothelial cells during the initial stages of attachment, beta-catenin disappeared from the heterotypic contacts during transmigration of melanoma cells. Immunolocalization and immunoprecipitation studies indicate that N-cadherin became tyrosine-phosphorylated, resulting in the dissociation of beta-catenin from these contact regions. Concomitantly, an increase in the nuclear level of beta-catenin occurred in melanoma cells, together with a sixfold increase in beta-catenin-dependent transcription. Transendothelial migration was compromised in cells expressing a dominant-negative form of beta-catenin, thus supporting a regulatory role of beta-catenin signaling in this process.

Interleukin-8 secreted by endothelial cells induces chemotaxis of melanoma cells through the chemokine receptor CXCR1.

There is increasing evidence that both cell adhesion molecules and soluble factors are involved in tumor metastasis. We have found that endothelial cells secrete chemoattractants that can induce melanoma cell chemotaxis. Protein separation on an ion-exchange column shows the association of IL-8 with fractions that contain the chemoattractant activity. This activity is completely lost from the conditioned medium after immunoprecipitation with anti-IL-8 antibodies, indicating that IL-8 is the major melanoma chemoattractant secreted by endothelial cells. IL-877, the predominant endothelial IL-8 isoform that contains 77 amino acids, is found to be twice as potent as the more common 72-amino acid isoform IL-872. Antibody inhibition studies indicate that the chemotactic response of melanoma cells is mediated by the CXC-chemokine receptor CXCR1 and not by the more promiscuous CXCR2. When stimulated by tumor necrosis factor alpha, the nonresponsive WM35 melanoma cells synthesize a higher level of CXCR1 and become chemotactic toward interleukin (IL)-8. Pretreatment of cells with pertussis toxin nullifies their chemotactic response, suggesting the involvement of G proteins. Antibodies against either IL-8 or CXCR1 inhibit melanoma transendothelial migration in a coculture assay by 30%. These results are consistent with a role for IL-8-induced chemotaxis in the transendothelial migration of melanoma cells.

Ca(2+) -calmodulin interacts with DdCAD-1 and promotes DdCAD-1 transport by contractile vacuoles in Dictyostelium cells.

The Ca(2+) -dependent cell-cell adhesion molecule DdCAD-1, encoded by the cadA gene of Dictyostelium discoideum, is synthesized at the onset of development as a soluble protein and then transported to the plasma membrane by contractile vacuoles. Calmodulin associates with contractile vacuoles in a Ca(2+) -dependent manner, and co-localizes with DdCAD-1 on the surface of contractile vacuoles. Bioinformatics analysis revealed multiple calmodulin-binding motifs in DdCAD-1. Co-immunoprecipitation and pull-down studies showed that only Ca(2+) -bound calmodulin was able to bind DdCAD-1. Structural integrity of DdCAD-1, but not the native conformation, was required for its interaction with calmodulin. To investigate the role of calmodulin in the import of DdCAD-1 into contractile vacuoles, an in vitro import assay consisting of contractile vacuoles derived from cadA(-) cells and recombinant proteins was employed. Prior stripping of the bound calmodulin from contractile vacuoles by EGTA impaired import of DdCAD-1, which was restored by addition of exogenous calmodulin. The calmodulin antagonists W-7 and compound 48/80 blocked the binding of calmodulin onto stripped contractile vacuoles, and inhibited the import of DdCAD-1. Together, the data show that calmodulin forms a complex with DdCAD-1 and promotes the docking and import of DdCAD-1 into contractile vacuoles. STRUCTURED DIGITAL ABSTRACT: CaM physically interacts with DdCAD-1 by pull down (View Interaction: 1, 2) DdCAD-1 binds to CaM by far western blotting (View interaction) DdCAD-1 physically interacts with CaM by anti bait coimmunoprecipitation (View interaction).

An activated Ras protein alters cell adhesion by dephosphorylating Dictyostelium DdCAD-1.

RasG-regulated signal transduction has been linked to a variety of growth-specific processes and appears to also play a role in the early development of Dictyostelium discoideum. In an attempt to uncover some of the molecular components involved in Ras-mediated signalling, several proteins have been described previously, including the cell adhesion molecule DdCAD-1, whose phosphorylation state was affected by the expression of the constitutively activated RasG, RasG(G12T). Here it has been shown that a cadA null strain lacks the phosphoproteins that were tentatively identified as DdCAD-1, confirming its previous designation. Further investigation revealed that cells expressing RasG(G12T) exhibited increased cell-cell cohesion, concomitant with reduced levels of DdCAD-1 phosphorylation. This increased cohesion was DdCAD-1-dependent and was correlated with increased localization of DdCAD-1 at the cell surface. DdCAD-1 phosphorylation was also found to decrease during Dictyostelium aggregation. These results revealed a possible role for protein phosphorylation in regulating DdCAD-1-mediated cell adhesion during early development. In addition, the levels of DdCAD-1 protein were substantially reduced in a rasG null cell line. These results indicate that RasG affects both the expression and dephosphorylation of DdCAD-1 during early development.

Reciprocal raft-receptor interactions and the assembly of adhesion complexes.

Cell adhesion complexes are critical for the physical coordination of cell-cell interactions and the morphogenesis of tissues and organs. Many adhesion receptors are anchored to the plasma membrane by a glycosylphosphatidylinositol (GPI) moiety and are thereby partitioned into membrane rafts. In this review, we focus on reciprocal interactions between rafts and adhesion molecules, leading to receptor clustering and raft expansion and stability. A model for a three-stage adhesion complex assembly process is also proposed. First, GPI-anchored adhesion molecules are recruited into rafts, which in turn promote receptor cis-oligomerization and thereby produce precursory complexes primed for avid trans-interactions. Second, trans-interactions of the receptors cross-link and stabilize large amalgams of rafts at sites of adhesion complex assembly. Finally, the enlarged and stabilized rafts acquire enhanced abilities to recruit the cytoskeleton and induce signaling. This process exemplifies how the domain structure of the plasma membrane can impact the function of its receptors.

Regulation of cell-cell adhesion during Dictyostelium development.

During development of Dictyostelium, four adhesion systems have been identified and adherens junction-like structures have been discovered in the fruiting body. The temporal and spatial expression of cell adhesion molecules (CAMs) is under stringent developmental control, corresponding to major shifts in morphological complexity. Genetic manipulations, including over-expression and knockout mutations, of the adhesion genes, cadA (encoding DdCAD-1), csaA (gp80) and lagC (gp150), have shed light on new roles for cell adhesion molecules in aggregate size regulation, cell-type proportioning, cell differentiation and cell sorting. As cell-cell interactions remain highly dynamic within cell streams and aggregates, mechanisms must exist to facilitate the rapid assembly and disassembly of adhesion complexes. Studies on gp80 have led to a model for the rapid assembly of adhesion complexes via lipid rafts.

Cytoskeleton interactions involved in the assembly and function of glycoprotein-80 adhesion complexes in dictyostelium.

Adhesion complexes typically assemble from clustered receptors that link to the cytoskeleton via cytoplasmic adapter proteins. However, it is unclear how phospholipid-anchored adhesion molecules, such as the Dictyostelium receptor gp80, interact with the cytoskeleton. gp80 has been found to form adhesion complexes from raftlike membrane domains, which can be isolated as a Triton X-100-insoluble floating fraction (TIFF). We report here that the actin-binding protein ponticulin mediates TIFF-cytoskeleton interactions. Analysis of gp80-null cells revealed that these interactions were minimal in the absence of gp80. During development, gp80 was required to enhance these interactions as its adhesion complexes assembled. Whereas ponticulin and gp80 could partition independently into TIFF, gp80 was shown to recruit ponticulin to cell-cell contacts and to increase its partitioning into TIFF. However, these proteins did not co-immunoprecipitate. Furthermore, sterol sequestration abrogated the association of ponticulin with TIFF without affecting gp80, suggesting that sterols may mediate the interactions between ponticulin and gp80. In ponticulin-null cells, large gp80 adhesion complexes assembled in the absence of ponticulin despite the lack of cytoskeleton association. We propose that such nascent gp80 adhesion complexes produce expanded raftlike domains that recruit ponticulin and thereby establish stable cytoskeleton interactions to complete the assembly process.

Disruption of the gene encoding the cell adhesion molecule DdCAD-1 leads to aberrant cell sorting and cell-type proportioning during Dictyostelium development.

The cadA gene in Dictyostelium encodes the Ca2+-dependent cell adhesion molecule DdCAD-1, which is expressed soon after the initiation of development. To investigate the biological role of DdCAD-1, the cadA gene was disrupted by homologous recombination. The cadA-null cells showed a 50% reduction in EDTA-sensitive cell adhesion. The remaining EDTA-sensitive adhesion sites were resistant to dissociation by anti-DdCAD-1 antibody, suggesting that they were distinct adhesion sites. Cells that lacked DdCAD-1 were able to complete development and form fruiting bodies. However, they displayed abnormal slug morphology and culmination was delayed by approximately 6 hours. The yield of spores was reduced by approximately 50%. The proportion of prestalk cells in cadA(-) slugs showed a 2.5-fold increase over the parental strain. When cadA(-) cells were transfected with pcotB::GFP to label prespore cells, aberrant cell-sorting patterns in slugs became apparent. When mutant prestalk cells were mixed with wild-type prespore cells, mutant prestalk cells were unable to return to the anterior position of chimeric slugs, suggesting defects in the sorting mechanism. The wild-type phenotype was restored when cadA(-) cells were transfected with a cadA-expression vector. These results indicate that, in addition to cell-cell adhesion, DdCAD-1 plays a role in cell type proportioning and pattern formation.