Contactin associates with Na+ channels and increases their functional expression.

Contactin (also known as F3, F11) is a surface glycoprotein that has significant homology with the beta2 subunit of voltage-gated Na(+) channels. Contactin and Na(+) channels can be reciprocally coimmunoprecipitated from brain homogenates, indicating association within a complex. Cells cotransfected with Na(+) channel Na(v)1.2alpha and beta1 subunits and contactin have threefold to fourfold higher peak Na(+) currents than cells with Na(v)1.2alpha alone, Na(v)1.2/beta1, Na(v)1.2/contactin, or Na(v)1.2/beta1/beta2. These cells also have a correspondingly higher saxitoxin binding, suggesting an increased Na(+) channel surface membrane density. Coimmunoprecipitation of different subunits from cell lines shows that contactin interacts specifically with the beta1 subunit. In the PNS, immunocytochemical studies show a transient colocalization of contactin and Na(+) channels at new nodes of Ranvier forming during remyelination. In the CNS, there is a particularly high level of colocalization of Na(+) channels and contactin at nodes both during development and in the adult. Contactin may thus significantly influence the functional expression and distribution of Na(+) channels in neurons.

Localization of Caspr2 in myelinated nerves depends on axon-glia interactions and the generation of barriers along the axon.

Cell recognition proteins of the contactin-associated protein (Caspr) family demarcate distinct domains along myelinated axons. Caspr is present at the paranodal junction formed between the axon and myelinating glial cells, whereas Caspr2 is localized and associates with K(+) channels at the adjacent juxtaparanodal region. Here we investigated the distribution of Caspr2 during development of peripheral nerves of normal and galactolipids-deficient [ceramide galactosyl transferase (CGT)-/-] mice. This mutant exhibits paranodal abnormalities, lacking all putative adhesion components of this junction, including Caspr, contactin, and neurofascin 155. In sciatic nerves of this mutant, Caspr2 was not found at the juxtaparanodal region but was concentrated instead at the paranodes with Kv1.2. Similar distribution of Caspr2 was found in the PNS of contactin knock-out mice, which also lack Caspr in their paranodes. During development of wild-type peripheral nerves, Caspr2 and Kv1.2 were initially detected at the paranodes before relocating to the adjacent juxtaparanodal region. This transition was not observed in CGT mice, where Caspr2 and Kv1.2 remained paranodal. Double labeling for Caspr and Caspr2 demonstrated that these two related proteins occupied mutually excluding domains along the axon and revealed the presence of both paranodal and internodal barrier-like structures that are delineated by Caspr. Finally, we found that the disruption of axon-glia contact in CGT-/- nerves also affects the localization of the cytoskeleton-associated protein 4.1B along the axon. Altogether, our results reveal a sequential appearance of members of the Caspr family at different domains along myelinated axons and suggest that the localization of Caspr2 may be controlled by the generation of Caspr-containing barriers along the axon.

Contactin orchestrates assembly of the septate-like junctions at the paranode in myelinated peripheral nerve.

Rapid nerve impulse conduction depends on specialized membrane domains in myelinated nerve, the node of Ranvier, the paranode, and the myelinated internodal region. We report that GPI-linked contactin enables the formation of the paranodal septate-like axo-glial junctions in myelinated peripheral nerve. Contactin clusters at the paranodal axolemma during Schwann cell myelination. Ablation of contactin in mutant mice disrupts junctional attachment at the paranode and reduces nerve conduction velocity 3-fold. The mutation impedes intracellular transport and surface expression of Caspr and leaves NF155 on apposing paranodal myelin disengaged. The contactin mutation does not affect sodium channel clustering at the nodes of Ranvier but alters the location of the Shaker-type Kv1.1 and Kv1.2 potassium channels. Thus, contactin is a crucial part in the machinery that controls junctional attachment at the paranode and ultimately the physiology of myelinated nerve.

Cadherins: molecular codes for axon guidance and synapse formation.

The formation of the myriad of neuronal connections within the vertebrate nervous system relies on expression of molecular tags that match extending axon populations with synaptic target sites. Recent work suggests that cadherins, a group of calcium-dependent cell adhesion molecules, are candidates to serve such a role. The diversity of the cadherin family in the nervous system allows for a multitude of interactions to specify neuronal connections. Specific cadherin types demarcate subpopulations of developing axons that interconnect within neuronal circuits. Expression of different cadherin species at select synapse populations raises exciting prospects for this molecule class in controlling adhesive interactions during synaptogenesis and plasticity. Regulation of cadherin-mediated adhesive strength is an attractive mechanism to explain the different cadherin functions in axon growth and at synapses.

Ataxia and abnormal cerebellar microorganization in mice with ablated contactin gene expression.

Axon guidance and target recognition depend on neuronal cell surface receptors that recognize and elicit selective growth cone responses to guidance cues in the environment. Contactin, a cell adhesion/recognition molecule of the immunoglobulin gene superfamily, regulates axon growth and fasciculation in vitro, but its role in vivo is unknown. To assess its function in the developing nervous system, we have ablated contactin gene expression in mice. Contactin-/- mutants displayed a severe ataxic phenotype consistent with defects in the cerebellum and survived only until postnatal day 18. Analysis of the contactin-/- mutant cerebellum revealed defects in granule cell axon guidance and in dendritic projections from granule and Golgi cells. These results demonstrate that contactin controls axonal and dendritic interactions of cerebellar interneurons and contributes to cerebellar microorganization.

Immunohistochemical evidence for the brevican-tenascin-R interaction: colocalization in perineuronal nets suggests a physiological role for the interaction in the adult rat brain.

Brevican is one of the most abundant chondroitin sulfate proteoglycans in the adult rat brain. We have recently shown that the C-type lectin domain of brevican binds fibronectin type III domains 3-5 of tenascin-R. Here we report strong evidence for a physiological basis for this interaction. Substantial brevican immunoreactivity was detected in a number of nuclei and in the reticular formations throughout the midbrain and hindbrain, including, but not limited to, the deep cerebellar nuclei, the trapezoid body, the red nucleus, the oculomotor nucleus, the vestibular nucleus, the cochlear nucleus, the gigantocellular reticular nucleus, the motor trigeminal nucleus, and the lateral superior olive. Most of the brevican immunoreactivity exhibited pericellular and reticular staining patterns. In almost all of these sites, brevican immunoreactivity colocalized with that of tenascin-R, which was also substantially codistributed with versican, another member of the lectican family. Detailed analysis revealed that the pericellular staining of brevican resembled that in perineuronal nets in which tenascin-R has been localized. Immunoelectron microscopy identified brevican immunoreactivity in the intercellular spaces surrounding presynaptic boutons and on their surfaces, but not in the synaptic clefts or in their immediate vicinity, a distribution pattern consistent with perineuronal nets. Taken together, our results provide strong evidence that the previously reported interactions between brevican and tenascin-R may play a functional role within the perineuronal nets.

Expression of multiple cadherins and catenins in the chick optic tectum.

Cadherins form a large family of homophilic cell adhesion molecules that are involved in numerous aspects of neural development. The best-studied neural cadherin, N-cadherin, is concentrated at synapses made by retinal axons in the chick optic tectum and is required for the arborization of retinal axons in their target (retinorecipient) laminae. By analogy, other cadherins might mediate arborization or synaptogenesis in other tectal laminae. Here we consider which cadherins are expressed in tectum, which cells express them, and how their expression is regulated. First, using N-cadherin as a model, we show that synaptic input regulates both cadherin gene expression and the subcellular distribution of cadherin protein. Second, we demonstrate that N-, R-, and T-cadherin are each expressed in distinct laminar patterns during retinotectal synaptogenesis and that N- and R- are enriched in nonoverlapping synaptic subsets. Third, we show that over 20 cadherin superfamily genes are expressed in the tectum during the time that synapses are forming and that many of them are expressed in restricted groups of cells. Finally, we report that both beta-catenin and gamma-catenin (plakoglobin), cytoplasmic proteins required for cadherin signaling, are enriched at synapses and associated with N-cadherin. However, beta- and gamma-catenins are differentially distributed and regulated, and form mutually exclusive complexes. This result suggests that cadherin-based specificity involves multiple cadherin-dependent signaling pathways as well as multiple cadherins.

T-cadherin is a major glycophosphoinositol-anchored protein associated with noncaveolar detergent-insoluble domains of the cardiac sarcolemma.

Sucrose-density flotation analysis of Triton-insoluble membrane domains isolated from highly purified sheep ventricular sarcolemma revealed the presence of two major 120- and 100-kDa proteins. Both species migrated in two-dimensional isoelectric focussing/SDS gels with an apparent pI of approximately 4.3, suggesting that they might be related. Microsequence analysis of peptides derived from the 100-kDa protein yielded amino acid sequences with high homology to T-cadherin, a truncated cadherin lacking a cytoplasmic domain. The similarity was confirmed using antibodies to chicken T-cadherin that reacted with both proteins on immunoblots. T-cadherin was released from the detergent-insoluble sarcolemmal fraction by phospholipase C treatment indicating that it is linked to the membrane by a glycophosphoinositol anchor. T-cadherin could be ADP-ribosylated by a transferase that was also present in the caveolin-enriched Triton-insoluble fraction. T-cadherin-containing membrane fragments cofractionated on sucrose gradients with caveolin-3, a marker protein for myocyte caveolae. However, immunopurified caveolin-3-containing membranes contained no associated T-cadherin. Immunocytochemical analysis of cultured rat atrial myocytes revealed that T-cadherin and caveolin have related but nonoverlapping staining patterns. These results suggest that T-cadherin is a major glycophosphoinositol-linked protein in cardiac myocytes and that it may be located in plasma membrane "rafts" distinct from but possibly adjacent to caveolae.

High affinity binding and overlapping localization of neurocan and phosphacan/protein-tyrosine phosphatase-zeta/beta with tenascin-R, amphoterin, and the heparin-binding growth-associated molecule.

We have studied the interactions of the nervous tissue-specific chondroitin sulfate proteoglycans neurocan and phosphacan with the extracellular matrix protein tenascin-R and two heparin-binding proteins, amphoterin and the heparin-binding growth-associated molecule (HB-GAM), using a radioligand binding assay. Both proteoglycans show saturable, high affinity binding to tenascin-R with apparent dissociation constants in the 2-7 nM range. Binding is reversible, inhibited in the presence of unlabeled proteoglycan, and increased by approximately 60% following chondroitinase treatment of the proteoglycans, indicating that the interactions are mediated via the core (glyco)proteins rather than by the glycosaminoglycan chains, which may in fact partially shield the binding sites. In contrast to their interactions with tenascin-C, in which binding was decreased by approximately 75% in the absence of calcium, binding of phosphacan to tenascin-R was not affected by the absence of divalent cations in the binding buffer, although there was a small but significant decrease in the binding of neurocan. Neurocan and phosphacan are also high affinity ligands of amphoterin and HB-GAM (Kd = 0.3-8 nM), two heparin-binding proteins that are developmentally regulated in brain and functionally involved in neurite outgrowth. The chondroitin sulfate chains on neurocan and phosphacan account for at least 80% of their binding to amphoterin and HB-GAM. The presence of amphoterin also produces a 5-fold increase in phosphacan binding to the neural cell adhesion molecule contactin. Immunocytochemical studies showed an overlapping localization of the proteoglycans and their ligands in the embryonic and postnatal brain, retina, and spinal cord. These studies have therefore revealed differences in the interactions of neurocan and phosphacan with the two major members of the tenascin family of extracellular matrix proteins, and also suggest that chondroitin sulfate proteoglycans play an important role in the binding and/or presentation of differentiation factors in the developing central nervous system.

The brain chondroitin sulfate proteoglycan brevican associates with astrocytes ensheathing cerebellar glomeruli and inhibits neurite outgrowth from granule neurons.

Brevican is a nervous system-specific chondroitin sulfate proteoglycan that belongs to the aggrecan family and is one of the most abundant chondroitin sulfate proteoglycans in adult brain. To gain insights into the role of brevican in brain development, we investigated its spatiotemporal expression, cell surface binding, and effects on neurite outgrowth, using rat cerebellar cortex as a model system. Immunoreactivity of brevican occurs predominantly in the protoplasmic islet in the internal granular layer after the third postnatal week. Immunoelectron microscopy revealed that brevican is localized in close association with the surface of astrocytes that form neuroglial sheaths of cerebellar glomeruli where incoming mossy fibers interact with dendrites and axons from resident neurons. In situ hybridization showed that brevican is synthesized by these astrocytes themselves. In primary cultures of cerebellar astrocytes, brevican is detected on the surface of these cells. Binding assays with exogenously added brevican revealed that primary astrocytes and several immortalized neural cell lines have cell surface binding sites for brevican core protein. These cell surface brevican binding sites recognize the C-terminal portion of the core protein and are independent of cell surface hyaluronan. These results indicate that brevican is synthesized by astrocytes and retained on their surface by an interaction involving its core protein. Purified brevican inhibits neurite outgrowth from cerebellar granule neurons in vitro, an activity that requires chondroitin sulfate chains. We suggest that brevican presented on the surface of neuroglial sheaths may be controlling the infiltration of axons and dendrites into maturing glomeruli.

Human STX polysialyltransferase forms the embryonic form of the neural cell adhesion molecule. Tissue-specific expression, neurite outgrowth, and chromosomal localization in comparison with another polysialyltransferase, PST.

PST and STX are polysialyltransferases that form polysialic acid in the neural cell adhesion molecule (N-CAM), although it is not known why these two polysialyltransferases exist. In the present study, we have first isolated cDNA encoding human STX, which includes 5'-untranslated sequence. Northern blot analysis, using this cDNA and PST cDNA previously isolated by us, demonstrated that PST and STX are expressed in different fetal and adult tissues. STX is primarily expressed in embryonic tissues, but only modestly in adult heart, brain, and thymus. PST, on the other hand, is continuously expressed in adult heart, brain, thymus, spleen, small and large intestines, and peripheral blood leukocytes. In various parts of adult brain, the relative amount of PST and STX appears to be substantially different depending on the regions. The analysis by in situ hybridization of mouse adult brain, however, suggests that polysialic acid in the hippocampal formation is synthesized by both STX and PST. HeLa cells doubly transfected with the isolated STX cDNA and N-CAM cDNA supported neurite outgrowth much better than HeLa cells expressing N-CAM alone. However, polysialic acid synthesized by PST appears to be a better substratum than that synthesized by STX. Moreover, the genes for PST and STX were found to reside at chromosome 5, band p21 and chromosome 15, band q26, respectively. These results, taken together, strongly suggest that PST and STX are expressed distinctly in tissue-specific and cell-specific manners and that they apparently have distinct roles in development and organogenesis.

Mutational analysis of the L1 neuronal cell adhesion molecule identifies membrane-proximal amino acids of the cytoplasmic domain that are required for cytoskeletal anchorage.

The preferential localization of the L1 cell adhesion molecule in the axons and growth cones of differentiating neurons suggests the existence of a mechanism for targeting or anchoring the molecule to these locations. We have used B28 glioma cells, which have an extremely flattened morphology, as a model system to study the organization of L1 on the cell structure. Transfection of L1 cDNA into B28 cells results in expression of the L1 protein in organized linear cell surface arrays which are codistributed with cytoskeletal stress fibers, but not with microtubles or intermediate filaments. Transfection studies with L1 deletion mutants identify the juxtamembrane segment of the cytoplasmic domain as the critical entity for arrangement of L1 into ordered cell surface arrays. The seventh cytoplasmic amino acid of L1, lysine 1150, and to a lesser extent the fourth cytoplasmic amino acid, lysine 1147, appear to be critical residues for maintaining normal L1 anchorage and distribution.

Differential targeting of T- and N-cadherin in polarized epithelial cells.

To test whether glycosyl phosphatidylinositol-linked T-cadherin is a component of cell junctions like classical cadherins, we have examined its distribution and targeting in polarized epithelial cells. In vivo, T-cadherin was detected on the apical cell surface of the chick intestinal epithelium. In cultures of transfected Madin-Darby canine kidney cells, T-cadherin was also expressed apically, whereas classical N-cadherin resided basolaterally. Both cadherins were directly targeted to their respective membrane domains. Mutant proteins were expressed in Madin-Darby canine kidney cells to identify the regions responsible for differential cadherin localization. NDeltacyt, an N-cadherin cytoplasmic domain deletion mutant, was stably distributed basolaterally. This mutant was transported to both the apical and basolateral membrane compartments, followed by preferential removal from the apical surface. T-NDeltacyt, a T-cadherin mutant with the N-cadherin cytoplasmic domain deletion, was localized basolaterally, whereas N-TGPI, a GPI-anchored N-cadherin mutant, resided at the apical domain. The T-cadherin carboxyl-terminal 76 amino acids contain the apical targeting signal and include the signal for GPI anchor attachment. Basolateral localization of N-cadherin is achieved through targeting signals in the cytoplasmic domain. Thus, GPI-linked T-cadherin is not a component of cell junctions, consistent with a function as a recognition rather than a cell adhesion molecule.

Inhibition of motor axon growth by T-cadherin substrata.

As spinal motor neurons project to their hindlimb targets, their growth cones avoid particular regions along their pathway. T-cadherin is discretely distributed in the avoided caudal sclerotome and on extrasynaptic muscle surfaces (B. J. Fredette and B. Ranscht (1994) J. Neurosci. 14, 7331-7346), and therefore, the ability of T-cadherin to inhibit neurite growth was tested in vitro. T-cadherin inhibited neurite extension from select neuron populations both as a substratum, and as a soluble recombinant protein. Anti-T-cadherin antibodies neutralized the inhibition. Spinal motor neurons were inhibited only during the stages of axon growth across the sclerotome and muscle innervation. Inhibitory responses corresponded to neuronal T-cadherin expression, suggesting a homophilic binding mechanism. These results suggest that T-cadherin is a negative guidance cue for motor axon projections.

Contactin/F11 and tenascin-C co-expression in the chick retina correlates with formation of the synaptic plexiform layers.

The neural immunoglobulin-like cell adhesion molecule contactin/F11 and the extracellular matrix glycoprotein tenascin-C are prominent molecules in the developing nervous system which interact in in vitro assays (Zisch et al., J. Cell Biol. 119, 203-213). To determine their potential role in neural development, the distribution of tenascin-C and contactin/F11 was examined in the developing chick retina. The onset of both tenascin-C and contactin/F11 expression coincides with the appearance of ganglion cell dendrides and neurites from bipolar and amacrine cells in the inner layer (IPL) at E8, and the extension of bipolar and horizontal cell processes in the outer plexiform layer (OPL) at E9. Contactin/F11 expression is co-ordinately upregulated with the TN190 and TN200 tenascin-C isoforms between embryonic day 8 (E8) and E17, while little, if any, of the TN220 isoform, which does not bind contactin/F11, is detected. In situ hybridization reveals that tenascin-C and contactin/F11 mRNAs are synthesized by different neuronal types. Tenascin-C mRNA probes hybridize to amacrine and displaced amacrine neurons, and horizontal neurons. In cultured retinal cells, tenascin-C is also present on process-bearing neurofilament-positive cells. Contactin/F11 mRNA is detected in bipolar cells or their precursors from E8-9, and later in horizontal and ganglion neurons. The highest levels and greatest overlap in the synaptic IPL and OPL are reached at E17, when the stratification of the retina is nearly complete. These results are consistent with a putative role for contactin/F11-tenascin-C interactions in the establishment of synaptic layers in the retina.

Expression cloning of a human polysialyltransferase that forms the polysialylated neural cell adhesion molecule present in embryonic brain.

Polysialic acid is a developmentally regulated posttranslational modification of the neural cell adhesion molecule (N-CAM). It has been suggested that this large anionic carbohydrate modulates the adhesive property of N-CAM, but the precise function of polysialic acid is not known. Here we describe the isolation and functional expression of a cDNA encoding a human polysialyltransferase. For this expression cloning, COS-1 cells were cotransfected with a human fetal brain cDNA library and a cDNA encoding human N-CAM. Transfected COS-1 cells were stained with a monoclonal antibody specific for polysialic acid and enriched by fluorescence-activated cell sorting. Sibling selection of recovered plasmids resulted in a cDNA clone that directs the expression of polysialic acid on the cell surface. The deduced amino acid sequence indicates that the polysialyltransferase shares a common sequence motif with other sialyltransferases cloned so far. The polysialyltransferase is, however, distinct by having two clusters of basic amino acids. The amount of the polysialyltransferase transcripts correlates well with the formation of polysialic acid in various human tissues, and is abundant in the fetal brain but not in the adult brain. Moreover, HeLa cells stably expressing polysialic acid and N-CAM promoted neurite outgrowth and sprouting. These results indicate that the cloned polysialyltransferase forms polysialylated, embryonic N-CAM, which is critical for plasticity of neural cells.

The glypiated neuronal cell adhesion molecule contactin/F11 complexes with src-family protein tyrosine kinase Fyn.

Glycosyl phosphatidylinositol-anchored glycoproteins of the immunoglobulin superfamily play an important role in the formation of neuronal networks during development. The mechanism whereby neuronal GPI-linked molecules transduce recognition signals remains to be established. Analysis of detergent-resistant immune-complexes reveals that the glypiated neuronal cell adhesion molecule contactin/F11 specifically complexes with the cytoplasmic, nonreceptor type src-family tyrosine kinase Fyn. Antibody-mediated cross-linking of contactin/F11 on embryonic chick neuronal cells leads to an increase of the Fyn-activity coprecipitated with contactin/F11, and elevates phosphorylation of an additional 75/80 K component within the contactin/F11-immune-complex. Additionally, binding of ligands, i.e., contactin/F11-specific antibody or tenascin-R, a natural ligand of contactin/F11, to the surface of HeLa transfectants expressing contactin/F11, causes capping of contactin/F11 and a concomitant change in the distribution of the intracellular kinase Fyn, thus confirming their physical association. This indicates that contactin/F11-mediated signaling requires Fyn.

T-cadherin expression delineates specific regions of the developing motor axon-hindlimb projection pathway.

T-cadherin is a unique member of the cadherin family anchored to the membrane by a glycosyl phosphatidylinositol moiety (Ranscht and Dours-Zimmermann, 1991). T-cadherin's distribution in the developing motor axon pathway was mapped by immunocytochemistry in the chick lumbosacral region as spinal neurons project to and innervate hindlimb muscle. On growing motor axons, T-cadherin was expressed biphasically. Initially, uniform T-cadherin expression occurred on motor neurons as they projected between the spinal cord and the base of the hindlimb (stage 21-24), and then decreased as the axons sorted to form dorsal, ventral and muscle nerve trunks (stage 25-27). Later, as motor axons entered and formed terminal axon arbors and synapses in muscle (stages 28-36), expression reoccurred heterogeneously among motor neuron pools. Thus, T-cadherin may guide the growth and fasciculation of all motor neurons during early axon extension, but only affect particular populations during the later expression period. In the mesenchyme of the motor axon pathway, T-cadherin was restricted to regions avoided by growing axons: the posterior-half sclerotome before and during the projection of motor axons through the T-cadherin-negative anterior half, and the extrasynaptic surfaces of developing muscle. The temporal and spatial expression patterns of T-cadherin and neurite outgrowth-promoting N-cadherin were complementary both in nerve and muscle tissues. Thus, in the posterior sclerotome and in maturing muscle, T-cadherin may act as a negative regulator that works in concert with neurite growth-promoting molecules to guide motor axons to their peripheral targets.

Cadherins and catenins: interactions and functions in embryonic development.

During the past year, the family of cadherin cell-adhesion molecules has increased in number and diversity. Recent studies have also emphasized how cadherin activity can be regulated by the dynamic association with intracellular components, the catenins, and with extracellular molecules that are linked to different cell-signaling pathways. Finally, the initial steps have been taken towards identifying the function of cadherins in vivo, including their potential roles in early embryonic development.