PubMed Health⌕ Search

Biomedical subjects

U Rutishauser

Publications and source records attributed to U Rutishauser.

At least 91 records · Page 5Linked to original sources
PubMed1986

Induction of neural cell adhesion molecule (NCAM) in Xenopus embryos.

Using a classical neural induction protocol (H. Spemann and H. Mangold (1924). Roux' Arch. Entwicklungsmech. Org. 123, 389-517), it has been demonstrated that the sustained presence of NCAM in Xenopus embryos, as detected by immunohistochemistry, was confined to the experimentally induced nervous system and the primary host nervous system. Furthermore, in vitro NCAM expression by dorsal blastopore lip and animal pole tissue was detected only when the two tissues were cultured in contact. These and other results show that readily detected and sustained levels of NCAM expression in Xenopus can be used as a marker for neural tissue and an early positive indicator that neural induction has occurred. They suggest that the observed levels of NCAM are a consequence of and not a prerequisite for induction. Using NCAM expression in vitro to determine the minimum time necessary for this induction to occur in vivo, it was found that NCAM was first detected in cultured animal pole that had been removed at stage 10.75 or later. Thus, an inductive step necessary and sufficient for stimulation of NCAM expression in animal pole tissues had not occurred or was reversible prior to the first 2 to 2.5 hr of gastrulation.

Age Factors↗
PubMed1986

The distribution of NCAM in the chick hindlimb during axon outgrowth and synaptogenesis.

We have determined the distribution and form of the neural cell adhesion molecule (NCAM) in the chick hindlimb from initial axon outgrowth (stage 17 1/2) until 3 days posthatching by immunohistological staining and sodium dodecyl sulfate-polyacrylamide gel electrophoresis immunoblots. Axons stained intensely for NCAM at all ages, whereas nonneuronal limb components exhibited dynamic changes in staining. Mesenchymal cells in the sclerotome adjacent to the neural tube developed NCAM immunoreactivity in an anterior-posterior sequence which correlated with the sequence of axonal outgrowth. Low to moderate amounts of NCAM were detected within and surrounding presumptive nerve pathways, consistent with a permissive role for NCAM in axon extension, but not with a precise delineation of pathway boundaries. On myotubes immunoreactivity for NCAM remained low from stage 26 to 30 when it increased dramatically in both aneural and control limbs, indicating that its appearance is not triggered by nerve-dependent activity or trophic interactions. The increase was temporally associated with muscle cleavage and may encourage subsequent axon ramification as well as synaptogenesis. Staining remained high on muscle fibers during secondary myotube formation and only declined during the week before hatching when polyneuronal innervation is withdrawn and the mature synaptic pattern becomes stabilized. This loss of muscle NCAM occurred first on fast and then on slow muscle fibers. Together these results suggest that the timing of innervation may be controlled by the muscle, through NCAM expression, but that the subsequent suppression of muscle NCAM may occur as a result of nerve-mediated activity.

Animals↗
PubMed1986

Expression and function of neural cell adhesion molecule during limb regeneration.

The neural cell adhesion molecule (NCAM) has been detected in regenerating limb bud of adult newts in addition to brain and peripheral nerves. In the regenerating tissue, NCAM was found primarily on mesenchymal cells and also in wound epidermis. Infusion of Fab fragments of antibodies to NCAM into limb buds at the early blastema stage delayed the regenerative process. Previous studies have indicated that NCAM serves as a homophilic ligand for adhesion among cells that express this molecule and, in doing so, can influence the interaction of nerves with their environment. The expression of NCAM in regenerating limb and the effects of antibody infusion are therefore consistent with the observation that limb regeneration requires interactions among axons and mesenchymal cells.

Animals↗
PubMed1986

Growth cone localization of neural cell adhesion molecule on central nervous system neurons in vitro.

Ultrastructural analysis of colloidal gold immunocytochemical staining and immunofluorescence microscopy has been used to study the presence of neural cell adhesion molecule (NCAM) on the surface of neuronal growth cones. The studies were carried out with cultures of rat hypothalamic and ventral mesencephalic cells, using morphology and expression of tyrosine hydroxylase, neurofilaments, and glial fibrillary acidic protein as differential markers for neurons and glia. NCAM was found on all plasmalemmal surfaces of neurons including perikarya and neurites. The density of NCAM varied for different neurons growing in the same culture dish, and neurons had at least 25 times more colloidal gold particles on their plasmalemmal membranes than astroglia. Of particular interest in the present study was a strong labeling for NCAM on all parts of neuritic growth cones, including the lamellar and filopodial processes that extend from the tip of the axon. The density of NCAM was similar on different filopodia of the same growth cone. Therefore, in situations where homophilic (NCAM-NCAM) binding might contribute to axon pathfinding, a choice in direction is more likely to reflect differences in the NCAM content of the environment, rather than the distribution of NCAM within a growth cone. On the other hand, the variation in NCAM levels between single neurons in culture was significant and could provide a basis for selective responses of growing neurites.

Animals↗
PubMed1986

Topography of N-CAM structural and functional determinants. I. Classification of monoclonal antibody epitopes.

12 distinct neural cell adhesion molecule (N-CAM) epitopes, each recognized by a different monoclonal antibody (mAb), have been characterized in terms of the major structural and functional features of the molecule. Seven antibodies, each recognizing the amino-terminal region of the molecule, altered the rate of N-CAM-mediated adhesion. Four of these were inhibitors, two of which also recognized a heparin-binding N-CAM fragment. The other three antibodies specifically enhanced the rate of N-CAM-mediated adhesion. Three epitopes, one polypeptide- and two carbohydrate-dependent, were associated with the sialic acid-rich central portion of the molecule. The remaining two antibodies were found to react with intracellular determinants, and are specific for the largest of the three major N-CAM polypeptide forms. Studies on the ability of one antibody to hinder recognition of native N-CAM by another antibody suggested that the epitopes associated with N-CAM binding functions are in close proximity compared with the other determinants. The classification of these mAb epitopes has allowed the topographical placement of key N-CAM features, as described in the following paper, and provides valuable probes for analysis of both the structure and function of N-CAM.

Animals↗
PubMed1986

Topography of N-CAM structural and functional determinants. II. Placement of monoclonal antibody epitopes.

The accompanying report (Watanabe, M., A. L. Frelinger III, and U. Rutishauser, 1986, J. Cell Biol., 103:1721-1727) describes a set of monoclonal antibodies (mAbs) directed against N-CAM epitopes representing the known major structural and functional domains of the molecule. In this study, we have generated and separated a variety of peptide fragments from N-CAM, and then used their size and reactivity with each antibody to position the antigenic sites along the peptide chain. This epitope map, together with the biological properties of the antibodies and previous studies on N-CAM, have been used to construct a topographical model for the molecule in the cell membrane.

Amino Acid Sequence↗
PubMed1986

Rapid axonal transport of the neural cell adhesion molecule.

The neural cell adhesion molecule (NCAM) is a cell-surface glycoprotein that mediates cell-cell interactions in the nervous system during development. In the present study, we demonstrate that NCAM is axonally transported in 3-d-old chick retinal ganglion cells and that it travels within the fast component of axonal transport (FC). Proteins were radiolabeled in retinal ganglion cell bodies after intraocular injection of 35S-methionine. The presence of radiolabeled NCAM in the optic nerves and contralateral tecta was detected by specific immunoadsorption to a monoclonal antibody. Major radioactive polypeptide bands at relative mobilities of approximately 200,000, 150,000, and 120,000 Mr (after SDS-PAGE) were recognized by the anti-NCAM antibody. These bands comigrated in 1-dimensional gels with components of purified NCAM from chick brain. The 2 largest NCAM polypeptides (at 200,000 and 150,000 Mr) were found to be transported in this system, while the 120,000 Mr form was apparently not transported. The ratio and electrophoretic profiles of the 2 transported forms of NCAM remained similar in the retina, optic nerve, chiasm, tract, and tectum, suggesting that there is no interconversion of the 2 major polypeptides. The fraction of NCAM in the 35S-labeled FC proteins appears to be at least an order of magnitude less than in the plasma membrane, suggesting that the turnover rate of NCAM at this age is slower than for other membrane proteins of the CNS.

Animals↗
PubMed1985

Influences of the neural cell adhesion molecule on axon growth and guidance.

The neural cell adhesion molecule (NCAM) has been shown to be a ligand in the formation of cell-cell bonds. This molecule is present on essentially the entire surface of differentiated nerve cells, including the cell body, neurite shaft, and growth cone. In mediating membrane-membrane adhesion, NCAM appears to ligate with itself, and one of its most obvious functions is in the self-association of nerve fibers to form fascicles. In most cases fasciculation occurs by the successive elongation of axons along other axons and, therefore, is likely to represent a growth cone-neurite shaft interaction as well as a shaft-to-shaft adhesion. Competition between neurite shafts and the surrounding substrate for growth cone adhesion probably represents a major factor in the branching of nerve bundles. In addition to neurons, NCAM appears on some glial and muscle cells. Recent experiments suggest that this molecule is involved in growth cone guidance along adhesive pathways on glial precursors in the vertebrate central nervous system, and in the initial interaction of axons with muscle prior to synaptogenesis.

Animals↗
PubMed1985

Phylogeny of a neural cell adhesion molecule.

The phylogeny of adhesion among cells derived from neural tissue has been examined using a combination of functional and immunological analyses. The presence of the neural cell adhesion molecule (NCAM) was evaluated with respect to NCAM-specific antigenic determinants attached to a polypeptide chain with appropriate electrophoretic properties. By these criteria, NCAM-like molecules were detected in all embryonic and adult vertebrates tested, and an adult mollusc, but not in an adult insect, crustacean, or nematode. The functional assays measured adhesiveness by simple aggregation of neural membrane vesicles, as well as by NCAM-specific binding between membranes from different species. The presence of the NCAM antigen in vertebrate membranes correlated with binding activity in both the NCAM-specific and general adhesion assays, implying that the adhesiveness of these membranes largely reflects NCAM-mediated binding. The results also indicate that NCAM function has been conserved during the evolution of vertebrates, and supports the possibility that mechanisms of nerve-nerve, nerve-muscle, and nerve-glial interaction, which have been demonstrated previously to involve NCAM, may be similar for many chordates. Whereas NCAM was not detected in adult fly and worm, these species did express NCAM-like antigens transiently during early development. These results are consistent with the hypothesis that NCAM is required during several periods of development, and that the functions of this molecule in nematodes and insects may be distinct from or a subset of those that occur in vertebrates. The expanded role of the molecule represented by its expression during later stages of vertebrate development may thus have been an important contribution to the evolution of chordates.

Animals↗
PubMed1985

Specific alteration of NCAM-mediated cell adhesion by an endoneuraminidase.

A phage endoneuraminidase that specifically cleaves alpha-2, 8-linked polysialic acid has been found to be a useful probe for examining the biological role of this sugar moiety on the neural cell adhesion molecule (NCAM). The enzyme caused a 3.3-fold increase in the rate of NCAM-dependent aggregation of membrane vesicles from chicken embryonic brain, without the nonspecific effects previously encountered with the use of exoneuraminidases. The enhancement of aggregation was closely correlated with removal of sialic acid as assessed by electrophoretic mobility. Extension of this analysis to cultures of spinal ganglia indicated that removal of sialic acid by the endoneuraminidase results in an increase in the thickness of neurite bundles. This enhancement of fasciculation was reversed by addition of anti-NCAM Fab, suggesting that the enzyme treatment was not toxic and did not produce nonspecific effects on adhesion. Injection of the enzyme into the eyes of 3.5-d chicken embryos consistently produced a striking array of abnormalities in those parts of the neural retina that contained the highest concentrations of NCAM at the time of injection. These perturbations included a dramatic thickening of the neural epithelium in the posterior eye, a failure of cells in this region to elongate radially, formation of an ectopic optic fiber layer, and an incomplete association of the presumptive pigmented epithelium with the neural retina. These results provide the first direct evidence that the polysialic acid on NCAM has a regulatory effect on adhesion between living cells, and that the amount of this carbohydrate is critical for the normal morphogenesis of nerve tissue.

Animals↗
PubMed1984

Comparison of two cell surface molecules involved in neural cell adhesion.

Two cell surface molecules found in mouse brain, N-CAM and the L1 antigen, were compared in terms of their cell adhesion function, polypeptide structures, antigenic determinants and distribution in cerebellar tissue. Fab fragments of polyclonal antibodies to either N-CAM or L1 antigen only partially inhibited the rate of calcium-independent aggregation of neuroblastoma N2A cells, whereas complete and more efficient inhibition was obtained when they were used in combination. Despite the functional similarity, comparison of the electrophoretic behaviour of the purified molecules and of their proteolytic fragments shows that the L1 antigen polypeptide is distinct from that of N-CAM. In addition, no antigenic cross-reactivity was detected between the two molecules. In cryostat sections of cerebellum from young post-natal mice, N-CAM was found to be present in all cell and neurite layers, whereas L1 antigen was expressed only in regions containing post-mitotic cells. These results indicate that two chemically and histochemically distinct cell surface polypeptides can contribute to the calcium-independent adhesiveness of neural cells, and suggest that their differential expression might cause adhesive specificity among cells of developing neural tissues.

Animals↗
PubMed1984

Guidance of optic axons in vivo by a preformed adhesive pathway on neuroepithelial endfeet.

Antibodies against the neural cell adhesion molecule (NCAM) were used in vivo both to localize NCAM antigenic determinants in developing tissues of the chicken visual system and to perturb cell-cell adhesion during growth of optic fibers to the tectum. The immunohistochemical studies revealed a staining pattern on neuroepithelial cells which coincided with certain regions of the presumptive route for optic axons, not only with respect to the overall pathway from the eye to the tectum, but also in the preferential distribution of the antigen on the marginal endfeet which are contacted by optic axon growth cones. The antibody-perturbation studies, which involved intraocular injection of anti-NCAM Fab at embryonic Day 3.5, demonstrated that inhibition of NCAM-mediated adhesion results in a dramatic distortion of growth cone-neuroepithelial cell relationships and consequently of the optic pathway. Together, these studies suggest that guidance of optic axons along the margin of the brain is at least in part influenced by a preformed adhesive pathway on neuroepithelial cells associated with NCAM antigens.

Animals↗
PubMed1984

Fiber-fiber interaction and tectal cues influence the development of the chicken retinotectal projection.

The development of the retinotectal projection has been studied by a new experimental approach combining antibodies against the nerve cell adhesion molecule (NCAM), and techniques for mapping neuronal pathways using rhodamine B isothiocyanate (RITC) crystals. Anti-NCAM Fab', which specifically inhibits neurite fasciculation, was injected into the eye cup of 4-day-old chicken embryos. After 4-6 days of development, a small RITC crystal was placed on the neural retina to stain selectively axons arising from a localized region. One day later the retina, optic nerve, and tectum were examined and the paths of the fluorescent retinal ganglion cell axons were traced. These studies have led to four observations: (i) The presence of anti-NCAM Fab' causes the axons to form a disordered nerve bundle at the optic fissure. (ii) Disorder produced in the optic nerve persists throughout the optic pathway up to the tectum. (iii) Many of the misrouted fibers growing on or near the tectal surface can at least partially correct their position. (iv) Late axons grow in straight tracks along other fibers and do not correct their position. Together the results suggest that formation of the retinotectal projection involves both reading of positional cues on the tectum by growth cones of early arriving retinal axons and the tracking of growth cones along preexisting fibers that normally belong to neighboring retinal ganglion cells.

Animals↗
PubMed1983

Neuron-glia adhesion is inhibited by antibodies to neural determinants.

Suspensions of embryonic chick neuronal cells adhered to monolayers of glial cells, but few neurons bound to control monolayers of fibroblastic cells from meninges or skin. Neuronal cell-glial cell adhesion was inhibited by prior incubation of the neurons with Fab' fragments of antibodies to neuronal membranes. In contrast, antibodies to the neural cell adhesion molecule (N-CAM) did not inhibit the binding. These results suggest that a specific adhesive mechanism between neurons and glial cells exists and that it is mediated by CAM's that differ from those so far identified.

Animals↗
PubMed1983

Molecular topography of the neural cell adhesion molecule N-CAM: surface orientation and location of sialic acid-rich and binding regions.

Chemical analyses and binding studies have been correlated to clarify the relationship of structure to function in the neural cell adhesion molecule (N-CAM) from embryonic chicken brain. N-CAM isolated from the cell surface appears to include two closely related polypeptide chains. Treatment with neuraminidase of such preparations of N-CAM bound by antibodies on solid supports yielded components of Mr 140,000 and 170,000. These components each had the same amino-terminal sequence as N-CAM and gave nearly identical profiles on peptide maps. Immunoprecipitation of N-CAM from 9-day brain cells treated with tunicamycin yielded corresponding components of Mr 130,000 and 160,000, suggesting that the differences between these two components of N-CAM are in the polypeptide rather than the carbohydrate portions of the molecules. N-CAM appears to be oriented with the amino terminus extending away from the cell surface and with the bulk of the sialic acid near the middle of the peptide chain. As shown previously, incubation of N-CAM at 37 degrees C generates a fragment (Fr1) of Mr 65,000 that lacks most of the sialic acid. Treatment of membranes with Staphylococcus aureus V-8 protease released a fragment (Fr2) of N-CAM that contained most of the sialic acid; this fragment had an Mr of 108,000 after neuraminidase treatment. Both of these fragments contain the amino-terminal portion of the polypeptide chain. At least a portion of the N-CAM binding site was found to be located in the amino-terminal region of the peptide chain. Most or all of the sialic acid was not directly involved in binding, although it can influence binding, as indicated by the finding that neuraminidase-treated N-CAM (desialylated-N-CAM) bound to cells to a greater extent than untreated N-CAM. The Fr1 and the Fr2 fragments in solution did not bind to cells but were as effective as N-CAM and desialylated-N-CAM as competitors for N-CAM binding to cells. When fixed covalently to beads, N-CAM, desialylated-N-CAM, and the Fr1 and Fr2 fragments bound specifically to cells. In contrast, the N-CAM autolysis products released along with Fr1 neither bound to cells nor competed for N-CAM binding. In addition to suggesting a location for the N-CAM binding region, the accumulated results raise the possibility that valence may play a key role in N-CAM binding.

Animals↗
PubMed1983

Neural cell adhesion molecule mediates initial interactions between spinal cord neurons and muscle cells in culture.

Previous studies in this laboratory have described a cell surface glycoprotein, called neural cell adhesion molecule or N-CAM, that appears to be a ligand in the adhesion between neural membranes. N-CAM antigenic determinants were also shown to be present on embryonic muscle and an N-CAM-dependent adhesion was demonstrated between retinal cell membranes and muscle cells in short-term assays. The present studies indicate that these antigenic determinants are associated with the N-CAM polypeptide, and that rapid adhesion mediated by this molecule occurs between spinal cord membranes and muscle cells. Detailed examination of the effects of anti-(N-CAM) Fab' fragments in cultures of spinal cord with skeletal muscle showed that the Fab' fragments specifically block adhesion of spinal cord neurites and cells to myotubes. The Fab' did not affect binding of neurites to fibroblasts and collagen substrate, and did not alter myotube morphology. These results indicate that N-CAM adhesion is essential for the in vitro establishment of physical associations between nerve and muscle, and suggest that binding involving N-CAM may be an important early step in synaptogenesis.

Animals↗
PubMed1982

Chemical characterization of a neural cell adhesion molecule purified from embryonic brain membranes.

A neural cell adhesion molecule (N-CAM) was purified in milligram quantities from detergent extracts of embryonic chick brain membranes. N-CAM has an unusual carbohydrate content and structure, is polydisperse in solution, and is associated with proteolytic activity leading to its spontaneous cleavage. The carbohydrate composition of N-CAM includes 13 mol of sialic acid but only 1.4 mol of galactose/100 mol of amino acids, suggesting the presence of a sialic acid to protein linkage not previously observed in higher organisms. N-CAM appears to be an integral membrane protein in that its extraction from membranes required detergent. Although soluble, the purified molecule was aggregated (Mr = 0.5 to 1.2 X 10(6)) and polydisperse in detergent-free solutions. N-CAM from brain also migrated as a broad but continuously stained region from Mr = 200,000 to Mr = 250,000 on sodium dodecyl sulfate-polyacrylamide gel electrophoresis; the molecule from retina was similar but had a somewhat faster mobility. Desialation of N-CAM did not significantly change its behavior in solution, but converted both brain and retinal N-CAM to components migrating on sodium dodecyl sulfate-polyacrylamide gel electrophoresis as material of about Mr = 140,000. Despite the apparent heterogeneity, amino acid sequence analysis and comparison of proteolytic fragments suggest that all forms of the glycoprotein are derived from the same polypeptide chain. On prolonged incubation at neutral pH, N-CAM undergoes apparent proteolysis to yield a polypeptide that contains little sialic acid and has a Mr = 65,000 on sodium dodecyl sulfate-polyacrylamide gel electrophoresis, a separate sialic acid-rich component, and a variety of small peptides. The 65,000-dalton polypeptide appeared to contain all of the antigenic determinants of intact N-CAM that neutralize the adhesion-blocking ability of anti-retinal cell Fab' fragments.

Amino Acid Sequence↗