Purification and characterization of a glucuronyltransferase involved in the biosynthesis of the HNK-1 epitope on glycoproteins from rat brain

The glucuronyltransferase involved in the biosynthesis of the HNK-1 epitope on glycoproteins was purified to an apparent homogeneity from the Nonidet P-40 extract of 2-week postnatal rat forebrain by sequential chromatographies on CM-Sepharose CL-6B, UDP-GlcA-Sepharose 4B, asialo-orosomucoid-Sepharose 4B, Matrex gel Blue A, Mono Q, HiTrap chelating, and HiTrap heparin columns. The purified enzyme migrated as a 45-kDa protein upon SDS-polyacrylamide gel electrophoresis under reducing conditions, but eluted as a 90-kDa protein upon Superose gel filtration in the presence of Nonidet P-40, suggesting that the enzyme forms homodimers under non-denatured conditions. The enzyme transferred glucuronic acid to various glycoprotein acceptors bearing terminal N-acetyllactosamine structure such as asialo-orosomucoid, asialo-fetuin, and asialo-neural cell adhesion molecule, whereas little activity was detected to paragloboside, a precursor glycolipid of the HNK-1 epitope on glycolipids. These results suggested that the enzyme is specifically associated with the biosynthesis of the HNK-1 epitope on glycoproteins. Sphingomyelin was specifically required for expression of the enzyme activity. Stearoyl-sphingomyelin (18:0) was the most effective, followed by palmitoyl-sphingomyelin (16:0) and lignoceroyl-sphingomyelin (24:0). Interestingly, activity was demonstrated only for sphingomyelin with a saturated fatty acid, i.e. not for that with an unsaturated fatty acid, regardless of the length of the acyl group.     

HNK-1 antigens on uveal and cutaneous melanoma cell lines

The HNK-1 epitope has been associated with the metastatic behaviour of uveal melanomas. We characterized HNK-1 antigens on four human uveal (primary and metastatic) and two primary cutaneous melanoma cell lines by immunocytochemistry and Western blot analysis. We also determined the involvement of the HNK-1 epitope in cell-cell interactions on a matrigel layer. Three uveal melanoma cell lines (one primary and two metastatic) and one cutaneous melanoma cell line showed HNK-1 expression by immunocytochemistry. On matrigel, only the HNK-1-positive cutaneous melanoma cell line Bowes grew in a honeycomb-like structure which disappeared after adding HNK-1 antibodies to the culture medium. Immunoblot analysis of the primary uveal melanoma cell line EOM-3 revealed five HNK-1-positive protein bands with apparent molecular weights of 200, 160, 115, 95 and 75 kDa. The cutaneous melanoma cell line Bowes showed three HNK-1-positive protein bands with apparent molecular weights of 150, 135 and 90 kDa. This study shows that two uveal (primary and metastatic) and one primary cutaneous melanoma cell lines express HNK-1 antigens on immunoblot. Only in the HNK-1-positive cutaneous melanoma cell line Bowes did the HNK-1 epitope have a function in intercellular adhesion. Although the primary uveal melanoma cell line EOM-3 showed a similar HNK-1 immunoreactivity, we could not demonstrate HNK-1-mediated cell adhesion. On immunoblot, the two cell lines displayed different HNK-1 antigens, which may explain the difference in cell adhesion.     

MR volumetric analysis of the human entorhinal, perirhinal, and temporopolar cortices

Cell-adhesion molecules (CAMs) are thought to play crucial roles in development and plasticity in the nervous system. This study tested for a role for cell adhesion and in particular, the recognition of two glycosyl epitopes (HNK-1 and oligomannoside) in the activity-driven sharpening of the retinotopic map formed by the regenerating retinal fibers of goldfish. HNK-1 is a prominent glycosyl epitope on many CAMs and extracellular matrix (ECM) molecules, including NCAM, L1, ependymin, and integrins, which have all been implicated in synaptic plasticity. To test for a role of HNK-1 in the sharpening process, we used osmotic minipumps to infuse HNK-1 antibodies for 7-21 days into the tectal ventricle starting at 18 days after optic nerve crush. Retinotopic maps recorded at 76-86 days postcrush showed a lack of sharpening similar to that seen previously with two antibodies to ependymin, an HNK-1-positive ECM component present in cerebrospinal fluid. The multiunit receptive fields at each point averaged 26 degrees versus 11-12 degrees in regenerates infused with control antibodies or Ringer's alone. The HNK-1 epitope also binds to the G2 domain of laminin to mediate neuron-ECM adhesion. To test for a role for laminin, a polyclonal antibody was similarly infused and also prevented sharpening to approximately the same degree. The results support a role for the HNK-1 epitope and laminin in retinotectal sharpening. The oligomannoside epitope (recognized by monoclonal antibody L3) on the CAM L1 interacts with NCAM on the same cell to promote stronger L1 homophilic interactions between cells. Both an L1-like molecule and NCAM are prominently reexpressed in the regenerating retinotectal system of fish. Infusion of oligomannosidic glycopeptides resulted in decreased sharpening, with multiunit receptive fields that averaged 22.7 degrees. Infusions of mannose-poor glycopeptides less prominently disrupted sharpening, with average multiunit receptive fields of 18 degrees. Thus, oligomannosidic glycans in particular may play a role in retinotopic sharpening. Blocking glycan-mediated interactions between CAMs and ECM molecules could decrease the extent of exploratory growth of retinal axon collaterals, preventing them from finding their retinotopic sites, or could interfere with L1 or NCAM and laminin binding at the synaptic densities preventing stabilization of retinotopically appropriate synapses. Together, these results support a prominent role for cell adhesion and glycan epitopes in visual synaptic plasticity.     

Characteristics of cefdinir uptake by rabbit small intestinal brush-border membrane vesicles

PURPOSE: To search for changes in the presence and distribution of the cell-adhesion-related HNK-1 carbohydrate epitope after cataract extraction. METHODS: Twenty-five pseudophakic and two aphakic human autopsy eyes and, for comparison, one anterior subcapsular cataract obtained at surgery were studied with monoclonal antibodies (MAbs) HNK-1 and NC-1 to the HNK-1 epitope using the avidin-biotinylated peroxidase complex method. RESULTS: MAbs to the HNK-1 epitope constantly immunolabelled the inner connective tissue layer of the ciliary body in all pseudophakic and aphakic eyes studied. The distribution of the immunoreaction was similar to that reported for normal eyes. They also labelled the extracellular matrix in each of 18 plaques of secondary cataract on the posterior capsule, in each of 13 plaques at the rim of the capsular bag, and in the anterior subcapsular cataract. Bladder cells in each of 16 Soemmering's rings remained unlabelled. CONCLUSION: The distribution of the HNK-1 epitope in the ciliary body does not appreciably change after cataract extraction, although the accommodative demand of the eye is altered. Its presence in an anterior subcapsular cataract suggests that the epitope may be locally produced by lens epithelial cells also in secondary cataract. The epitope is associated with cell adhesion and migration, both of which may play a role in the pathogenesis of secondary cataract.     

Three time windows for amnestic effect of antibodies to cell adhesion molecule L1 in chicks

The L1/NgCAM cell adhesion molecule is involved in neurite outgrowth, axonal fasciculation and cell migration in the nervous system. We studied the effects of antibodies against L1 injected intracranially at various times before and after training 2-day-old chicks in a visual categorization task. Memory retention was tested 24 h post-training. Anti-L1 antibodies impaired task retention only when administered in three restricted time windows: immediately before training, at about 5.5 h after training and from 15 to 18 h after training. No amnesia was produced by injections before, between or after these sensitive periods (from -1 to +21 h relative to training). These results indicate that there are multiple post-training periods during which L1 is involved in the formation of long-term memory.     

Multiple roles of neural cell adhesion molecule, neural cell adhesion molecule-polysialic acid, and L1 adhesion molecules during sensory innervation of the otic epithelium in vitro

To explore the role of cell adhesion molecules in the innervation of the inner ear, antibody perturbation was used on histotypic co-cultures of the ganglionic and epithelial anlagen derived from the otocyst. When unperturbed, these tissues survived and differentiated in this culture system with outgrowth of fasciculated neuronal fibers which expressed neural cell adhesion molecule and L1. The fibers exhibited target choice and penetration, then branching and spreading within the otic epithelium as individual axons. Treatment of the co-cultures, or of the ganglionic anlagen alone, with anti-neural cell adhesion molecule or anti-L1 Fab fragments produced a defasciculation of fibers but did not affect neurite outgrowth. In the co-cultures this defasciculation was accompanied by a small increase in the number of fibers found in inappropriate tissues. However, the antibodies did not prevent fiber entry to the otic epithelium. In contrast, removal of polysialic acid from neural cell adhesion molecule with endoneuraminadase-N, while producing a similar fiber defasciculation, also increased the incidence of fibers entering the epithelium. Nevertheless, once within the target tissue, the individual fibers responded to either Fab or to desialylation by spreading out more rapidly, branching, and growing farther into the epithelium. The findings suggest that fasciculation is not essential for specific sensory fibers to seek out and penetrate the appropriate target, although it may improve their tracking efficiency. Polysialic acid on neural cell adhesion molecule appears to limit initial penetration of the target epithelium. Polysialic acid as well as neural cell adhesion molecule and L1 function are involved in fiber-target interactions that influence the arborization of sensory axons within the otic epithelium.     

Differential distribution of the HNK-1 carbohydrate epitope in the vertebrate retina

The expression of the cell adhesion-related HNK-1 carbohydrate epitope in the retina and ciliary body was studied in different vertebrates and in man. A series of eyes from 4 fish, 5 bird, and 9 mammalian species was analyzed by immunohistochemistry with monoclonal antibodies (MAb) HNK-1 and VC1.1 to the HNK-1 epitope, and with MAb SY38 to synaptophysin. Additionally, 7 morphologically normal human eyes were studied. In all fishes, as well as in baboons and man, the radial glia and all retinal layers except the photoreceptor cell layer were immunoreactive for the HNK-1 epitope. In all birds, the nerve fiber layer and both plexiform layers were labelled. In nonprimate mammals only the plexiform layers were immunoreactive. Fine differences in this general immunoreaction pattern were seen in different species. Mab SY38 labeled both plexiform layers of mammals only. In the ciliary body, immunoreaction for the HNK-1 epitope was seen in the inner connective tissue layer only in man, but the ciliary nerves were labelled in all species except the mouse and rat. The HNK-1 epitope seems to be phylogenetically conserved in the retina, where the HNK-1 immunoreactive plexiform layers possibly are overlapped with HNK-1 reactive radial glial cells in fishes and primates. Instead in the inner connective tissue layer of the ciliary body, the HNK-1 epitope is not phylogenetically conserved.     

Glycobiology of the olfactory system

The olfactory system is a highly plastic region of the nervous system. Continuous remodeling of neuronal circuits in the olfactory bulb takes place throughout life as a result of constant turnover of primary sensory olfactory neurons in the periphery. Glycoconjugates are very important in olfactory development, regeneration and function. This article deals with different aspects of glycobiology relevant for the olfactory system. Various anatomical, developmental and functional subdivisions of the olfactory system have been labeled with exogenous lectins. The application of reverse lectin histochemistry resulted in the visualization of endogenous lectins, involved in fasciculation of olfactory axons. Numerous glycoproteins, among them members of the immunoglobulin superfamily, the cadherins and integrins as well as different glycolipids and proteoglycans can act as surface adhesion molecules in the olfactory system. The olfactory-specific form of the sialoglycoprotein neural cell adhesion molecule is implicated in olfactory neuronal and axonal guidance. Glycoconjugates including laminin, fibronectin and proteoglycans are abundant components of the olfactory extracellular matrix, influencing neurite outgrowth and cellular migration. Immunohistochemical labeling has revealed occurrence of the carbohydrate differentiation antigen, playing a role in neurulation and morphogenesis of the very early olfactory system. The synaptic vesicle glycoprotein, appearing also early in olfactory development, is used as a marker of olfactory tumors. Finally, membrane and transmembrane glycoconjugates as well as secreted glycoconjugates may act as olfactory receptor molecules.     

Vascular cell adhesion molecule--a new approach to detect endothelial cell activation in MS and encephalitis in vivo

INTRODUCTION: Leukocyte migration into inflammatory lesions is controlled by adhesion molecules on activated vascular endothelium. Pivotal among these are E-selectin and the vascular cell adhesion molecule-1 (VCAM-1), which are found on very few cell types other than activated endothelium. METHODS: We determined the presence of the soluble form of these adhesion molecules (sE-selectin and sVCAM-1) in serum and CSF of patients with multiple sclerosis (MS), viral encephalitis, and controls, using enzyme-linked immunosorbent assays. RESULTS: MS patients with active, Gadolinium-DTPA-enhancing lesions on magnetic resonance imaging had significantly higher sVCAM-1 serum levels than normal controls. Patients with viral encephalitis had significantly higher levels of sVCAM-1 in serum and cerebrospinal fluid than controls. sE-selectin levels showed no significant variations. CONCLUSION: Activated vascular endothelium controlling leukocyte migration may be demonstrated in MS patients in vivo by determining sVCAM-1 in serum. Furthermore, sVCAM-1 may be useful for monitoring inflammatory activity in central nervous system inflammatory disease.     

Glial cell interactions with tenascin-C: adhesion and repulsion to different tenascin-C domains is cell type related

The multimodular glycoprotein tenascin-C is transiently expressed, predominantly by glial cells, during the development of the central and peripheral nervous systems. This extracellular matrix glycoprotein is involved in the control of cell adhesion, neuron migration and neurite outgrowth. Distinct functional properties for neuronal cell types have been attributed to separate tenascin-C domains using antibody perturbation studies and in vitro experiments on tenascin-C fragments. In order to study potential roles of tenascin-C for glial cell biology, a library of recombinant tenascin-C domains was used in a bioassay in vitro. Embryonic day 14 astrocytes, various astroglial-derived cell lines (C6, A7 and Neu7) and oligodendroglial-derived cell types (Oli-neu and G26-20) were examined in an adhesion assay and compared to the neuroblastoma cell line N2A. A binding site for most cell types, except for A7 and N2A, could be assigned to the first three fibronectin type III domains. Repulsive properties could be mapped to three different sites the epidermal growth factor-like repeats, fibronectin type III repeats 4 and 5 and to the alternatively spliced region of the molecule. The responses to these repulsive sites varied according to the cell type. These data are consistent with the interpretation that different cell types express distinct sets of tenascin-C receptors which might regulate cellular responses via distinct second messenger pathways.     

Selective neural cell adhesion molecule signaling by Src family tyrosine kinases and tyrosine phosphatases

Nerve growth cone guidance is a highly complex feat, involving coordination of cell adhesion molecules, trophic factor gradients, and extracellular matrix proteins. While navigating through the developing nervous system, the growth cone must integrate diverse environmental signals into a singular response. The repertoire of growth cone responses to these extracellular cues includes axonal growth, fasciculation, and synaptic stabilization, which are achieved through dynamic changes in the cytoskeleton and modulation of gene expression. It has become evident that interactions between cell adhesion molecules can activate intracellular signaling pathways in neurons. Such signaling pathways are just beginning to be defined for the axonal growth promoting molecules L1 and NCAM which are members of the immunoglobulin (Ig) superfamily. Recent findings have revealed that L1 and NCAM induce neurite outgrowth by activating intracellular signaling pathways in the growth cone mediated by two different members of the src family of nonreceptor protein tyrosine kinases (PTKs), pp60(c-src) and p59(fyn5,6). Growth cones display diverse morphologies and variable motility on these different cell adhesion molecules, which are likely to be generated by src kinases. In this review we will address novel features of nonreceptor PTKs of the src family which dictate their distinctive molecular interactions with cell adhesion molecules and signaling components.     

Polysialic acid and the regulation of cell interactions

Polysialic acid, a unique glycosylation of the neural cell adhesion molecule, is highly regulated in its expression. Its function is manifested in the modulation of cell interactions, probably through its unusual physical properties. Recent advances have clarified the enzymatic mechanism of polysialic acid biosynthesis, expanded its role in cell migration and axon guidance, and suggested that it promotes plasticity in the adult nervous system.     

GP55 inhibits both cell adhesion and growth of neurons, but not non-neuronal cells, via a G-protein-coupled receptor

There is compelling evidence for the role of inhibitory molecules in guiding neurons to their appropriate targets. Furthermore, continued expression of these molecules in the adult could explain why there is little regeneration of neurons in the central nervous system. We have previously identified a family of glycosyl phosphatidylinositol-linked glycoproteins (GP55) from adult chicken brain that has been shown to inhibit neurite outgrowth from dorsal root ganglion and forebrain neurons. GP55 consists of two or more glycoproteins and belongs to a subgroup of the lg superfamily which contains OBCAM, LAMP, neurotrimin and CEPU-1. We now show that GP55 is anti-adhesive, blocking the adhesion of neurons to normally adhesive substrata in a concentration dependent manner. The anti-adhesive effect can be blocked using antiserum raised against GP55 and pertussis toxin (PTX) but not the beta oligomer alone. In contrast, the adhesion of fibroblasts and Schwann cells to the substrata is not affected by GP55. Indeed, non-neuronal cells spread and grow normally. These results would suggest that both the anti-adhesive effect and the inhibition of outgrowth by GP55 is specific to neurons and is mediated by a PTX sensitive, G-protein-coupled receptor.     

Identification and characterization of the murine homologue of CD22, a B lymphocyte-restricted adhesion molecule

The human B lymphocyte-specific Ag, CD22, is a cell adhesion molecule expressed on the surface during a narrow window of B cell development, coincident with surface IgD. A ligand for CD22 has recently been identified on human T cells as the low molecular mass isoform of the leukocyte common Ag, CD45RO. CD22 has been reported to function in the regulation of both T and B cell activation in vitro. In this study, we report the isolation and expression of a molecular cDNA clone encoding the murine homologue of CD22, mCD22. Within their predicted protein sequences, murine and human sequences overall have 62% identity, which includes 18 of 20 extracellular cysteines and six of six cytoplasmic tyrosines. BHK cells transfected with mCD22 cDNA specifically adhere to resting and activated T lymphocytes and in addition bound activated, but not resting, B cells. Five Th clones were analyzed for their ability to adhere to mCD22; two Th0 clones and one Th1 clone bound CD22+ BHK transfectants, but not all T cell clones bound CD22+ cells: another Th1 clone and a Th2 clone did not. mCD22+ BHK transfectants were also specifically bound by the B cell-specific mAb, NIM-R6, demonstrating that this mAb is specific for murine CD22. Human cell lines expressing the counter-receptors for human CD22 were also examined for adhesion to the murine CD22 homologue; the epitope responsible for B cell adhesion to CD22 is conserved, whereas the T cell epitope binding to CD22 is not. The cDNA and mAb to murine CD22 will be useful for defining the in vivo function of CD22.     

Immunoreactivity of PMP-22, P0, and other 19 to 28 kDa glycoproteins in peripheral nerve myelin of mammals and fish with HNK1 and related antibodies

Mammalian peripheral nervous system (PNS) myelin contains several glycoproteins with molecular weights of 19 to 28 kDa, including the major 28 kDa P0 glycoprotein and a recently cloned protein called PMP-22. Some glycoproteins in this M(r) range in humans, cats and some other mammals react with HNK1, a mouse monoclonal antibody that identifies a carbohydrate epitope shared between the immune system and a number of adhesion proteins in the nervous system. A variety of antibodies to P0, PMP-22, and the carbohydrate determinants reacting with HNK1 were used to characterize immunochemically these 19 to 28 kDa glycoproteins of cat PNS myelin. The HNK1-reactive components include P0 and two slightly smaller 23 to 26 kDa proteins that are immunologically related to P0. However, HNK1 reacts most strongly with a lower molecular weight glycoprotein that does not react with the antibodies to P0 and was identified as PMP-22. Since the carbohydrate structure reacting with HNK1 is generally expressed on adhesion molecules, this result suggests that PMP-22 may function in cell-cell or membrane-membrane interactions. Furthermore, the related human anti-MAG monoclonal IgM antibodies from patients with neuropathy also react strongly with PMP-22, suggesting that it may be a target antigen in the pathogenesis of this disease. Purified PNS and CNS myelin from bony fish (toadfish and trout) were also shown to contain major glycoproteins, in the same 19 to 28 kDa M(r) range, that react very strongly with HNK1. It is shown that fish myelin has major proteins of this size that are immunologically and structurally related to mammalian P0, and it is demonstrated here that one of the strongly HNK1-positive proteins reacted well with an antiserum raised to bovine P0. The presence of high levels of the adhesion-related HNK1 epitope on these major myelin proteins of fish suggests that this carbohydrate structure may have played a role in the molecular evolution of myelin.     

Enhancement of neurite outgrowth by the soluble form of human L1 (neural cell adhesion molecule)

L1, a neural cell adhesion molecule, is involved in neurite outgrowth, migration and fasciculation. Although L1 is a membrane glycoprotein expressed on neural cells, the soluble form of L1 is generated in vivo by proteolysis. In the present study, a stable transfectant of Chinese hamster ovary (CHO) cells secreting human L1 without cytoplasmic and membrane spanning domains was generated, and the function of the secreted L1 was examined. Explants from embryonic chick brain stem were cultured on a substrate coated with polyethylenimine (PEI) alone, on substrate-bound L1 or in medium containing soluble L1. The neurites induced by L1, both the substrate-bound form and the soluble form, were 2-3 times longer than those cultured on PEI. The ability of the soluble L1 to induce neurite formation was slightly greater than that of the substrate L1. The present results demonstrated that neurite outgrowth was induced not only by substrate-bound L1 but also by soluble L1. Soluble L1 could be a pharmaceutical candidate for the promotion of nerve regeneration.     

Spatio-temporal diversity in the microenvironments for neural cell adhesion molecule, neural cell adhesion molecule-polysialic acid, and L1-cell adhesion molecule expression by sensory neurons and their targets during cochleo-vestibular innervation

Sixteen phases in the microenvironments were defined for the structural development and innervation of the cochleo-vestibular ganglion and its targets. In each phase the cell adhesion molecules, neural cell adhesion molecule, neural cell adhesion molecule-polysialic acid, and L1-cell adhesion molecule, were expressed differentially by cochleo-vestibular ganglion cells, their precursors, and the target cells on which they synapse. Detected by immunocytochemistry in staged chicken embryos, in the otocyst, neural cell adhesion molecule, but not L1-cell adhesion molecule, was localized to the ganglion and hair cell precursors. Ganglionic precursors, migrating from the otocyst, only weakly expressed neural cell adhesion molecule. Epithelial hair cell precursors, remaining in the otocyst, expressed neural cell adhesion molecule, but not L1-cell adhesion molecule. Post-migratory ganglion cell processes expressed both molecules in all stages. The cell adhesion molecules were most heavily expressed by axons penetrating the otic epithelium and accumulated in large amounts in the basal lamina. In the basilar papilla (cochlea), cell adhesion molecule expression followed the innervation gradient. Neural cell adhesion molecule and L1 were heavily concentrated on axonal endings peripherally and centrally. In the rhombencephalon, primitive epithelial cells expressed neural cell adhesion molecule, but not L1-cell adhesion molecule, except in the floorplate. The neuroblasts and their axons expressed L1-cell adhesion molecule, but not neural cell adhesion molecule, when they began to migrate and form the dorsal commissure. There was a stage-dependent, differential distribution of the cell adhesion molecules in the floorplate. Commissural axons expressed both cell adhesion molecules, but their polysialic acid disappeared within the floorplate at later stages. In conclusion, the cell adhesion molecules are expressed by the same cells at different times and places during their development. They are positioned to play different roles in migration, target penetration, and synapse formation by sensory neurons. A multiphasic model provides a morphological basis for experimental analyses of the molecules critical for the changing roles of the microenvironment in neuronal specification.     

Cytoskeleton and cell adhesion molecules: critical targets of toxic agents

Normal development of the nervous system depends upon complex physical interactions between cells and their local environment. These interactions are mediated by several families of cell adhesion molecules (CAMs). Differential expression and function of CAMs are operative in neural tube formation, neuron migration, in post-migratory differentiation, and maintenance of mature neural structure. CAMs also facilitate contact-dependent cell processes, such as formation of cell junctions. Temporal regulation of these molecules during development may provide "windows of vulnerability" to toxicants. In addition to their extracellular binding activities, some CAMs have membrane-spanning domains by which they communicate directly with the cytoskeleton, permitting extracellular signals to be rapidly translated into cell responses via modifications in cytoskeletal organization. These cytologic changes are particularly critical during migration, neurite formation and synaptogenesis. Toxic perturbation of adhesion molecules can have catastrophic effects on morphogenetic processes both directly and via events which depend upon cytoskeletal rearrangement. Toxicants can also act directly upon the cytoskeleton, resulting secondarily in changes of the membrane distribution and function of CAMs. Toxicant-induced changes in CAMs and cytoskeleton may occur contemporaneously. Interference of cell adhesion-cytoskeleton interactions may be a pivotal molecular event dictating developmental consequences of neurotoxicant exposure.     

Orientation of cell adhesion and growth on patterned heterogeneous polystyrene surface

Studies of neurite outgrowth or cell migration, two important processes in neuronal networks formation, are facilitated by cell culture models capable of orientating cellular growth and of designing a well-defined cellular pattern. Heterogeneous polystyrene surfaces composed of oxygen plasma-treated stripes (PSox) with a low hydrophobicity separated by non-treated areas (PS) have these properties. In this study, to guide cell growth, we developed a cell culture model using these supports and we identified the molecular factors involved in cellular orientation. When the heterogeneous supports were not coated, proteins from a serum culture medium were required for cells to line up on PSox. On the other hand, cell orientation on coated surfaces was clearly influenced by competitive adsorption of adhesive proteins such as fibronectin or collagen and anti-adhesive molecules as pluronic F68 or albumin. Attachment factors were adsorbed on PSox stripes while adsorption of anti-adhesive molecules on the most hydrophobic PS areas prevented cell adhesion or growth. Thus, we describe the preparation of a cell culture substrate that succeeded in orientating cell growth and that led to a line of cells on adhesive PSox stripes ranging from 2 to 100 microns width.