Ligatin from embryonic chick neural retina

Ligatin, a filamentous protein previously found in suckling rat ileum, has been purified from plasma membranes of embryonic chick neural retina. The isolated plasma membranes are covered in part by 4.5-nm filaments that can be released from the membranes by treatment with Ca++. Subsequent dialysis against EGTA followed by sieve chromatography results in purification of the 10,000-dalton ligatin monomer. When labeled either with radioisotopes or with fluorescamine, the monomer is shown to electrophorese as a single discrete band in polyacrylamide gels. However, during standard fixing and staining procedures it diffuses from the gels and thus is not visualized. Ligatin's amino acid composition is distinguished by its high content of polar residues, especially Glx and Asx, and by the presence of phosphorylated serine. Upon re-addition of Ca++, purified ligatin monomers polymerize to form filaments 3 nm in Diam, identical to those formed by purified ileal ligatin. However, in both retina and ileum, the filaments observed on plasma membranes are greater than 3 nm in Diam. In ileum, this enlargement results from ligatin's function as a baseplate for the attachment of another protein, a beta-N-acetylhexosaminidase, to the cell surface. In retina, a corresponding difference in diameter between filaments seen in vivo and those formed from repolymerized ligatin alone and the co-solubilization of other proteins with ligatin suggest that ligatin may also function there as a baseplate for other cell surface proteins. The proteins associated with ligatin in retina differ morphologically from beta-N-acetylhexosaminidase and do not possess this enzymatic activity.     

Gangliosides support neural retina cell adhesion

Cell surface carbohydrates and complementary carbohydrate receptors may mediate cell-cell recognition during neuronal development. To model such interactions, we developed a technique to test the ability of cell surface lipids (particularly glycosphingolipids) to mediate specific cell recognition and adhesion (Blackburn, C.C., and Schnaar, R.L. (1983) J. Biol. Chem. 258, 1180-1188). When cells were incubated on plastic microwells adsorbed with various glycolipids, carbohydrate-specific cell adhesion was readily detected. We report here the use of this method to study adhesion of embryonic chick neural retina cells to purified cell surface lipids. Rapid and specific cell adhesion was observed when the neural retina cells were incubated on surfaces adsorbed with gangliosides (an important class of neuronal cell surface glycoconjugates) but not on surfaces adsorbed with various neutral glycosphingolipids, phospholipids, or sulfatide. This suggests that the observed cell adhesion was specific for the carbohydrate moiety of the adsorbed ganglioside and was not due to nonspecific ionic or hydrophobic interactions. Although the surface density of adsorbed lipid required to support cell adhesion was the same for all gangliosides examined, the extent of adhesion varied when different purified gangliosides were used. Ganglioside-specific adhesion was not dependent on the presence of calcium (at 37 degrees C) and was attenuated by pretreatment of the cells with trypsin. The extent of ganglioside-directed neural retinal cell adhesion varied with embryonic age. These results imply that gangliosides may play a role in cell-cell recognition in the developing nervous system.     

Expression of v-src in embryonic neural retina alters cell adhesion, inhibits histogenesis, and prevents induction of glutamine synthetase.

Using Rous sarcoma virus as the vector, v-src or c-src genes were introduced into 6-day chicken embryo retina tissue in organ culture and their effects on retina development were investigated. Overexpression of c-src in many of the cells had no noticeable effect on retina development. In contrast, infection with v-src resulted in abnormal histogenesis and inhibition of differentiation. Although only a portion of the cells in infected tissue expressed the oncogene and displayed the transformation phenotype, the other cells were also hindered from becoming normally positioned and organized. Therefore, presence of oncogene-transformed cells within the tissue hindered organization and development of adjacent nontransformed cells. Failure of normal cell relationships impeded induction by cortisol of glutamine synthetase in Muller glia, which requires contact associations of the glia cells with neurons. The transformed cells tended to assemble into chaotic clusters, suggesting that their adhesiveness and contact affinities had become altered. This was confirmed by aggregation experiments with dissociated cells which showed that adhesiveness of transformed cells was greatly reduced and that they had lost the ability to cohere with nontransformed cells. In binary mixtures of transformed and nontransformed cells, the two sorted out into separate aggregates. Transformed cells formed loose clusters devoid of tissue architecture; aggregates of nontransformed cells became organized into retinotypic structures, and glutamine synthetase was inducible. Our findings suggest that the mechanisms of cell adhesion and cell affinities are a key target of v-src activity in infected cells and that modification of the cell surface may be a leading factor in other cellular changes characteristic of the v-src transformation phenotype.     

Spontaneous and lectin-induced redistribution of cell surface receptors on embryonic chick neural retina cells.

The mobility of plant lectin receptors in the plane of the membrane is examined for cells prepared from embryonic chick neural retinas by a variety of procedures. Cells liberated from the intact tissue by trypsin treatment followed by mechanical dissociation are able to redistribute their receptors into 'caps' both spontaneously and in the presence of a multivalent lectin. These cells, dispersed by trypsinization, upon repair in culture for a suitable period of time lose their ability to redistribute lectin receptors. Cells dispersed by mechanical means without prior trypsin treatment are unable to undergo 'cap' formation. In addition, cells within intact tissues are also unable to redistribute their lectin receptors into 'caps.' Based on these observations we propose that within solid tissues which have assumed their characteristic architecture, cell surfaces are immobilized, and that this phenomenon may be a critical parameter in determining the potential of a cell to undergo morphogenetic rearrangements.     

Emergence of flat cells from glia in stationary cultures of embryonic chick neural retina

Summary When embryonic retina is dissociated into a single cell suspension and maintained in stationary culture, a population of flat cells is found on the culture dish. We have carried out a morphologic and immunologic study of the emergence of this population in vitro. Ten- and fourteen-day-old chick embryo retinas were dissociated with trypsin, seeded on glass cover slips for various times, and prepared for scanning electron microscopy (SEM) and immunofluorescence (IF) for Vimentin, an intermediate filament protein. SEM indicates that the characteristic flat cell morphology is initiated in some cells in as little as 30 min after the start of the culture. Not all of the cells that attach flatten. As incubation proceeds, small clusters of cells that had formed in suspension attach to the substrate, and flat cells emerge from them. The flattened cells are positive for Vimentin by IF within 10 min of attachment. The percent of fluorescent cells found on the substrate is constant during the time in culture. This suggests that flat cells do not attach first, followed by neural cells, but that the neural cells and flat cells attach to the dish at the same rate. When aggregates that had formed in suspension attach to the substrate, they are anchored by flat cells that migrate out of the aggregate. Since Vimentin appears in the cultured cells within 10 min, it is unlikely that it has been newly synthesized. Thus, the same cells that contained Vimentin in the retina now express it as flat cells. this supports the hypothesis that flat cells derive from the same cells in the retina that give rise to Müller cells. We have also observed the emergence of a population of cells with short (0.5μm) microvilli that appear within 8 h of culture. They seem to be a distinct subpopulation of the cells on the upper portion of attached clusters. This research was supported in part by grant EY-04892 and RR-0715 from the National Institutes of Health, Bethesda, MD, and a grant from the W.W. Smith Foundations     

Interferon suppresses steroid-inducible glutamine synthetase biosynthesis in embryonic chick neural retina

Effect of chick interferon on the biosynthesis of glutamine synthetase (L-glutamate: ammonia ligase (ADP-forming), EC 6.3.1.2) was studied in the embryonic chick neural retina cultures induced for the enzyme activity by hydrocortisone. The retinal enzyme radioactively labelled with [³H]leucine was precipitated by specific antibody against the enzyme isolated from adult chick liver. The immunological determination offered evidence that the suppressive effect of interferon on the hormonal induction of the enzyme was primarily due to reduced rate of its synthesis and accumulation.     

Interferon suppresses glutamine synthetase induction in chick embryonic neural retina.

Two different proteins precipitable with antiserum to albumin exist in liver. One is albumin, the other is precursor albumin. Liver cells in suspension contain mainly precursor, but secrete only albumin. In subcellular fractions isolated from liver homogenate, 95.3% of anti-albumin precipitable protein in the rough endoplasmic reticulum, 51.4% in the smooth endoplasmic reticulum, 33.5% in the Golgi apparatus and 0% in the supernatant fraction was precursor albumin. The results suggest that albumin precursor is synthesized in the rough endoplasmic reticulum and converted into albumin in the smooth endoplasmic reticulum and the Golgi apparatus.     

Zinc inhibits p75NTR-mediated apoptosis in chick neural retina.

It has previously been documented that Zn2+ inhibits TrkA-mediated effects of NGF. To evaluate the ability of Zn2+ to attenuate the biological activities of NGF mediated by p75NTR, we characterized the effects of this transition metal cation on both binding and the pro-apoptotic properties of the NGF-p75NTR interaction. Binding of NGF to p75NTR displayed higher affinity in embryonic chick retinal cells than in PC12 cells. NGF induced apoptosis in dissociated cultures of chick neural retina. The addition of 100 microM Zn2+ inhibited binding and chemical cross-linking of 125I-NGF to p75NTR, and also attenuated apoptosis mediated by this ligand-receptor interaction. These studies lead to the conclusion that Zn2+ antagonizes NGF/p75NTR-mediated signaling, suggesting that the effect of this transition metal cation can be either pro- or anti-apoptotic depending on the cellular context.     

Inhibition of embryonic neural retina cell-substratum adhesion with a monoclonal antibody

The C1H3 monoclonal antibody recognizes two distinct developmentally regulated cell surface antigens, with molecular masses of 170,000 and 140,000 daltons, in embryonic chick neural retina (Cole, G. J., and Glaser, L. (1984) Proc. Natl. Acad. Sci. U. S. A., in press). In vitro, the 170,000-dalton polypeptide is released by retinal cells into the surrounding culture medium and is present in material sedimentable at 100,000 X g. This pelletable material contains particles designated as adherons (Schubert, D., LaCorbiere, M., Klier, F. G., and Birdwell, G. (1983) J. Cell Biol. 96, 990-998) which promote cell-substratum adhesion of chick neural retina cells. In the present study, evidence is provided that the C1H3 monoclonal antibody inhibits cell adhesion to adheron-coated dishes when bound either to cells or to the adherons. The failure of other monoclonal antibodies, that bind to retinal cells with equal abundance, to disrupt adhesion demonstrates that the effect is specific. These data suggest that the neural-specific 170,000-dalton C1H3 polypeptide is the neural cell-adhesion molecule which is responsible for the ability of adherons to bind to cells.     

Interferon suppresses steroid-inducible glutamine synthetase biosynthesis in embryonic chick neural retina.

Effect of chick interferon on the biosynthesis of glutamine synthetase (L-glutamate: ammonia ligase (ADP-forming), EC 6.3.1.2) was studied in the embryonic chick neural retina cultures induced for the enzyme activity by hydrocortisone. The retinal enzyme radioactively labelled with [3H]leucine was precipitated by specific antibody against the enzyme isolated from adult chick liver. The immunological determination offered evidence that the suppressive effect of interferon on the hormonal induction of the enzyme was primarily due to reduced rate of its synthesis and accumulation.     

Glycoprotein differences among cells of the 14-day embryonic chick neural retina

In order to test the hypothesis that the progressive layering and differentiation of cell types during the development of the neural retina are associated with cell surface alterations we have separated distinct cell populations from the 14-day embryonic chick retina. Cells of these populations have been shown to differ in associative behavior and intramembrane particle content. We now report that these cells differ in cell surface glycoproteins. Proteins were labeled with two different extrinsic labels and one metabolic label. We used enzymatic transfer of galactose from UDP-gal to cellular acceptors, and borotritide reduction after galactose oxidation as extrinsic labels. Glucosamine incorporation was used as the metabolic label. In all these cases, we were able to identify bands on electrophoretic gels which were unique to individual populations.     

A role for adherons in neural retina cell adhesion

Embryonic chick neural retina cells release glycoprotein complexes, termed adherons, into their culture medium. When absorbed onto the surface of petri dishes, neural retina adherons increase the initial rate of neural retina cell adhesion; they also stimulate the rate of cell-cell aggregation. Adheron-stimulated adhesion is tissue specific, and the spontaneous aggregation of neural retina cells is inhibited by monovalent Fab' fragments prepared from an antiserum against neural retina adherons. Therefore cell surface antigenic determinants shared with adherons are involved in normal cell-cell adhesions. The particles from the heterogeneous neural retina population contain many proteins and several glycosaminoglycans. The adherons migrate as a symmetrical 12S peak on sucrose gradients and are predominantly 15-nm spheres when examined by electron microscopy. Finally, the specific activity of neural retina adherons increases from embryonic days 7 through 12 and then declines. These results suggest that glycoprotein particles may be involved in some of the adhesive interactions between neural retina cells and between the cells and their environment.     

pp60c-src expression in transdifferentiating cultures of embryonic chick neural retina cells

Chick embryo neural retinal cells transdifferentiate extensively into lens cells when cultured in Eagle's MEM containing horse and fetal calf sera (FHMEM). Such cultures express elevated levels of pp60c-src-associated tyrosine kinase activity relative to parallel cultures prevented from transdifferentiating by the addition of supplementary glucose (FHGMEM) or replacement of MEM by medium 199 (F199). Northern blotting and in vitro translation studies suggest that c-src mRNA levels are only slightly higher in late transdifferentiating (FHMEM) cultures as compared to parallel blocked (FHGMEM or F199) cultures. By immunocytochemical staining, we show that pp60c-src protein is largely localized in cell groups undergoing conversion into lens (i.e. expressing delta crystallin) in late FHMEM cultures. Initial studies of pp60c-src in chick lens tissues during development indicate that higher kinase activity is found in the epithelial cells relative to mature lens fibres. Thus pp60c-src may be expressed both during the differentiation of lens cells in vivo and during the transdifferentiation of neural retina cells into lens in vitro.     

A decay of gap junctions in association with cell differentiation of neural retina in chick embryonic development.

Ultrastructural studies of thin-sectioned and freeze-cleaved materials were performed on developing retinal tissues of 3- to 9-day-old chick embryos to clarify the junctional structures between neural retinal cells and between neural retinal cells and cells of the pigmented epithelium. Frequency, size and position of gap junctions in developing neural retina are different at each stage of development. In 3-day-old embryos, some cells adhere to each other by gap junctions immediately below the outer limiting membrane of neural retinae. The size and number of gap junctions increase remarkably during 5-6 days of incubation. In this period of development, well developed gap junctions consisting of subcompartments of intramembrane particles are found between cell surfaces at both the outer limiting membrane region and the deeper portion of the neural retina. Gap junctions disappear thereafter, and at 7-5 days of incubation, small gap junctions are predominant between cell surfaces at the outer limiting membrane region, while the frequency of gap junctions in the deeper portion is very low. At 9 days of incubation, gap junctions are rarely found. Typical gap junctions are always found between neural retinal cells and those of the pigmented epithelium in embryos up to 7-5 days of incubation. Tight junctions are not found in the neural retina or between neural retina and pigmented epithelium throughout the stages examined.