Bruce A. Cunningham

Ontogenetic expression of cell adhesion molecules: L-CAM is found in epithelia derived from the three primary germ layers☆

Immunofluorescence techniques using specific antibodies against the liver cell adhesion molecule, L-CAM, were used to explore the appearance of L-CAM during early embryogenesis and organogenesis, as well as in adult tissue. Immunoblots of L-CAM from embryonic and adult organs indicated that molecules detected in each tissue were L-CAM, and that the antibodies were not simply detecting cross-reacting molecules. L-CAM was found in low levels on pregastrulation embryos. During gastrulation, the molecule remained present on ectoderm but was not detected on mesodermal and definitive endodermal cells. During neurulation, L-CAM disappeared from the neural ectoderm, in which staining for the neural cell adhesion molecule, N-CAM, had previously been shown to increase markedly. During organogenesis, L-CAM appeared in all endodermal structures, in ectoderm other than neural derivatives, in placodes, in extraembryonic ectoderm and endoderm, and in some mesodermal structures such as Wolffian ducts, oviduct, and kidney epithelium. Other mesodermal derivatives were not stained and the molecule was not detected in hemangioblastic areas of the lateral plate mesoderm nor in splanchnopleural derivatives such as spleen, adrenal glands, and gonads. During embryonic induction, for example, neurulation and in early kidney development, changes in L-CAM distribution were correlated with both locations and times of induction events. Analysis of distribution in the adult revealed that L-CAM was present in the stratum germinativum of the skin, in endodermally derived epithelia, in the female reproductive tract, and in the kidneys. In several fully differentiated glandular organs, L-CAM staining was restricted to basal or apical parts of the cell surface. When correlated with previous results obtained for N-CAM, these findings support the idea that local cell surface modulation of a small number of cell adhesion molecules may regulate other primary processes of development to yield specific patterns, both in early development and in organogenesis. Reflections of these patterns remain in adult life.

Cell adhesion molecules as morphoregulators

Many significant advances have been made recently in our understanding of the structure and function of cell adhesion molecules (CAMs). The most provocative, however, are those that indicate that CAM-mediated adhesion may lead to changes in gene expression and those that suggest that the expression of CAM genes may be regulated by the products of Hox and related genes.

Cultured rat hippocampal neural progenitors generate spontaneously active neural networks

We previously demonstrated that the neural cell adhesion molecule (N-CAM) inhibited the proliferation of cultured rat hippocampal progenitor cells and increased the number of neurons generated. We demonstrate here that the continued presence of fibroblast growth factor 2 along with N-CAM or brain-derived neurotrophic factor over 12 days of culture greatly increased the number of both progenitors and neurons. These progenitor-derived neurons expressed neurotransmitters, neurotransmitter receptors, and synaptic proteins in vitro consistent with those expressed in the mature hippocampus. Progenitor cells cultured on microelectrode plates formed elaborate neural networks that exhibited spontaneously generated action potentials after 21 days. This activity was observed only in cultures grown in the presence of fibroblast growth factor 2 and either N-CAM or brain-derived neurotrophic factor. Analysis of neuronal activity after various pharmacological treatments indicated that the networks formed functional GABAergic and glutamatergic synapses. We conclude that mitogenic growth factors can synergize with N-CAM or neurotrophins to generate spontaneously active neural networks from neural progenitors.

Two isoforms of the cold-inducible mRNA-binding protein RBM3 localize to dendrites and promote translation

A diverse set of mRNA‐binding proteins (BPs) regulate local translation in neurons. However, little is known about the role(s) played by a family of cold‐inducible, glycine‐rich mRNA‐BPs. Unlike neuronal mRNA‐BPs characterized thus far, these proteins are induced by hypothermia and are comprised of one RNA recognition motif and an adjacent arginine‐ and glycine‐rich domain. We studied the expression and function of the RNA‐binding motif protein 3 (RBM3), a member of this family, in neurons. RBM3 was expressed in multiple brain regions, with the highest levels in cerebellum and olfactory bulb. In dissociated neurons, RBM3 was observed in nuclei and in a heterogeneous population of granules within dendrites. In sucrose gradient assays, RBM3 cofractionated with heavy mRNA granules and multiple components of the translation machinery. Two alternatively spliced RBM3 isoforms that differed by a single arginine residue were identified in neurons; both were post‐translationally modified. The variant lacking the spliced arginine exhibited a higher dendritic localization and was the only isoform present in astrocytes. When overexpressed in neuronal cell lines, RBM3 isoforms‐enhanced global translation, the formation of active polysomes, and the activation of initiation factors. These data suggest that RBM3 plays a distinctive role in enhancing translation in neurons.

Palmitoylation of the Cytoplasmic Domain of the Neural Cell Adhesion Molecule N-CAM Serves as an Anchor to Cellular Membranes

The neural cell adhesion molecule N-CAM is expressed at key sites during embryonic development and mediates homophilic adhesion between cells both in the embryo and in the adult. N-CAM is expressed in multiple forms and two of the major isoforms differ in their cytoplasmic domains, one (ld form) having an insert of 261 amino acids that is missing in the other (sd form). N-CAM has been previously shown to be palmitoylated, but the sites of acylation have not been localized. We show here that the cytoplasmic domain of the N-CAM became palmitoylated after transfection of a cDNA encoding N-CAM into COS-7 cells, and that this acylation occurs on the four closely spaced cysteines in the cytoplasmic domain of N-CAM. Moreover, when a cDNA encoding only the cytoplasmic domain was transfected into cells, the protein was palmitoylated and associated with membranes even though it lacked a membrane spanning segment. Site directed mutagenesis of the four cysteine residues to serines at positions 5, 11, 16, and 22 in the cytoplasmic domain (723, 729, 734, and 740 in the native protein) eliminated both the palmitoylation and association with the membrane fraction. Mutagenesis of the cysteines individually, in pairs, and in groups of three indicated that C5 is not acylated with either palmitate or oleate, but the other three cysteines are acylated to different extents. Cytoplasmic domains with single cysteine mutations localized primarily in the membrane fraction, while those with three mutations were found primarily in the cytoplasm. Proteins containing two mutated cysteines were found in both the cytoplasm and the membrane fraction with C11 and C16 having the most influence on the distribution in accord with their higher level of acylation. Mutation of the cysteines did not affect the ability of full-length N-CAM to promote aggregation when transfected into COS-7 cells. Based on these results we suggest that the primary role of palmitoylation is to provide a second anchor in the plasma membrane to direct the protein to discrete membrane microdomains or to organize the cytoplasmic region for interaction with factors that affect signaling events resulting from N-CAM mediated adhesion.

Circular permutation of amino acid sequences among legume lectins

Abstract Lectins, the carbohydrate-binding proteins found in a variety of organisms, are a diverse group of proteins. Many lectins from leguminous plants, however, are closely related to each other and are related to the jack bean lectin Concanavalin A by an unusual circular permutation of amino acid sequences.

Expression patterns of the cell adhesion molecule Nr-CAM during histogenesis of the chick nervous system

Neuron-glia-related cell adhesion molecule (Nr-CAM) is a recently characterized cell adhesion molecule in the family of immunoglobulin-related molecules of which the neural cell adhesion molecule, N-CAM, is the prototype. Nr-CAM shares structural properties with another member of this family (neuron-glia CAM, Ng-CAM) and both molecules exhibit homophilic and heterophilic binding properties. To understand better the role of such molecules in development, we have examined the sites of synthesis and expression of Nr-CAM by means of in situ hybridization and immunohistochemistry. Both methods indicated that Nr-CAM is expressed only in the nervous system. The molecule was observed on neurons in both the peripheral and central nervous systems and on epithelial floor plate cells in the spinal cord, but it was absent in the germinal zones. The protein was present on perikarya, but was found preferentially on axonal tracts. As observed for messenger RNAs specifying other cell adhesion molecules, messenger RNA for Nr-CAM was localized in the perikarya. The temporal expression of Nr-CAM was correlated with various neural morphoregulatory events, including cell proliferation and migration, axonal outgrowth and myelination. The molecule was expressed during the onset of neurogenesis at embryonic day 3 in the floor plate epithelium, and then on postmitotic ventral horn motor neurons of the spinal cord. At later stages, it was expressed throughout the spinal cord but disappeared from the floor plate. In the cerebellum, Nr-CAM was found on granule and Purkinje neurons and afferent fibers. Both local and projection neurons in the optic tectum, as well as axonal pathways throughout the telencephalon, expressed Nr-CAM. In the peripheral nervous system, Nr-CAM was expressed strongly in sensory and autonomic ganglia and in the enteric nervous system. At the onset of myelination, there was a general decrease in staining for Nr-CAM protein in the central nervous system but not in the periphery. Comparison of the expression of Nr-CAM to that of the structurally related Ng-CAM showed considerable overlap in their distributions, although there were differences in the levels at which each CAM was observed in particular structures. For example, sympathetic ganglia stained more intensely for Nr-CAM protein than for Ng-CAM. This differential but co-distributed pattern is consistent with the idea that although similar cell adhesion molecules have independent binding specificities, they may have related functions that act synergistically in the development of the nervous system.

Structure and Function of Concanavalin A

Lectins have been extensively used to analyze a variety of fundamental processes in cell biology. In conjuntion with our studies on the cell surface and mitosis, we have determined the amino acid sequence and three-dimensional struction of concanavalin A (Con A), the mitogenic lectin from the jack bean. Knowledge of the structure has been helpful in interpreting experiments on lymphocyte mitogenesis and the effects of Con A on cell surface receptor mobility. Con A subunits for molecular weight 25,500 are folded into dome-like structures of maximum dimensions 42 times 40 times 39 A. The domes are related by 222 symmetry to form roughly tetrahedral tetramers. Each subunit contains two large antiparallel pleated sheets, and subunits are joined to form dimers and tetramers by interactions involving one of these pleated sheets. We have examined the binding of a variety of carbohydrates to Con A and have obtained preliminary data which suggest that there are differences in the saccharide-binding behavior of Con A in solution and in the crystalline state. Dimeric chemical derivatives of Con A have been prepared and shown to have biological activities different from those of the native tetrameric protein. Under different conditions, native Con A exhibits two antagonistic activities on the lymphoid cell surface: the induction of cap formation by its own receptors and the inhibition of the mobility of a variety of receptors, including its own receptors. The dimeric derivative, succinyl-Con A, is just as effective a mitogen as the native lectin, but it lacks the ability to modulate cell surface receptor mobility. The data suggest that neither extensive immobilization of cell surface receptors nor cap formation is required for cell stimulation. Further studies on modulation of receptor translocation suggest that hypothesis that there exists a connecting network of colchicine-sensitive proteins that links receptors of different kinds and mediates their rearrangement. The degree of connectivity of this postulated network appears to be altered by changes in the state of attachment of various surface receptors to the network. Thus the network might provide the cell with a means of transmitting signals such as the stimulus for mitosis by lectins or antigens.

Association between the first two immunoglobulin-like domains of the neural cell adhesion molecule N-CAM

The extracellular domain of N‐CAM contains five immunoglobulin‐like (Ig) and two fibronectin type III‐like domains and facilitates cell‐cell binding through multiple, weak interdomain interactions. NMR spectroscopy indicated that the two N‐terminal Ig‐like domains from chicken N‐CAM (Ig I and Ig II) interact with millimolar affinity. Physico‐chemical studies show that this interaction is significantly amplified when the domains are covalently linked, consistent with an antiparallel domain arrangement. The binding of the two individual domains and the dimerization of the concatenated protein were essentially independent of salt, up to a concentration of 200 mM. The residues in Ig I involved in the interaction map to the BED strands of the β sandwich, and delineate a largely hydrophobic patch.

Cell adhesion molecules: a new perspective on molecular embryology

Abstract Identification and characterization of cell adhesion molecules (CAMs) have suggested new views of the role of cell adhesion in development and have provided an important new way of analysing the molecular mechanisms of embryogenesis. Rapidly developing protein-chemical and molecular biological studies promise to extend these findings in many new directions.