Interactions of Schwann cells with neurites and with other Schwann cells involve the calcium-dependent adhesion molecule,N-cadherin

During embryogenesis, Schwann cells interact with axons and other Schwann cells, as they migrate, ensheath axons, and participate in organizing peripheral nervous tissues. The experiments reported here indicate that the calcium-dependent molecule, N-cadherin, mediates adhesion of Schwann cells to neurites and to other Schwann cells. Cell cultures from chick dorsal root ganglia and sciatic nerves were maintained in media containing either 2mM Ca⁺⁺ or 0.2 mM Ca⁺⁺, a concentration that inactivates calcium-dependent cadherins. When the leading lamellae of Schwann cells encountered migrating growth cones in medium with 2 mM Ca⁺⁺, they usually remained extended, and the growth cones often advanced onto the Schwann cell upper surface. In the low Ca⁺⁺ medium, the frequency of withdrawal of the Schwann cell lamella after contact with a growth cone was much greater, and withdrawal was the most common reaction to growth cone contact in medium with 2 mM Ca⁺⁺ and anti-N-cadherin. Similarly, when motile leading margins of two Schwann cells touched in normal Ca⁺⁺ medium, they often formed stable areas of contact. N-cadherin and vinculin were co-concentrated at these contact sites between Schwann cells. However, in low Ca⁺⁺ medium or in the presence of anti-N-cadherin, interacting Schwann cells usually pulled away from each other in a behavior reminiscent of contact inhibition between fibroblasts. In cultures of dissociated cells in normal media, Schwann cells frequently were aligned along neurites, and ultrastructural examination showed extensive close apposition between plasma membranes of neurites and Schwann cells. When dorsal root ganglia explants were cultured with normal Ca⁺⁺, Schwann cells migrated away from the explants in close association with extending neurites. All these interactions were disrupted in media with 0.2 mM Ca⁺⁺. Alignment of Schwann cells along neurites was infrequent, as were extended close apposition between axonal and Schwann cell plasma membranes. Finally, migration of Schwann cells from ganglionic explants was reduced by disruption of adhesive contact with neurites. The addition of antibodies against N-cadherin to medium with normal Ca⁺⁺ levels had similar effects as lowering the Ca⁺⁺ concentration, but antibodies against the neuronal adhesive molecule, L1, had no effects on interactions between Schwann cells and neurites.     

Interactions of Schwann cells with neurites and with other Schwann cells involve the calcium-dependent adhesion molecule, N-cadherin.

During embryogenesis, Schwann cells interact with axons and other Schwann cells, as they migrate, ensheath axons, and participate in organizing peripheral nervous tissues. The experiments reported here indicate that the calcium-dependent molecule, N-cadherin, mediates adhesion of Schwann cells to neurites and to other Schwann cells. Cell cultures from chick dorsal root ganglia and sciatic nerves were maintained in media containing either 2 mM Ca++ or 0.2 mM Ca++, a concentration that inactivates calcium-dependent cadherins. When the leading lamellae of Schwann cells encountered migrating growth cones in medium with 2 mM Ca++, they usually remained extended, and the growth cones often advanced onto the Schwann cell upper surface. In the low Ca++ medium, the frequency of withdrawal of the Schwann cell lamella after contact with a growth cone was much greater, and withdrawal was the most common reaction to growth cone contact in medium with 2 mM Ca++ and anti-N-cadherin. Similarly, when motile leading margins of two Schwann cells touched in normal Ca++ medium, they often formed stable areas of contact. N-cadherin and vinculin were co-concentrated at these contact sites between Schwann cells. However, in low Ca++ medium or in the presence of anti-N-cadherin, interacting Schwann cells usually pulled away from each other in a behavior reminiscent of contact inhibition between fibroblasts. In cultures of dissociated cells in normal media, Schwann cells frequently were aligned along neurites, and ultrastructural examination showed extensive close apposition between plasma membranes of neurites and Schwann cells. When dorsal root ganglia explants were cultured with normal Ca++, Schwann cells migrated away from the explants in close association with extending neurites. All these interactions were disrupted in media with 0.2 mM Ca++. Alignment of Schwann cells along neurites was infrequent, as were extended close apposition between axonal and Schwann cell plasma membranes. Finally, migration of Schwann cells from ganglionic explants was reduced by disruption of adhesive contact with neurites. The addition of antibodies against N-cadherin to medium with normal Ca++ levels had similar effects as lowering the Ca++ concentration, but antibodies against the neuronal adhesive molecule, L1, had no effects on interactions between Schwann cells and neurites.     

Nerve growth factor enhances expression of neuron-glia cell adhesion molecule in PC12 cells

The neuron-glia cell adhesion molecule (Ng-CAM) has been identified in mammalian brain tissue and PC12 pheochromocytoma cells as Mr 200,000 and Mr 230,000 species, respectively. When PC12 cells were treated with nerve growth factor (NGF), the amount of Ng-CAM at the cell surface was increased approximately threefold, whereas the amount of the neural cell adhesion molecule (N-CAM) remained unchanged. An NGF-inducible large external glycoprotein (NILE) has been previously identified by its enhanced expression in NGF-treated PC12 cells. Ng-CAM and NILE are similar in molecular weight, expression during development, and responsiveness to NGF in PC12 cells, suggesting that the two molecules are related. In addition, antibodies to Ng-CAM and NILE cross-reacted and the molecules had similar peptide maps after limited proteolysis. Moreover, antibodies to Ng-CAM inhibited fasciculation of neurites, a functional property shared with NILE. The results show that cell adhesion molecules can respond selectively to growth factors and suggest that NILE is, in fact, mammalian Ng-CAM.     

Regulation of neural cell adhesion molecule expression on cultured mouse Schwann cells by nerve growth factor.

Schwann cells from early postnatal mouse sciatic nerve were obtained as a homogenous population and shown by indirect immunofluorescence to express the neural cell adhesion molecules L1, N-CAM and J1 and their common carbohydrate epitope L2/HNK-1. L1 and N-CAM are synthesized in molecular forms that are slightly different from those expressed by small cerebellar neurons or astrocytes. As in astrocytes, the J1 antigen is expressed by Schwann cells in multiple forms generally ranging from 160 to 230 kd in the reduced state. J1 is secreted by Schwann cells in a 230-kd mol. wt form. Expression of L1 by Schwann cells can be regulated by nerve growth factor (NGF). L1 expression on the cell surface is increased 1.6-fold in the presence of NGF after 3 days of maintenance in vitro and 3-fold after 16 days. NGF does not change expression of N-CAM. The glia-derived neurite-promoting factor (GdNPF) increases L1 expression by a factor of 1.9 and decreases N-CAM expression by a factor of 0.4 after 3 days in vitro. J1 expression on Schwann cell surfaces remains unchanged in the presence of NGF or GdNPF. Antibodies to NGF abolish the influence of NGF on L1 expression. Addition of NGF antibodies to the Schwann cell cultures without exogenously added NGF decreases L1 expression, indicating that Schwann cells secrete NGF that may influence L1 expression by an autocrine mechanism. Our experiments show for the first time that cell adhesion molecule expression on a non-neuronal cell, the Schwann cell, can be directly regulated by the neurotrophic factor NGF. These observations indicate a considerable degree of 'plasticity' of peripheral glia in regulating cell adhesion molecule expression.     

Interplay between adhesion turnover and cytoskeleton dynamics in the control of growth cone migration

The migration of neuronal growth cones, driving axon extension, is a fascinating process which has been subject of intense investigation over several decades. Many of the key underlying molecules, in particular adhesion proteins at the cell membrane which allow for target recognition and binding, and cytoskeleton filaments and motors which power locomotion have been identified. However, the precise mechanisms by which growth cones coordinate, in time and space, the transmission of forces generated by the cytoskeleton to the turnover of adhesion proteins are still partly unresolved. To get a better grasp at these processes, we put here in relation the turnover rate of ligand/receptor adhesions and the degree of mechanical coupling between cell adhesion receptors and the actin rearward flow. These parameters were obtained recently for N-cadherin and IgCAM based adhesions using ligand-coated microspheres in combination with optical tweezers and photo-bleaching experiments. We show that the speed of growth cone migration requires both a fairly rapid adhesion dynamics and a strong physical connection between adhesive sites and the cytoskeleton.Key words: actin retrograde flow, molecular clutch, myosin, N-cadherin, IgCAMGrowth cones are motile structures at the distal extremity of axons responsible for pathfinding and neurite extension during nervous system development and repair (Fig. 1A). Growth cone advance relies on two coupled processes. First, an internal dynamics of the cytoskeletal network, with actin polymerization occurring at the leading edge, depolymerization in the central region, and myosin activity pulling on lamellipodial actin filaments.¹ These integrated mechanisms altogether result in a continuous retrograde flow of actin (Fig. 1B). This flow provides the mechanical tension that drives axonal extension, through a connection to the dynamic array of microtubules that fills the axon and invades the growth cone central domain.² Second, there is repeated formation and dissociation of transient contacts between growth cones and the extracellular matrix or adjacent cells. These contacts are mediated by trans-membrane cell adhesion molecules (CAMs), e.g. integrins,³ immunoglobulin CAMs (IgCAMs)^4,5 and cadherins,^6,7 which form specific ligand/receptor bonds with variable lifetimes. A still open question is how these two processes, i.e. actin flow and adhesion dynamic, are coordinated at the growth cone level and contribute to set migration speed. A thorough understanding of these mechanisms is important both from a fundamental perspective and for the design of new compounds to foster axon regeneration after injury.Open in a separate windowFigure 1Growth cone advance and actin flow. (A) Growth cone from a 2 DIV rat hippocampal neuron plated on N-cadherin coated glass. This growth cone moved forward at a speed of about 1 µm/min (B) Raw fluorescence image of transfected actin-GFP. (C) Sequential actin-GFP images were subtracted, giving rise to intensity variations that display the movement of newly assembled actin (black). Note the rapid retrograde movement of actin spots (arrowheads), at a velocity of several µm/min.The coupling between actin-based motility and substrate adhesion has been shown for certain adhesion molecules such as NCAM and N-cadherin to involve a “molecular clutch” (Fig. 2). This mechanism implies a direct transmission of traction forces from the cytoskeleton to the substrate through a strong physical connection between the actin flow and ligand-bound adhesion receptors.^8,9 The connection is likely provided by adaptor proteins that can make transient bridges between actin filaments and the cytoplasmic domain of adhesion molecules, i.e. α-and β-catenin in the case of N-cadherin,¹⁰ ankyrin and ezrin in the case of IgCAMs such as L1.^11–14 These purely mechanical connections can also be accompanied by signalling events such as Rac-1 activation by N-cadherin liganding¹⁵ and phosphorylation of the L1 intracellular tail that regulates binding to ankyrin.^11,12 When only few molecular bonds are formed, e.g. at low ligand density, coupling to the actin flow is not strong enough, resulting in “slippage.” In this process, transient bonds can be formed and broken repeatedly between ligand-occupied adhesion receptors and the actin network. This is how the speed of growth cone translocation usually reaches at most 1 µm/min, whereas the internal actin flow rate proceeds at a rate of several µm/min (Fig. 1A and B). Such slippage is best demonstrated by the use of optical tweezers to impose low forces on ligand-coated microspheres presented to the growth cone dorsal surface (Fig. 3A). Beads tend to move rearward as they couple to the actin flow, and then suddenly snap back into the trap center, when receptor-cytoskeleton bonds break¹⁶ (the force of optical tweezers is usually not enough to rupture ligand-receptor bonds, which remain intact at the cell surface). Thus, a step in which a nucleating cluster of adhesion receptors recruits a minimal number of intracellular partners allowing coupling to the actin flow, can be a rate-limiting factor in growth cone progression.Open in a separate windowFigure 2Molecular components involved in growth cone migration. (A) Top view diagram showing filopodia which sense the environment, a flat lamellipodium which is the site of actin dynamics and the thicker central domain and axon which contain dynamic microtubules. The plus signs are sites of actin polymerization and the minus signs indicate actin depolymerization. (B) Side view showing the life cycle of ligand/receptor adhesions.Open in a separate windowFigure 3Optical tweezers and FRAP experiments to measure ligand/receptor and receptor/cytoskeleton dynamics. (A) Optical tweezers experiments performed on ligand-coated beads placed on the growth cone dorsal surface.¹⁶ (B) The distance traveled rearward with respect to the trap center is measured. A 2 min trajectory is indicated in red. A pooled parameter called coupling index taking into account the latency for bead escape, as well as the mean velocity and lateral diffusion of the bead, measures the strength of receptor/cytoskeleton interactions. (C) FRAP experiments on membrane GFP-tagged molecules accumulated at ligand-coated microspheres having sedimented on growth cones. The fluorescence intensity is normalized to represent the receptor enrichment level at the bead contact. (D) The recovered intensity is fit by a diffusion/reaction model, which yields a collective equilibrium turnover rate of ligand/receptor bonds. In red is the average of a series of individual curves (grey).In contrast, when strong connection is formed and if the substrate is resistant enough, then the molecular clutch engages and the cell reacts. In growth cones from Aplysia bag cells, forces were imposed on microspheres coated with ApCAM (the homolog of vertebrate NCAM) using a microneedle to locally block the retrograde actin flow. This was systematically followed by a protrusion of the microtubule-rich central domain towards those stiff contacts and forward expansion of the actin-rich lamellipodium.⁹ These phenomena were later shown to be controlled by a src protein kinase.¹⁷ In the case of rat hippocampal neurons, a dramatic accumulation of actin at N-cadherin coated microspheres is observed when the latter are restrained from moving rearward by a microneedle.¹⁶ This phenomenon is mediated by a connection between N-cadherin and α-catenin, likely triggering local actin polymerization. By careful analysis of the bead trajectories at varying ligand densities and computation of the latency for bead escape when the optical trap is applied continuously, one can extract a quantitative index of receptor-cytoskeleton coupling (Fig. 3B). Overall, a strong correlation was observed between such coupling index and the velocity of growth cone migration on N-cadherin substrates, both by varying N-cadherin ligand density and by expressing mutated N-cadherin molecules, supporting the clutch concept.¹⁶ This mechanism is consistent with in vivo experiments showing that overexpression of the N-cadherin intracellular tail in retinal ganglion cells results in severely impaired axon outgrowth.¹⁸ As a negative example of the clutch model, beads coated with fibronectin (our unpublished data) or anti-α1 integrin antibodies³ couple weakly to the actin flow in growth cones while, in parallel, the migration of growth cones on fibronectin- or collagen-coated substrates is rather limited.^6,19Molecular mechanisms parallel to the “clutch” can also be involved in growth cone migration. For example, IgCAM adhesions can not only couple to the rearward actin flow but also to static components of the cytoskeleton. Indeed, a 30% fraction of TAG-1 or anti-L1 coated beads can stay immobile on the growth cone surface.^12,20 These contrasting behaviors are likely mediated by interactions between the IgCAM intracellular domain and different binding partners (ankyrin vs. ERM),¹³ and may be responsible for the pauses which alternate with phases of growth cone advance. Also, homophilic adhesions between molecules of cadherin-11 couple very weakly to the actin flow, but promote substantial growth cone migration when cadherin-11 is presented as a substrate. This effect seems to be mediated by an independent interaction with the FGF receptor, which triggers actin dynamic through a signaling cascade.^21,22As growth cones migrate, adhesion sites must be recycled at a rate that somehow matches the speed of migration. Adhesion turnover can be schematically decomposed in several sequential phases (Fig. 2B). (1) Initiation of a first single ligand/receptor bond powered by membrane diffusion²³ and followed by trapping through a key/lock interaction; (2) Formation of small adhesion clusters through the recruitment of more ligand/receptor pairs, and possibly stabilized by cis-oligomerization (cadherins through the same interface as the trans-dimer, IgCAMs through FnIII domains). These clusters might form very transiently and serve as sites of actin recruitment, as demonstrated for N-cadherin;¹⁶ (3) contact maturation and possible reinforcement by connection to the cytoskeleton (as demonstrated for integrins in fibroblasts²⁴); (4) Adhesion rupture, which can proceed through ligand/receptor dissociation triggered by cytoskeleton tension. Indeed, the intrinsic lifetime of ligand/receptor bonds such as cadherins, is sensitive to the mechanical force applied on them.²⁵ Furthermore, the loosening of receptor/cytoskeketon connections can cause inside-out rupture of ligand/receptor bonds. This was demonstrated for fibronectin/integrin interactions by the fact that when fibronectin coated-beads reach the base of a fibroblast lamellipodium, they spontaneously detach from the cell surface.²⁶ In the case of very sticky ligand/receptor interactions such as SynCAM homophilic adhesions,²⁷ this process can actually be a limiting step that slows down growth cone advance. Indeed, SynCAM couples very well to the actin flow, but is unable to support growth cone migration.¹⁶ Finally, adhesion rupture might also proceed through membrane rupture, the adhesion receptors being extracted from the cell membrane and left behind on the substrate (demonstrated for integrins at the tail of fibroblasts²⁸).These basic processes can be accompanied by more complex and active phenomena, e.g. involving forward surface transport as shown for NCAM²⁹ or internal trafficking in the case of L1.³⁰ By interacting with the clathrin adaptor AP-2 through a specific RSLE motif in its intracellular tail, L1 can undergo endocytosis in the central domain and exocytosis at the periphery of the growth cone.³⁰ This mechanism generates a density gradient of L1 molecules which accelerates the formation of bonds with a variety of ligands, including L1 itself. The use of an L1-GFP construct in which the N-terminal GFP could be rapidly cleaved off by thrombin, together with L1-Fc microspheres manipulated by optical tweezers showed that local exocytosis of L1-rich vesicles at the growth cone periphery indeed participates in enhancing the formation of L1 homophilic contacts.³¹ We did not observe such internal traffic for N-cadherin within the growth cone, partly because of a difficulty to introduce a fluorescent protein tag in the ectodomain, which otherwise perturbs the adhesive function. However, the use of an N-cadherin molecule with triple mutation in the juxta-membrane domain that abolishes binding to p120 catenin, involved in the export of N-cadherin to the cell surface, suggested that recycling events might also play a role.³²By measuring the fluorescence recovery after photobleaching (FRAP) of GFP-tagged receptors transiently trapped at ligand-coated microspheres and analyzing the curves using a diffusion/reaction model, we were able to compute the equilibrium turnover rates of ligand/receptor pairs in controlled adhesive contacts involving many simultaneous bonds (Fig. 3C and D). We found that mature L1 homophilic adhesions recycle fast compared to other IgCAMs such as TAG-1/NrCAM adhesions,²⁰ likely owing to the specific internalization motif present in L1. Indeed, the recycling rate was reduced by a factor of 3 after truncation of the L1 intracellular tail, which prevented endocytosis.³¹ N-cadherin homophilic adhesions have an intermediate turnover rate, which is sensitive to the binding to catenin partners.³² Using these measurements as well as data from the literature, we plotted the impact of both receptor-cytoskeleton coupling and adhesion turnover rate on neurite outgrowth (Fig. 4A and B), which is strongly proportional to growth cone velocity.¹⁶ The graphs show that a strong coupling between ligand-occupied receptors and the actin flow is necessary, but not sufficient for neurite extension (Fig. 4C). Another requirement is that the turnover of ligand/receptor adhesions lies in an optimal range: not too high, otherwise bonds detach before coupling can occur, and not too slow either, since sticky bonds which do not rupture paralyze growth cone progression (Fig. 4D). A similar bell-shape curve between the strength of cell-substrate adhesion and cell migration speed was demonstrated for fibroblasts³³ and keratocytes,³⁴ indicating that these coupled mechanisms are fundamental to cell migration. To fully understand the quantitative relationship between adhesion turnover and the clutch process, it would be helpful to add data to this preliminary graph. For example, the extracellular matrix molecule laminin is known to support axon growth very efficiently but, to our knowledge, neither the coupling to the actin flow in growth cones or the adhesive turnover rate of integrins has been evaluated yet. Conversely, NCAM was shown to couple well to the actin flow³⁵ and induce neurite outgrowth,^4,36 but measurements of the lifetime of NCAM homophilic adhesions within growth cones are still lacking.Open in a separate windowFigure 4Impact of ligand/receptor turnover rate and receptor/cytoskeletal coupling on neurite outgrowth. (A and B) Example of 2 DIV rat hippocampal neurons plated on N-cadherin-Fc coated substrate and transfected at 1 DIV with N-cadherin-GFP. (A) DIC image. (B) Fluorescence image. The longest neurite, most likely the axon, is outlined by arrowheads. (C and D) In both graphs, the y-axis represents the longest neurite length after two days plating on ligand-coated glass. (Red) Rat hippocampal neurons transfected with either wild type or mutated N-cadherin molecules, interacting with purified N-cadherin ligands.^16,32 The scale in red intensity represents from dark to light: wild type N-cadherin, N-cadherin deleted of the whole ectodomain, N-cadherin truncated in the C-terminal region binding to β-catenin, N-cadherin with triple mutation in the juxta-membrane domain interacting with p120, and wild type N-cadherin in the presence of cytochalasin D. (Blue) Neurons transfected with either wild type L1 (dark) or L1 truncated in the intracellular tail (light), interacting with purified L1.³¹ Neurite growth on L1 was estimated from references.^7,14,30 (Grey) Interaction between endogenous SynCAM1 molecules expressed on growth cones and SynCAM-Fc ligands coated on microspheres or flat glass.¹⁶ The turnover of SynCAM homophilic interactions was estimated from SynCAM-coated Quantum dots detaching from neurons transfected with SynCAM1.⁴² (Green) Neuroblastoma cells expressing NrCAM-GFP in contact with TAG-1 coated microspheres.²⁰ DRG neurite growth on TAG-1 was taken from reference.⁴³ (Orange) The coupling index was taken from optical tweezers experiments using anti-β1 integrin coated beads interacting with DRG neurons,³ the turnover rate was inferred from FRAP experiments on fibroblast focal contacts⁴⁴ and neurite growth on fibronectin was estimated from reference.¹⁹ We omitted statistics for clarity. The SEM are usually in the order of 5–15% of the mean, for sample sizes of typically 20–30 beads (coupling index and turnover rate) and 40–100 transfected cells (neurite length).One important question is how these observations obtained from simplified in vitro systems using stiff substrates of well-defined geometry coated with specific purified proteins at controlled density, translate to the in vivo situation. There, the 3D substrate is comprised of extracellular matrix and multiple cell types, co-expressing many different CAMs that can bind simultaneously in various stoechiometries and also generating local gradients of chemo-attractant and chemo-repulsive signals.³⁷ Substrate flexibility is probably an important factor, since axons grow more slowly when neurons are plated on a layer of fibroblasts expressing CAMs²¹ than when molecules are immobilized on a substrate.^16,30 This preference for cells to move on stiff substrates, called durotaxis, has been well described for fibroblasts.³⁸ Another specific feature of the in vivo situation is the existence of decision points, often correlated with the presence of guidepost cells where growth cones make a pause and often change shape and reorient before turning to another direction.³⁹ This type of behavior has been successfully mimicked in vitro using artificial guideposts made of fibronectin or laminin coated microspheres.⁴⁰ Whereas growth cones display a fairly continuous displacement on a homogeneous substrate,¹⁶ the presence of these guideposts make growth cones either slow down, pause or even collapse, or conversely accelerate, depending on the CAM grafted on the bead.^40,41 Finally, the shape itself of the growth cone can be an indicator of its motile state:³⁹ this is also true in vitro where small growth cones are often the most rapid whereas large and flat growth cones stay rather immobile. Thus, although the in vivo situation seems at first sight awfully complex, some general trends can be explained given a small number of interacting molecular species and rather simple bio-chemical and mechanical models.In conclusion, the dynamic regulation of growth cone advance can take place at several levels: (1) the actin-associated proteins controlling actin dynamics (nucleation, polymerization, sequestering, branching); (2) the activity of motors pulling on the actin network, generating the retrograde flow; (3) the intracellular adaptor proteins that link actin to the CAMs; (4) the membrane delivery and retrieval of CAMs; (5) the ligand/receptor interaction properties themselves; and (6) the processes regulating microtubule assembly and microtubule/actin interactions at the base of the growth cone. The orchestration in time and space of all these processes generates the movement and reactivity of growth cones necessary to lead axons to their target cells.     

Interplay between adhesion turnover and cytoskeleton dynamics in the control of growth cone migration

The migration of neuronal growth cones, driving axon extension, is a fascinating process which has been subject of intense investigation over several decades. Many of the key underlying molecules, in particular adhesion proteins at the cell membrane which allow for target recognition and binding, and cytoskeleton filaments and motors which power locomotion, have been identified. However, the precise mechanisms by which growth cones coordinate, in time and space, the transmission of forces generated by the cytoskeleton to the turnover of adhesion proteins, are still partly unresolved. To get a better grasp at these processes, we put here in relation the turnover rate of ligand/receptor adhesions and the degree of mechanical coupling between cell adhesion receptors and the actin rearward flow. These parameters were obtained recently for N-cadherin and IgCAM based adhesions using ligand-coated microspheres in combination with optical tweezers and photo-bleaching experiments. We show that the speed of growth cone migration requires both a fairly rapid adhesion dynamics and a strong physical connection between adhesive sites and the cytoskeleton.     

Essential role of SRC suppressed C kinase substrates in Schwann cells adhesion, spreading and migration

Integrin-mediated substrate adhesion of endothelial cells leads to dynamic rearrangement of the actin cytoskeleton. Protein kinase C (PKC) stimulates reorganization of microfilaments and adhesion, while the responses of Schwann cells during adhesion and migration are unknown, so we examined the expression changes of SSeCKS and F-actin in Schwann cells after exposure to fibronectin. Src (sarcoma) suppressed C kinase substrate (SSeCKS) is a PKC substrate that may play an important role in regulating actin cytoskeleton. We found that SSeCKS was localized to focal adhesion sites soon after Schwann cells adhesion and that SSeCKS increased during the process of cell spreading. Using small interfering RNAs specific to SSeCKS, we showed that Schwann cells in which SSeCKS expression was inhibited reduced cellular adhesion, spreading and promoted cellular migration on fibronectin through reorganization of actin stress fibers and blocking formation of focal adhesions. These results demonstrated SSeCKS modulate Schwann cells adhesion, spreading and migration by reorganization of the actin cytoskeleton.     

MicroRNA-29a suppresses the growth,migration, and invasion of lung adenocarcinoma cells by targeting carcinoembryonic antigen-related cell adhesion molecule 6

Carcinoembryonic antigen-related cell adhesion molecule 6 (CEACAM6) is an important regulator of cell adhesion, invasion, and metastasis. The aim of this study was to evaluate the functional roles of CEACAM6 in lung adenocarcinoma and to identify miRNAs that inhibit the growth, migration, and invasion of lung adenocarcinoma cells by targeting CEACAM6. CEACAM6 expression is associated with poor prognosis of patients with lung adenocarcinoma, and CEACAM6 has important functional roles in controlling the growth, migration, and invasion of lung adenocarcinoma cells in vitro and in vivo. Furthermore, miR-29a can suppress the growth, migration, and invasion of lung adenocarcinoma cells by targeting CEACAM6. Therefore, miR-29a/CEACAM6 axis represents a potential therapeutic target for treatment of lung adenocarcinoma.     

A role for the Ca2(+)-dependent adhesion molecule, N-cadherin, in myoblast interaction during myogenesis

The formation of multinucleate skeletal muscle cells (myotubes) is a Ca2(+)-dependent process involving the interaction and fusion of mononucleate muscle cells (myoblasts). Specific cell-cell adhesion precedes lipid bilayer union during myoblast fusion and has been shown to involve both Ca2(+)-independent (CI)2 and Ca2(+)-dependent (CD) mechanisms. In this paper we present evidence that CD myoblast adhesion involves a molecule similar or identical to two known CD adhesion glycoproteins, N-cadherin and A-CAM. These molecules were previously identified by other laboratories in brain and cardiac muscle, respectively, and are postulated to be the same molecule. Antibodies to N-cadherin and A-CAM immunoblotted a similar band with a molecular weight of approximately 125,000 in extracts of brain, heart, and pectoral muscle isolated from chick embryos and in extracts of muscle cells grown in vitro at Ca2+ concentrations that either promoted or inhibited myotube formation. In assays designed to measure the interaction of fusion-competent myoblasts in suspension, both polyclonal and monoclonal anti-N-cadherin antibodies inhibited CD myoblast aggregation, suggesting that N-cadherin mediates the CD aspect of myoblast adhesion. Anti-N-cadherin also had a partial inhibitory effect on myotube formation likely due to the effect on myoblast-myoblast adhesion. The results indicate that N-cadherin/A-CAM plays a role in myoblast recognition and adhesion during skeletal myogenesis.     

Platelet endothelial cell adhesion molecule, PECAM-1, modulates cell migration.

Cell migration is an important process in such phenomena as growth, development, and wound healing. The control of cell migration is orchestrated in part by cell surface adhesion molecules. These molecules fall into two major categories: those that bind to extracellular matrix and those that bind to adjacent cells. Here, we report on the role of a cell-cell adhesion molecule, platelet-endothelial cell adhesion molecule-1, (PECAM-1), a member of the lg superfamily, in the modulation of cell migration and cell-cell adhesion. PECAM-1 is a 120-130 kDa integral membrane protein that resides on endothelial cells and localizes at sites of cell-cell contact. Since endothelial cells express PECAM-1 constitutively, we studied the effects of PECAM-1 on cell-cell adhesion and migration in a null-cell population. Specifically, we transfected NIH/3T3 cells with the full length PECAM-1 molecule (two independent clones). Transfected cells containing only the neomycin resistance gene, cells expressing a construct coding for the extracellular domain of the molecule, and cells expressing the neu oncogene were used as controls. The PECAM-1 transfectants appeared smaller and more polygonal and tended to grow in clusters. Indirect immunofluorescence of PECAM-1 transfectants showed peripheral staining at sites of cell-cell contact, while the extracellular domain transfectants and the control cells did not. In two quantitative migration assays, the full-length PECAM-1 transfectants migrated more slowly than control cells. Thus, PECAM-1 transfected into a null cell appears to localize to sites of cell-cell contact, promote cell-cell adhesion, and diminish the rate of migration. These findings suggest a role for this cell-cell adhesion molecule in the process of endothelial cell migration.     

CEACAM1 inhibits cell-matrix adhesion and promotes cell migration through regulating the expression of N-cadherin

Carcinoembryonic antigen-related cell adhesion molecule 1 (CEACAM1) is a member of the immunoglobulin super family and has been observed to have two paradoxical functions: tumor suppression and the promotion of tumor invasion. In the present study, we discovered that CEACAM1 functions as an adhesion inhibitor and a migration promoter. The CEACAM1 transfected cells, either 293-CEACAM1 or LOVO/trans-CEACAM1, was proved to have lower adhesion rate. Furthermore, HT29/siRNA-CEACAM1 cells had a higher adhesion rate than HT29 cells. These results indicated that CEACAM1 was an inhibitor of cell-matrix adhesion. Additionally, 293-CEACAM1 LOVO/trans-CEACAM1 cells exhibited better motility in a trans-well migration assay. N-cadherin expression levels were positively correlated with CEACAM1 in 293-CEACAM1, LOVO/trans-CEACAM1 and HT29/siRNA-CEACAM1 cells. When blocked by a GC-4 antibody, the adhesive capacities of 293-CEACAM1 and LOVO/trans-CEACAM1 were recovered and the motilities of them were suppressed, which suggested that CEACAM1 functioned through N-cadherin.     

Polysialylation of the neural cell adhesion molecule: interfering with polysialylation and migration in neuroblastoma cells

Polysialic acid represents a unique posttranslational modification of the neural cell adhesion molecule (NCAM). It is built as a homopolymer of up to 150 molecules of alpha 2-8-linked sialic acids on N-glycans of the fifth immunoglobulin-like domain of NCAM. Besides its role in cell migration and axonal growth during development, polysialic acids are closely related to tumor malignancy as they are linked to the malignant potential of several tumors, such as undifferentiated neuroblastoma. Polysialic acid expression is significantly more frequent in high-grade tumors than in low-grade tumors. It is synthesized in the Golgi apparatus by the activity of two closely related enzymes, the polysialyltransferases ST8SiaII and ST8SiaIV. Interestingly, polysialylation of tumors is not equally synthesized by both polysialyltransferases. It has been shown that especially the ST8SiaII gene is not expressed in some normal tissue, but is strongly expressed in tumor tissue. Here we summarize some knowledge on the role of polysialic acid in cell migration and tumor progression and present novel evidence that interfering with polysialylation using unnatural sialic acid precursors decreases the migration of neuroblastoma cells.     

Effects of N-cadherin overexpression on the adhesion properties of embryonic stem cells

Constitutive overexpression of N-cadherin in mouse embryonic stem cells led to marked changes in the phenotype and adhesion properties of these cells. The changes included the formation of smaller embryonic bodies, elevated mRNA and total protein levels of N-cadherin, and increased amounts of p120 catenin and connexin-43. N-cadherin cells exhibited decreased attachment to non-cell surfaces, while their adhesiveness to each other and to rat neonatal cardiomyocytes was significantly elevated. The findings suggest that N-cadherin overexpression can facilitate electromechanical integration of stem cells into excitable tissues with endogenously high levels of N-cadherin, such as the heart and brain.Key words: stem cells, cardiomyocytes, N-cadherin, connexin 43, gap junctions     

Effects of N-cadherin overexpression on the adhesion properties of embryonic stem cells

Constitutive overexpression of N-cadherin in mouse embryonic stem cells led to marked changes in the phenotype and adhesion properties of these cells. The changes included the formation of smaller embryonic bodies, elevated mRNA and total protein levels of N-cadherin, and increased amounts of p120 catenin and connexin-43. N-cadherin cells exhibited decreased attachment to non-cell surfaces, while their adhesiveness to each other and to rat neonatal cardiomyocytes was significantly elevated. The findings suggest that N-cadherin overexpression can facilitate electromechanical integration of stem cells into excitable tissues with endogenously high levels of N-cadherin, such as the heart and brain.     

Transforming growth factor-beta-mediated chondrogenesis of human mesenchymal progenitor cells involves N-cadherin and mitogen-activated protein kinase and Wnt signaling cross-talk

The multilineage differentiation potential of adult tissue-derived mesenchymal progenitor cells (MPCs), such as those from bone marrow and trabecular bone, makes them a useful model to investigate mechanisms regulating tissue development and regeneration, such as cartilage. Treatment with transforming growth factor-beta (TGF-beta) superfamily members is a key requirement for the in vitro chondrogenic differentiation of MPCs. Intracellular signaling cascades, particularly those involving the mitogen-activated protein (MAP) kinases, p38, ERK-1, and JNK, have been shown to be activated by TGF-betas in promoting cartilage-specific gene expression. MPC chondrogenesis in vitro also requires high cell seeding density, reminiscent of the cellular condensation requirements for embryonic mesenchymal chondrogenesis, suggesting common chondro-regulatory mechanisms. Prompted by recent findings of the crucial role of the cell adhesion protein, N-cadherin, and Wnt signaling in condensation and chondrogenesis, we have examined here their involvement, as well as MAP kinase signaling, in TGF-beta1-induced chondrogenesis of trabecular bone-derived MPCs. Our results showed that TGF-beta1 treatment initiates and maintains chondrogenesis of MPCs through the differential chondro-stimulatory activities of p38, ERK-1, and to a lesser extent, JNK. This regulation of MPC chondrogenic differentiation by the MAP kinases involves the modulation of N-cadherin expression levels, thereby likely controlling condensation-like cell-cell interaction and progression to chondrogenic differentiation, by the sequential up-regulation and progressive down-regulation of N-cadherin. TGF-beta1-mediated MAP kinase activation also controls WNT-7A gene expression and Wnt-mediated signaling through the intracellular beta-catenin-TCF pathway, which likely regulates N-cadherin expression and subsequent N-cadherin-mediated cell-adhesion complexes during the early steps of MPC chondrogenesis.     

The cell adhesion molecule NrCAM is crucial for growth cone behaviour and pathfinding of retinal ganglion cell axons

We investigated the role of the cell adhesion molecule NrCAM for axonal growth and pathfinding in the developing retina. Analysis of the distribution pattern of NrCAM in chick embryo retina sections and flat-mounts shows its presence during extension of retinal ganglion cell (RGC) axons; NrCAM is selectively present on RGC axons and is absent from the soma. Single cell cultures show an enrichment of NrCAM in the distal axon and growth cone. When offered as a substrate in addition to Laminin, NrCAM promotes RGC axon extension and the formation of growth cone protrusions. In substrate stripe assays, mimicking the NrCAM-displaying optic fibre layer and the Laminin-rich basal lamina, RGC axons preferentially grow on NrCAM lanes. The three-dimensional analysis of RGC growth cones in retina flat-mounts reveals that they are enlarged and form more protrusions extending away from the correct pathway under conditions of NrCAM-inhibition. Time-lapse analyses show that these growth cones pause longer to explore their environment, proceed for shorter time spans, and retract more often than under control conditions; in addition, they often deviate from the correct pathway towards the optic fissure. Inhibition of NrCAM in organ-cultured intact eyes causes RGC axons to misroute at the optic fissure; instead of diving into the optic nerve head, these axons cross onto the opposite side of the retina. Our results demonstrate a crucial role for NrCAM in the navigation of RGC axons in the developing retina towards the optic fissure, and also for pathfinding into the optic nerve.     

Transepithelial migration of Toxoplasma gondii involves an interaction of intercellular adhesion molecule 1 (ICAM-1) with the parasite adhesin MIC2

Toxoplasma gondii crosses non-permissive biological barriers such as the intestine, the blood-brain barrier and the placenta thereby gaining access to tissues where it most commonly causes severe pathology. Herein we show that in the process of migration Toxoplasma initially concentrates around intercellular junctions and probably uses a paracellular pathway to transmigrate across biological barriers. Parasite transmigration required viable and actively motile parasites. Interestingly, the integrity of host cell barriers was not altered during parasite transmigration. As intercellular adhesion molecule 1 (ICAM-1) is upregulated on cellular barriers during Toxoplasma infection, we investigated the role of this receptor in parasite transmigration. Soluble human ICAM-1 and ICAM-1 antibodies inhibited transmigration of parasites across cellular barriers implicating this receptor in the process of transmigration. Furthermore, human ICAM-1 immunoprecipitated the mature form of the parasite adhesin MIC2 present on the parasite surface, indicating that this interaction may contribute to cellular migration. These findings reveal that Toxoplasma exploits the natural cell trafficking pathways in the host to cross cellular barriers and disseminate to deep tissues.