Binding of I-Concanavalin a and agglutination of embryonic neural retina cells : Age-dependent and experimental changes

Embryonic chick neural retina cells dissociated from retina tissue by treatment with EGTA (a calcium chelator) show an age-dependent decline in ability to agglutinate with concanavalin A (ConA). This developmental change in cell surface properties is not due to loss of ConA-binding sites, since mature retina cells can be rendered agglutinable by mild trypsinization. It is also not due to masking of ConA receptors, or to a decrease in their amount, since retina cells from late embryos (19 days) bind four times as much ¹²⁵I-ConA as cells from early embryos (8 days). Our findings lead us to suggest that, as the retina differentiates the lateral mobility of ConA receptors in the cell membrane decreases resulting in a reduction of cell agglutinability; trypsinization of late embryo retina cells increases the mobility of the receptors and thereby facilitates their clustering by the lectin into a configuration conducive to cell agglutination.The ability of late embryo (19 day) retina cells dispersed with EGTA to agglutinate with ConA could be increased by still other treatments: by pre-incubation of the cell suspension in Tyrode's balanced salt solution (1 h, 37 °C); and by brief pre-exposure to glutaraldehyde. These two treatments did not enhance cell agglutination with wheat germ agglutinin (WGA). Glutaraldehyde treatment of trypsinized cells made them agglutinable with ConA also at 4 °C; cells treated otherwise agglutinated only at higher temperature. Surface-saturation of monodispersed retina cells with ConA at 37 °C—but not at 4 °C—prevented their agglutination with this lectin, but not with WGA; this inhibition was reversible by methyl a-D-glucopyranoside (αMG).     

Cortisol receptors and inducibility of glutamine synthetase in embryonic retina

Glutamine synthetase (GS) is a marker enzyme for Müller glia cells in neural retina. In chick embryo retina GS begins to increase sharply on the 16th day of development, but can be precociously induced by premature supply of the inducer, cortisol, already on the 8th day. At this stage GS inducibility is low, but it increases progressively with embryonic age. We investigated whether there was a corresponding age-dependent increase of cortisol-binding molecules (cortisol receptors) and found that their level is highest in the early retina and decreases with development. In light of this inverse relationship, we examined whether functional characteristics of these receptors change with age, but detected no differences. In in vitro tests, receptors from older retina translocated cortisol into nuclei from young retina, and vice versa, with similar effectiveness. Also, cortisol receptors from liver cells (which differ from retina receptors) can translocate the hormone into retina nuclei, and vice versa. These findings indicate that translocation of cortisol receptors is neither tissue-specific or age-dependent, nor is it conditional on the total amount of receptors normally present in cells. Therefore, the age-dependent increase of GS inducibility in embryonic retina cannot be directly related to quantitative or functional differences of cortisol receptors and is evidently controlled primarily at the gene level. The very large amount of cortisol-binding molecules in early embryonic retina raises the possibility that they play some role in early differentiation of retina cells unrelated to hormone binding.     

Malformation of Embryonic Neural Retina Elicited by BrdU

Exposure of neural retina tissue from early chick embryos (5 and 6-days) to 5-bromo-2'-deoxyuridine (BrdU) for 24 h irreversibly prevented normal histogenesis and resulted in the formation of chaotically disorganized tissue. The sensitivity of the retina to this effect decreased with embryonic age and declined sharply after the commencement of cell stratification. Examination by electron microscopy revealed the following progressive morphologic changes resulting from BrdU treatment: complete breakdown of the outer limiting membrane due to disappearance of its constituent tight junctions which normally anchor cells at the outer retinal surface; collapse and endocytosis of cilia, resulting in the absence of photoreceptor processes; increasing disorganization of the cells which commenced at the outer surface of the retina and progressed inward, resulting in chaotic distortion of the histologic architecture of the retina. Ultrastructural differences were noted between cells in the malformed retina, indicating the presence of several cell types. Possible mechanisms of this BrdU-elicited malformation are considered in the Discussion.     

Photoreceptor number and outer segment disk membrane surface area in the retina of the rat: stereological data for whole organ and average photoreceptor cell

A random sampling scheme is employed to obtain stereological estimates of disk membrane surface area in the entire retina and in the average photoreceptor cell. The scheme involves the use of vertical sections with combined light and electron microscopy at several magnification levels. Left and right retinas from six albino animals were analysed. There were no significant lateral differences. On average, the retina had a volume of 16 mm3, thickness of 200 μm and surface area of 80 mm2 (representing about 56% of the external surface of the eyeball). Photoreceptor disk membranes within outer segments amplified total retinal surface by almost 1000-fold (final surface 770 cm2 per retina). The retina contained 3×107 photoreceptors (packing density 374 000 mm-2) with an average disk membrane surface area of 2600 μm2. Mean nuclear volume in photoreceptor cells was 59 μm3 and the coefficient of variation for the distribution of nuclear volumes was 57%. The data are consistent with an average of 700 disks per photoreceptor cell, a membrane area of 4 μm2 per disk and a convergence ratio of ～260 photoreceptors per optic nerve fibre. The basic scheme could be modified for other species and for direct cell counts conducted on rods and cones separately.     

Neuronal-Glial Interactions in Retina Development

The development of embryonic retinoblasts into phenotypicallymature Müller glial cells has been shown to be dependenton close juxtapositional relationships between heterotypicellsof the retina. In this report, I review experiments in whichwe have attempted to examine the role of actual cell contactin the regulation of biochemical differentiation of retinalglial cells. Probes which bind to cell surface components includingantibodies to the retina cell membrane and plant lectins weretested for their ability to interfere with normal histogenesisand glial maturation in a reaggregation-basedin vitro developmentassay. Data are discussed which show that antibodies to thecell surface and the succinylated derivative of the plant lectinconcanavalin A can markedly impair both histogenesis and glialmaturation potential if introduced into cultures of reaggregatingdissociated embryonic retina cells. Preliminary analyses ofmembrane components which react with the lectin have been performed.The results suggest that certain specific membrane glycopeptidesare expressed by dissociated retina cells in an age-dependentmanner. Also, the results show that decline in the ability ofthe embryonic cells to elaborate these surface components correlateswith the capacity of the cells toreform developmentally regulatoryneuronal-glial communication "linkages"     

Surface proteins on Cloudman melanoma cells

Lactoperoxidase-catalyzed cell surface radioiodination was used to study the size distribution and physiological characteristics of cell surface proteins of Cloudman melanoma cells maintained in vivo (An cell) or in vitro (TC cells). Slight qualitative and quantitative differences were observed between detergent-extracted radiolabeled membrane proteins obtained from An and TC cells when the proteins were resolved by polyacrylamide gel electrophoresis in sodium dodecyl sulfate. Striking differences were observed in the membrane residence times of radioiodinated proteins of the An and TC cells when labeled cells were cultured 24 hr. Many labeled surface proteins are lost more rapidly from An cells than from TC cells. The possibility that the release of surface proteins from tumor cell membranes may be an important factor which determines the nature of the host immune response against incipient cancers is considered.     

Freeze-fracturing and surface labelling of embryonic neural retina cells

Freeze-fracturing of dissociated and aggregating neural retina cells from 7-day chick embryos revealed on the inner faces (PF) of the cell membrane numerous particles 6–20 nm in size. In contrast, the PF faces of blebs and some of the lobopodia that project from the cell surface were practically devoid of such particles. However, the elongated filopodia that abound on these cells showed numerous particles on their PF faces. These regional differences in the distribution of particles on PF faces of these cells are interpreted as reflecting membrane activity that leads to the formation of blebs and lobopodia. The frequent presence of “pits” at the basis of blebs and lobopodia is described. It is suggested that the “pits” are associated with the formation of these membrane projections; they may represent anchoring sites for microfilaments and for microtubules involved in the dynamic structure of the cell surface. ConA-binding sites on these cells were studied by scanning electron microscopy, using labeling with hemocyanin. The distribution of these sites on different regions of the cell surface coincided with the regional differences in the distribution of the inner membrane particles.     

Calcium,cellular adhesion and aggregation competence in the cellular slime mold Polysphondylium violaceum

Cellular adhesion in Polysphondylium violaceum is mediated by Ca²⁺ ions. The extent of cell adhesion exhibited by developing P. violaceum is greater in the presence of 0.5 mM Ca²⁺ than in the absence of Ca²⁺. Vegetative amebae exhibit some adhesive properties, although the cellular interactions expressed by vegetative amebae are not as extensive as those exhibited by developing amebae. If the amebae are incubated in the presence of chelators (EGTA or EDTA) cellular adhesion is prevented and the amebae remain as single cells. Vegetative cell adhesion is blocked by 1 mM EGTA, whereas blocking adhesion in developing cells requires 5- to 10-fold greater concentrations of EGTA. The acquisition of developmental adhesive properties occurs even if the amebae are incubated in the presence of EGTA, suggesting that Ca²⁺ is required for interaction between adhesion sites but not for their formation. P. violaceum amebae become aggregationcompetent (aggregate immediately when placed on a solid surface) at the same time that the developmental adhesion sites are expressed, even when incubated in the presence of EGTA. Thus it seems unlikely that cellular adhesion is required to develop aggregation competence.     

Morphogenesis and pattern formation in the retina of the crayfish Procambarus clarkii

Pattern formation and ommatidial differentiation in the crayfish retina were analyzed using confocal, light and electron microscopy. Optic primordia first appear in the embryo as round elevations covered by a surface epithelial layer. Retinal differentiation begins with a wave of mitotic activity that moves across this epithelium from lateral to medial. Ommatidial cell clusters are visible at the surface along a transition zone, which lies at the interface of the medial undifferentiated retina and the lateral patterned retina. This zone is 8–10 cells wide and composed of small uniform cell profiles. Lateral to the transition zone the initial ommatidial cell clusters form staggered rows across the surface. Each first row cluster contains eight retinula cells surrounded by four cone, two corneagenous and two distal pigment cells. Ommatidial clusters in the first nine rows show significant changes in their organization, which are visible at the surface of the retina. In row 10 the retinula cells recede from the surface and the cone cells close in above them creating a constant cell pattern at the surface. Rhabdome development begins distally and extends downward as the retinula cluster recedes from the surface. Movement of the retinula cells inward and enlargement of the cone and corneagenous cells at the surface creates a two-tiered organization characteristic of each ommatidium. Comparison of retinal pattern formation and differentiation in the crayfish with retinal morphogenesis in Drosophila and other insects show several similarities between the two arthropod groups.     

Membrane dielectric changes indicate induced apoptosis in HL-60 cells more sensitively than surface phosphatidylserine expression or DNA fragmentation

The specific membrane capacitance and conductivity of mammalian cells, which reflect their surface morphological complexities and membrane barrier functions, respectively, have been shown to respond to cell physiologic and pathologic changes. Here, the effects of induced apoptosis on these membrane properties of cultured human promyelocytic HL-60 cells are reported. Changes in membrane capacitance and conductivity were deduced from measurements of cellular dielectrophoretic crossover frequencies following treatment with genistein (GEN). The apparent specific cell membrane capacitance of HL-60 cells fell from an initial value of 17.6±0.9 to 9.1±0.5 mF/m² 4 h after treatment. Changes began within minutes of treatment and preceded both the externalization of phosphatidylserine (PS), as gauged by the Annexin V assay, and the appearance of a sub-G1 cell subpopulation, as determined through ethidium bromide staining of DNA. Treatment by the broad spectrum caspase inhibitor N-benzyloxycarbony-Val-Ala-Asp(O-methyl)-fluoromethyketone (zVAD-fmk) did not prevent these early cell membrane dielectric responses, suggesting that the caspase system was not involved. Although membrane conductivity did not alter during the first 4 h of GEN treatment, it rose significantly and progressively thereafter. Finally, as the barrier function failed and the cells became necrotic, it increased by many orders of magnitude. The effective membrane capacitance and conductivity findings serve to focus attention on the membrane as a site for early participation in apoptosis. In conjunction with our prior reports of the use of dielectric methods for cell manipulation and separation, these results demonstrate that dielectrophoretic technologies should be applicable to the rapid detection, separation, and quantification of normal, apoptotic, and necrotic cells from cell mixtures.     

Alterations in Nicotiana tabacum L. cv Xanthi Cell Membrane Function following Treatment with an Ethylene Biosynthesis-Inducing Endoxylanase

An ethylene biosynthesis-inducing xylanase (EIX) produced by the fungus Trichoderma viride elicited enhanced ethylene biosynthesis and leakage of potassium and other cellular components when applied to leaf disks of tobacco (Nicotiana tabacum L. cv Xanthi). Suspension-cultured cells of Xanthi tobacco responded to EIX by rapid efflux of potassium, uptake of calcium, alkalization of the medium, inhibition of ethylene biosynthesis, and increased leakage of cellular components. EIX-treated cell suspensions released 1-aminocyclopropane-1-carboxylate (ACC) into the surrounding medium, resulting in a reduction of cellular pools of ACC. The responses of both cell suspensions and leaf disks were inhibited (50-80%) by the preincubation of the tissues with the calcium channel blocker La³⁺. High concentrations of EGTA inhibited the alkalization of the medium by cell suspensions responding to EIX, but EGTA alone caused extensive loss of K⁺ and ACC and inhibited ethylene biosynthesis by tobacco cells. Alterations in membrane function appear to be important in the mode of action of EIX in Xanthi cells.     

Separation of abscission zone cells in detached Azolla roots depends on apoplastic pH

In studies on the mechanism of cell separation during abscission, little attention has been paid to the apoplastic environment. We found that the apoplastic pH surrounding abscission zone cells in detached roots of the water fern Azolla plays a major role in cell separation. Abscission zone cells of detached Azolla roots were separated rapidly in a buffer at neutral pH and slowly in a buffer at pH below 4.0. However, cell separation rarely occurred at pH 5.0–5.5. Light and electron microscopy revealed that cell separation was caused by a degradation of the middle lamella between abscission zone cells at both pH values, neutral and below 4.0. Low temperature and papain treatment inhibited cell separation. Enzyme(s) in the cell wall of the abscission zone cells might be involved in the degradation of the pectin of the middle lamella and the resultant, pH-dependent cell separation. By contrast, in Phaseolus leaf petioles, unlike Azolla roots, cell separation was slow and increased only at acidic pH. The rapid cell separation, as observed in Azolla roots at neutral pH, did not occur. Indirect immunofluorescence microscopy, using anti-pectin monoclonal antibodies, revealed that the cell wall pectins of the abscission zone cells of Azolla roots and Phaseolus leaf petioles looked similar and changed similarly during cell separation. Thus, the pH-related differences in cell separation mechanisms of Azolla and Phaseolus might not be due to differences in cell wall pectin, but to differences in cell wall-located enzymatic activities responsible for the degradation of pectic substances. A possible enzyme system is discussed.     

EVIDENCE FOR CELL-SURFACE GLYCOSYLTRANSFERASES : Their Potential Role in Cellular Recognition

Intact chicken embryo neural retina cells have been shown to catalyze the transfer of galactose-¹⁴C from uridine diphosphate galactose (UDP-galactose) to endogenous acceptors of high molecular weight as well as to exogenous acceptors. Four lines of evidence indicate that the galactosyltransferases catalyzing these reactions are at least partly located on the outside surface of the plasma membrane: (a) there is no evidence for appreciable uptake of sugar-nucleotides by vertebrate cells nor did unlabeled galactose, galactose 1-phosphate, or UDP-glucose interfere with the radioactivity incorporated during the reaction; (b) the cells remained essentially intact during the course of the reaction; (c) there was insufficient galactosyltransferase activity in the cell supernatants to account for the incorporation of galactose-¹⁴C into cell pellets; and (d) the intact cells could transfer galactose to acceptors of 10⁶ daltons, and the product of this reaction was in the extracellular fluid. Appropriate galactosyl acceptors interfered with the adhesive specificity of neural retina cells; other compounds, which were not acceptors, had no effect. These results suggested that the transferase-acceptor complex may play a role in cellular recognition.     

An approach for the separation of mononuclear inflammatory cells from central nervous system tissue

It is not known whether currently available methods separate lymphoid cells from nervous system sites of immunologic disorders without the selective enrichment or depletion of particular cell subpopulations. To test the validity of a cell separation technique we developed, we applied this technique to a mixture of guinea pig blood mononuclear cells already characterized for surface membrane characteristics with autologous spinal cord tissue. Using this method, 10–20% of the original blood cell population was recovered. The percentages of E-rosetting and surface immunoglobulin cells in the separated cells was quite similar to that in the added blood cells. EAC-rosetting cell percentages were somewhat lower. This method shows promise for the in vitro study of cells obtained from lesions of autoimmune diseases in the central nervous system.     

Immunomagnetic Purification of Colletotrichum lindemuthianum Appressoria

We developed a method to purify appressoria of the bean anthracnose fungus Colletotrichum lindemuthianum for biochemical analysis of the cell surface and to compare appressoria with other fungal structures. We used immunomagnetic separation after incubation of infected bean leaf homogenates with a monoclonal antibody that binds strongly to the appressoria. Preparations with a purity of >90% could be obtained. Examination of the purified appressoria by transmission electron microscopy showed that most had lost their cytoplasm. However, the plasma membrane was retained, suggesting that there is some form of attachment of this membrane to the cell wall. The purified appressoria can be used for studies of their cell surface, and we have shown that there are clear differences in the glycoprotein constituents of cell walls of appressoria compared with mycelium.     

Cell disorganization and malformation in neural retina caused by antibodies to R-cognin: ultrastructural study

Retina tissue from 6-day chick embryos was organ-cultured for 3 days in the presence of antibodies to R-cognin, a surface antigen of retina cells. The antibodies which are known to bind to this antigen caused a striking malformation: interruption of the outer limiting membrane and extensive cell disorganization resulting in exteriorization of many cells and forming of chaotic masses on the surface of the tissue. Controls did not show these effects. These results further confirm that R-cognin is involved in the mechanism of histotypic contacts and recognition of retina cells, and that it plays an essential role in cell organization and histogenesis in the retina.     

Separation of human rosette- and nonrosette-forming lymphoid cells by countercurrent distribution in an aqueous two-phase system.

Mixtures of aqueous solutions of dextran and of poly(ethylene glycol) form immiscible two-phase systems suitable for the separation (by partition) of cells based on subtle differences in membrane surface properties. Lymphoid cells from human tonsils were subjected to countercurrent distribution in such a system. Analysis of cells from different parts of the countercurrent extraction train by the sheep red blood cell rosette test indicates a separation of rosette-forming and nonrosette-forming lymphocytes with the former having the higher partition coefficient. Since a major determinant of cell partition in these phases is a membrane-charge associated property it appears likely that the rosette-forming cells have a higher surface charge than the nonrosette-forming cells.     

Fibulin 2, a Tyrosine O-Sulfated Protein,Is Up-regulated Following Retinal Detachment

Retinal detachment is the physical separation of the retina from the retinal pigment epithelium. It occurs during aging, trauma, or during a variety of retinal disorders such as age-related macular degeneration, diabetic retinopathy, retinopathy of prematurity, or as a complication following cataract surgery. This report investigates the role of fibulin 2, an extracellular component, in retinal detachment. A major mechanism for detachment resolution is enhancement of cellular adhesion between the retina and the retinal pigment epithelium and prevention of its cellular migration. This report shows that fibulin 2 is mainly present in the retinal pigment epithelium, Bruch membrane, choriocapillary, and to a lesser degree in the retina. In vitro studies revealed the presence of two isoforms for fibulin 2. The small isoform is located inside the cell, and the large isoform is present inside and outside the cells. Furthermore, fibulin 2 is post-translationally modified by tyrosine sulfation, and the sulfated isoform is present outside the cell, whereas the unsulfated pool is internally located. Interestingly, sulfated fibulin 2 significantly reduced the rate of cellular growth and migration. Finally, levels of fibulin 2 dramatically increased in the retinal pigment epithelium following retinal detachment, suggesting a direct role for fibulin 2 in the re-attachment of the retina to the retinal pigment epithelium. Understanding the role of fibulin 2 in enhancing retinal attachment is likely to help improve the current therapies or allow the development of new strategies for the treatment of this sight-threatening condition.