Extended neck regions stabilize tetramers of the receptors DC-SIGN and DC-SIGNR

The human cell surface receptors DC-SIGN (dendritic cell-specific intercellular adhesion molecule-grabbing nonintegrin) and DC-SIGNR (DC-SIGN-related) bind to oligosaccharide ligands found on human tissues as well as on pathogens including viruses, bacteria, and parasites. The extracellular portion of each receptor contains a membrane-distal carbohydrate-recognition domain (CRD) and forms tetramers stabilized by an extended neck region consisting of 23 amino acid repeats. Cross-linking analysis of full-length receptors expressed in fibroblasts confirms the tetrameric state of the intact receptors. Hydrodynamic studies on truncated receptors demonstrate that the portion of the neck of each protein adjacent to the CRD is sufficient to mediate the formation of dimers, whereas regions near the N terminus are needed to stabilize the tetramers. Some of the intervening repeats are missing from polymorphic forms of DC-SIGNR. Two different crystal forms of truncated DC-SIGNR comprising two neck repeats and the CRD reveal that the CRDs are flexibly linked to the neck, which contains alpha-helical segments interspersed with non-helical regions. Differential scanning calorimetry measurements indicate that the neck and CRDs are independently folded domains. Based on the crystal structures and hydrodynamic data, models for the full extracellular domains of the receptors have been generated. The observed flexibility of the CRDs in the tetramer, combined with previous data on the specificity of these receptors, suggests an important role for oligomerization in the recognition of endogenous glycans, in particular those present on the surfaces of enveloped viruses recognized by these proteins.     

Direct measurements of multiple adhesive alignments and unbinding trajectories between cadherin extracellular domains

Direct measurements of the interactions between antiparallel, oriented monolayers of the complete extracellular region of C-cadherin demonstrate that, rather than binding in a single unique orientation, the cadherins adhere in three distinct alignments. The strongest adhesion is observed when the opposing extracellular fragments are completely interdigitated. A second adhesive alignment forms when the interdigitated proteins separate by 70 +/- 10 A. A third complex forms at a bilayer separation commensurate with the approximate overlap of cadherin extracellular domains 1 and 2 (CEC1-2). The locations of the energy minima are independent of both the surface density of bound cadherin and the stiffness of the force transducer. Using surface element integration, we show that two flat surfaces that interact through an oscillatory potential will exhibit discrete minima at the same locations in the force profile measured between hemicylinders covered with identical materials. The measured interaction profiles, therefore, reflect the relative separations at which the antiparallel proteins adhere, and are unaffected by the curvature of the underlying substrate. The successive formation and rupture of multiple protein contacts during detachment can explain the observed sluggish unbinding of cadherin monolayers. Velocity-distance profiles, obtained by quantitative video analysis of the unbinding trajectory, exhibit three velocity regimes, the transitions between which coincide with the positions of the adhesive minima. These findings suggest that cadherins undergo multiple stage unbinding, which may function to impede adhesive failure under force.     

The minimal essential unit for cadherin-mediated intercellular adhesion comprises extracellular domains 1 and 2

N-cadherin comprises five homologous extracellular domains, a transmembrane, and a cytoplasmic domain. The extracellular domains of N-cadherin play important roles in homophilic cell adhesion, but the contribution of each domain to this phenomenon has not been fully evaluated. In particular, the following questions remain unanswered: what is the minimal domain combination that can generate cell adhesion, how is domain organization related to adhesive strength, and does the cytoplasmic domain serve to facilitate extracellular domain interaction? To address these issues, we made serial constructs of the extracellular domains of N-cadherin and produced various cell lines to examine adhesion properties. We show that the first domain of N-cadherin alone on the cell surface fails to generate adhesive activity and that the first two domains of N-cadherin form the "minimal essential unit" to mediate cell adhesion. Cell lines expressing longer extracellular domains or N-cadherin wild type cells formed larger cellular aggregates than those expressing shorter aggregates. However, adhesion strength, as measured by a shearing test, did not reveal any differences among these aggregative cell lines, suggesting that the first two domains of N-cadherin cells generate the same strength of adhesive activity as longer extracellular domain cells. Furthermore, truncations of the first two domains of N-cadherin are also sufficient to form cisdimerization at an adhesive junction. Our findings suggest that the extracellular domains of N-cadherin have distinct roles in cell adhesion, i.e. the first two domains are responsible for homophilic adhesion activity, and the other domains promote adhesion efficiency most likely by positioning essential domains relatively far out from the cell surface.     

The role of extracellular and cytoplasmic splice domains of alpha7-integrin in cell adhesion and migration on laminins

The major laminin-binding integrin of skeletal, smooth, and heart muscle is alpha7beta1-integrin, which is structurally related to alpha6beta1. It occurs in three cytoplasmic splice variants (alpha7A, -B, and -C) and two extracellular forms (X1 and X2) which are developmentally regulated and differentially expressed in skeletal muscle. Previously, we have shown that ectopic expression of the alpha7beta-integrin splice variant in nonmotile HEK293 cells specifically induced cell locomotion on laminin-1 but not on fibronectin. To investigate the specificity and the mechanism of the alpha7-mediated cell motility, we expressed the three alpha7-chain cytoplasmic splice variants, as well as alpha6A- and alpha6B-integrin subunits in HEK293 cells. Here we show that all three alpha7 splice variants (containing the X2 domain), as well as alpha6A and alpha6B, promote cell attachment and stimulate cell motility on laminin-1 and its E8 fragment. Deletion of the cytoplasmic domain (excluding the GFFKR consensus sequence) from alpha7B resulted in a loss of the motility-enhancing effect. On laminin-2/4 (merosin), the predominant isoform in mature skeletal muscle, only alpha7-expressing cells showed enhanced motility, whereas cells transfected with alpha6A and alpha6B neither attached nor migrated on laminin-2. Adhesion of alpha7-expressing cells to both laminin-1 and laminin-2 was specifically inhibited by a new monoclonal antibody (6A11) specific for alpha7. Expression of the two extracellular splice variants alpha7X1 and alpha7X2 in HEK293 cells conferred different motilities on laminin isoforms: Whereas alpha7X2B promoted cell migration on both laminin-1 and laminin-2, alpha7X1B supported motility only on laminin-2 and not on laminin-1, although both X1 and X2 splice variants revealed similar adhesion rates to laminin-1 and -2. Fluorescence-activated cell sorter analysis revealed a dramatic reduction of surface expression of alpha6-integrin subunits after alpha7A or -B transfection; also, surface expression of alpha1-, alpha3-, and alpha5-integrins was significantly reduced. These results demonstrate selective responses of alpha6- and alpha7-integrins and of the alpha7 splice variants to laminin-1 and -2 and indicate differential roles in laminin-controlled cell adhesion and migration.     

Geometry of phase-separated domains in phospholipid bilayers by diffraction-contrast electron microscopy.

The sizes and shapes of solidus (gel) phase domains in the hydrated molecular bilayers of dilauroylphosphatidylcholine/dipalmitoylphasphatidylcholine (DLPC/DPPC) (1:1) and phosphatidylserine (PS)/DPPC (1:2) are visualized directly by low dose diffraction-contrast electron microscopy. The temperature and humidity of the bilayers are controlled by an environmental chamber set in an electron microscope. The contrast between crystalline domains is enhanced by electron optical filtering of the diffraction patterns of the bilayers. The domains are seen as a patchwork in the plane of the bilayer, with an average width of 0.2-0.5 micrometer. The percentage of solidus area measured from diffraction-contrast micrographs at various temperatures agrees in general with those depicted by known phase diagrams. The shape and size of the domains resemble those seen by freeze-fracture in multilamellar vesicles. Temperature-related changes in domain size and in phase boundary per unit area are more pronounced in the less miscible DLPC/DPPC mixture. No significant change in these geometric parameters with temperature is found in the PS/DPPC mixture. Mapping domains by their molecular diffraction signals not only verifies the existance of areas of different molecular packing during phase separation but also provides a quantitative measurement of structural boundaries and defects in lipid bilayers.     

Regulation of cell-substrate adhesion by proteoglycans immobilized on extracellular substrates

We have demonstrated previously that chick embryo fibroblasts synthesize and secrete a large chondroitin sulfate proteoglycan (designated PG-M) that binds to fibronectin. We now report the possibility that PG-M interactions with cell surfaces can modulate cell-substrate adhesion. When PG-M was added to the medium, various types of trypsinized cells failed to adhere not only to fibronectin-coated substrates but also to collagen- or vitronectin-coated substrates. Adhesion of the cells to laminin or glycyl-arginyl-glycyl-aspartyl-serine derivatized serum albumin (arginyl-glycyl-aspartic acid-containing molecules with no capacity to bind PG-M) was also inhibited by PG-M. Treatment of the proteoglycan with either proteolytic enzymes or chondroitinase abolished its inhibitory effects on the cell adhesion. These results suggest that direct binding between PG-M and fibronectin, if any, is not a cause of the inhibition by PG-M and that only the proteoglycan form is responsible for the activity. When the immobilization of added PG-M to available plastic surfaces of coated dishes was blocked by pretreating the dishes with serum albumin, the inhibitory effect of PG-M was abolished, suggesting that the immobilized fraction of PG-M can act as a cell adhesion inhibitor. In immobilized form, both cartilage chondroitin sulfate proteoglycan (designated PG-H) and chondroitin sulfate-derivatized serum albumin also inhibited cell adhesion. In contrast, heparan sulfate proteoglycan form LD and heparan sulfate-derivatized serum albumin had far lower inhibitory activities, indicating that the active site for the interaction between cells and PG-M is on the chondroitin sulfate chains.     

Different extracellular domains of the neural cell adhesion molecule (N- CAM) are involved in different functions

The neural cell adhesion molecule (N-CAM) engages in diverse functional roles in neural cell interactions. Its extracellular part consists of five Ig-like domains and two fibronectin type III homologous (type III) repeats. To investigate the functional properties of the different structural domains of the molecule in cell interactions and signal transduction to the cell interior, we have synthesized, in a bacterial expression system, the individual domains and tandem sets of individual domains as protein fragments. These protein fragments were tested for their capacity to influence adhesion and spreading of neuronal cell bodies, promote neurite outgrowth, and influence cellular migration patterns from cerebellar microexplants in vitro. Ig-like domains I and II and the combined type III repeats I-II were most efficient for adhesion of neuronal cell bodies, when coated as substrates. Neurite outgrowth was best on the substrate-coated combined type III repeats I- II, followed by the combined Ig-like domains I-V and Ig-like domain I. Spreading of neuronal cell bodies was best on substrate-coated combined type III repeats I-II, followed by Ig-like domain I and the combined Ig- like domains I-V. The cellular migration pattern from cerebellar microexplant cultures plated on a mixture of laminin and poly-L-lysine was modified by Ig-like domains I, III, and IV, while Ig-like domains II and V and the combined type III repeats I-II did not show significant modifications, when added as soluble fragments. Outgrowth of astrocytic processes from the explant core was influenced only by Ig- like domain I. Metabolism of inositol phosphates was strongly increased by Ig-like domain I and less by the Ig-like domains II, III, IV, and V, and not influenced by the combined type III repeats I-II. Intracellular concentrations of Ca2+ and pH values were increased only by the Ig-like domains I and II. Intracellular levels of cAMP and GMP were not influenced by any protein fragment. These experiments indicate that different domains of N-CAM subserve different functional roles in cell recognition and signal transduction, and are functionally competent without nervous system-derived carbohydrate structures.     

Limitation of cell adhesion by the elasticity of the extracellular matrix

Cell/matrix adhesions are modulated by cytoskeletal or external stresses and adapt to the mechanical properties of the extracellular matrix. We propose that this mechanosensitivity arises from the activation of a mechanosensor located within the adhesion itself. We show that this mechanism accounts for the observed directional growth of focal adhesions and the reduction or even cessation of their growth when cells adhere to a soft extracellular matrix. We predict quantitatively that both the elasticity and the thickness of the matrix play a key role in the dynamics of focal adhesions. Two different types of dynamics are expected depending on whether the thickness of the matrix is of order of or much larger than the adhesion size. In the latter situation, we predict that the adhesion region reaches a saturation size that can be tuned by the mechanical properties of the matrix.     

The evolution of extracellular fibrillins and their functional domains

Fibrillins constitute the major backbone of multifunctional microfibrils in elastic and non-elastic extracellular matrices, and are known to interact with several binding partners including tropoelastin and integrins. Here, we study the evolution of fibrillin proteins. Following sequence collection from 39 organisms representative of the major evolutionary groups, molecular evolutionary genetics and phylogeny inference software were used to generate a series of evolutionary trees using distance-based and maximum likelihood methods. The resulting trees support the concept of gene duplication as a means of generating the three vertebrate fibrillins. Beginning with a single fibrillin sequence found in invertebrates and jawless fish, a gene duplication event, which coincides with the appearance of elastin, led to the creation of two genes. One of the genes significantly evolved to become the gene for present-day fibrillin-1, while the other underwent evolutionary changes, including a second duplication, to produce present-day fibrillin-2 and fibrillin-3. Detailed analysis of several sequences and domains within the fibrillins reveals distinct similarities and differences across various species. The RGD integrin-binding site in TB4 of all fibrillins is conserved in cephalochordates and vertebrates, while the integrin-binding site within cbEGF18 of fibrillin-3 is a recent evolutionary change. The proline-rich domain in fibrillin-1, glycine-rich domain in fibrillin-2 and proline-/glycine-rich domain in fibrillin-3 are found in all analyzed tetrapod species, whereas it is completely replaced with an EGF-like domain in cnidarians, arthropods, molluscs and urochordates. All collected sequences contain the first 9-cysteine hybrid domain, and the second 8-cysteine hybrid domain with exception of arthropods containing an atypical 10-cysteine hybrid domain 2. Furin cleavage sites within the N- and C-terminal unique domains were found for all analyzed fibrillin sequences, indicating an essential role for processing of the fibrillin pro-proteins. The four cysteines in the unique N-terminus and the two cysteines in the unique C-terminus are also highly conserved.     

Substratum adhesion and gliding in a diatom are mediated by extracellular proteoglycans

Diatoms are unicellular microalgae encased in a siliceous cell wall, or frustule. Pennate diatoms, which possess bilateral symmetry, attach to the substratum at a slit in the frustule called the raphe. These diatoms not only adhere, but glide across surfaces whilst maintaining their attachment, secreting a sticky mucilage that forms a trail behind the gliding cells. We have raised monoclonal antibodies to the major cell surface proteoglycans of the marine raphid diatom Stauroneis decipiens Hustedt. The antibody StF.H4 binds to the cell surface, in the raphe and to adhesive trails and inhibits the ability of living diatoms to adhere to the substratum and to glide. Moreover, StF.H4 binds to a periodate-insensitive epitope on four frustule-associated proteoglycans (relative molecular masses 87, 112, and >200 kDa). Another monoclonal antibody, StF.D5, binds to a carbohydrate epitope on the same set of proteoglycans, although the antibody binds only to the outer surface of the frustule and does not inhibit cell motility and adhesion. Received: 2 December 1996 / Accepted: 6 March 1997     

Linker length dependent binding of a focal adhesion kinase derived peptide to the Src SH3-SH2 domains

The interaction between a peptide encompassing the SH3 and SH2 binding motifs of focal adhesion kinase (FAK) and the Src SH3-SH2 domains has been investigated with NMR spectroscopy and calorimetry. The binding to both motifs is anti-cooperative. Reduction of the long linker connecting the motifs does not lead to cooperativity. Short linkers that do not allow simultaneous intramolecular binding of the peptide to both motifs cause peptide-mediated dimerisation, even with a linker of only three amino acids. The role of the SH3 binding motif is discussed in view of the independent nature of the SH interactions.     

Length of mucin-like domains enhances cell-Ebola virus adhesion by increasing binding probability

The Ebola virus (EBOV) hijacks normal physiological processes by apoptotic mimicry to be taken up by the cell it infects. The initial adhesion of the virus to the cell is based on the interaction between T cell immunoglobulin and mucin domain protein, TIM, on the cell surface and phosphatidylserine (PS) on the viral outer surface. Therefore, it is important to understand the interaction between EBOV and PS and TIM, with selective blocking of the interaction as a potential therapy. Recent experimental studies have shown that for TIM-dependent EBOV entry, a mucin-like domain with a length of at least 120 amino acids is required, possibly because of the increase of area of the PS-coated surface sampled. We examine this hypothesis by modeling the process of TIM-PS adhesion using a coarse-grained molecular model. We find that the strength of individual bound PS-TIM pairs is essentially independent of TIM length. TIMs with longer mucin-like domains collectively have higher average binding strengths because of an increase in the probability of binding between EBOV and TIM proteins. Similarly, we find that for larger persistence length (less flexible), the average binding force decreases, again because of a reduction in the probability of binding.     

Contributions of galectin-3 and -9 to epithelial cell adhesion analyzed by single cell force spectroscopy

Galectins are widely expressed in epithelial tissues and have been implicated in a variety of cellular processes, including adhesion and polarization. Here we studied the contributions of galectins in cell adhesion and cyst formation of Madin-Darby canine kidney cells. Quantitative single cell force spectroscopy and standard adhesion assays were employed to study both early (<2 min) and long term (90 min) adhesion of cells to different extracellular matrix components. Inhibitors were used to examine the contribution of integrins and galectins in general and RNA interference to specifically address the role of two abundantly expressed galectins, galectin-3 and -9. We found that both galectin-3 and -9 were required for optimal long term cell adhesion to both collagen I and laminin-111. Early adhesion to laminin was found to be integrin-independent and was instead mediated by carbohydrate interactions and galectin-3 and -9. The opposite was observed for early adhesion to collagen. Although similar, the contributions of galectin-3 and -9 to adhesion appeared to be by distinct processes. These defects in adhesion of the two galectin knockdown cell lines may underlie the epithelial phenotypes observed in the cyst assays. Our findings emphasize the complex regulation of epithelial cell functions by galectins.     

Reversible and irreversible adhesion of motile Escherichia coli cells analyzed by total internal reflection aqueous fluorescence microscopy

The initial events in bacterial adhesion are often explained as resulting from electrostatic and van der Waals forces between the cell and the surface, as described by DLVO theory (developed by Derjaguin, Landau, Verwey, and Overbeek). Such a theory predicts that negatively charged bacteria will experience greater attraction toward a negatively charged surface as the ionic strength of the medium is increased. In the present study we observed both smooth-swimming and nonmotile Escherichia coli bacteria close to plain, positively, and hydrophobically coated quartz surfaces in high- and low-ionic-strength media by using total internal reflection aqueous fluorescence microscopy. We found that reversibly adhering cells (cells which continue to swim along the surface for extended periods) are too distant from the surface for this behavior to be explained by DLVO-type forces. However, cells which had become immobilized on the surface did seem to be affected by electrostatic interactions. We propose that the "force" holding swimming cells near the surface is actually the result of a hydrodynamic effect, causing the cells to swim at an angle along the glass, and that DLVO-type forces are responsible only for the observed immobilization of irreversibly adhering cells. We explain our observations within the context of a conceptual model in which bacteria that are interacting with the surface may be thought of as occupying one of three compartments: bulk fluid, near-surface bulk, and near-surface constrained. A cell in these compartments feels either no effect of the surface, only the hydrodynamic effect of the surface, or both the hydrodynamic and the physicochemical effects of the surface, respectively.     

Mapping and length measurements of restriction enzyme fragments by electron microscopy.

1. We have mapped by electron microscopy the DNA-fragments formed by the action of the restriction endonuclease from Arthrobacter luteus of phi X 174 replicative form DNA. These fragments were separated by polyacrylamide gel electrophoresis and hybridized to phiX 174 single stranded DNA. The partial duplex molecules were inspected in the electron microscope. In this way the relative order of eleven fragments ranging in size from approximately 100 to 1000 nucleotide pairs has been established and compared with that deduced from reciprocal digestion studies. 2. The measured lengths of the fragments agreed well with the lengths found by gel electrophoresis. 3. The purity of the isolated fragments was checked. Most of the contaminating fragments derive from nearest neighbours in the preparative polyacrylamide gels.     

Isolation of human basal keratinocytes by selective adhesion to extracellular matrix proteins

Epidermal human cells (keratinocytes) differently interact with extracellular matrix proteins of the skin basal membrane depending on the stages of their differentiation. The pool of basal keratinocytes commonly includes stem cells and transient amplifying cells. They directly attach to the skin basal membrane. Keratinocytes change their adhesive properties during differentiation, lose direct interaction with the basal membrane and move to suprabasal epidermal strata. From this, it is suggested that basal and primarily stem cells can be isolated from a heterogenous keratinocyte population due to their selective adhesion to the extracellular matrix proteins. In the current study, we analysed the specificity of interaction between primary keratinocytes and extracellular matrix proteins (collagens of I and IV types, laminin-2/4, fibronectin and matrigel). We have demonstrated that the basal keratinocytes extracted from the skin have different adhesive abilities. The rapidly spreading cells usually interacted with collagen and fibronectin rather that with laminin-2/4 or matrigel. The majority of these cells being represented by basal keratinocytes. Our data demonstrate that the applied method of keratinocyte selection may be directed for precise isolation of skin stem from a common cell population.     

Galectin-9 suppresses tumor metastasis by blocking adhesion to endothelium and extracellular matrices

We previously described an inverse correlation between galectin-9 (Gal-9) expression and metastasis in patients with malignant melanoma and breast cancer. This study verified the ability of Gal-9 to inhibit lung metastasis in experimental mouse models using highly metastatic B16F10 melanoma and Colon26 colon cancer cells. B16F10 cells transfected with a secreted form of Gal-9 lost their metastatic potential. Intravenous Gal-9 administration reduced the number of metastases of both B16F10 and Colon26 cells in the lung, indicating that secreted Gal-9 suppresses metastasis. Analysis of adhesive molecule expression revealed that B16F10 cells highly express CD44, integrin alpha1, alpha 4, alpha V, and beta1, and that Colon26 cells express CD44, integrin alpha2, alpha 5, alpha V, and beta1, suggesting that Gal-9 may inhibit the adhesion of tumor cells to vascular endothelium and the extracellular matrix (ECM) by binding to such adhesive molecules. Indeed, Gal-9 suppressed the binding of hyaluronic acid to CD44 on both B16F10 and Colon26 cells, and also suppressed the binding of vascular cell adhesion molecule-1 to very late antigen-4 on B16F10 cells. Furthermore, Gal-9 inhibited the binding of tumor cells to ECM components, resulting in the suppression of tumor cell migration. The present results suggest that Gal-9 suppresses both attachment and invasion of tumor cells by inhibiting the binding of adhesive molecules on tumor cells to ligands on vascular endothelium and ECM.     

The organization and genetics of rDNA length variants in peas

There is extensive repeat length variation in pea rDNA. Many lines of pea have more than one repeat length class, and those with two rDNA repeat length classes are the most common. No interspersion of rDNA repeat length classes has been found. The rDNA length classes show simple Mendelian inheritance. Length variation of pea rDNA is organized between long tandem arrays of rDNA repeat. Studies of trisomies have shown that rDNA length class dosage correlates with chromosome dosage.