Analysis of unconventional approaches for the rapid detection of surface lectin binding ligands on human cell lines.

For over a decade our laboratory has developed and used a novel histochemical assay using derivatized agarose beads to examine the surface properties of various cell types. Most recently, we have used this assay to examine lectin binding ligands on two human cell types, CCL-220, a colon cancer cell line, and CRL-1459, a non-cancer colon cell line. We found that CCL-220 cells bound specific lectins better than CRL-1459, and this information was used to test for possible differential toxicity of these lectins in culture, as a possible approach in the design of more specific anti-cancer drugs. Although we have examined the validity of the bead-binding assay in sea urchin cell systems, we have not previously validated this technique for mammalian cells. Here the binding results of the bead assay are compared with conventional fluorescence assays, using lectins from three species (Triticum vulgaris, Phaseolus vulgaris, and Lens culinaris) on the two colon cell lines. These lectins were chosen because they seemed to interact with the two cell lines differently. Binding results obtained using both assays were compared for frozen, thawed and fixed; cultured and fixed; and live cells. Both qualitative and quantitative fluorescence results generally correlated with those using the bead assay. Similar results were also obtained with all of the three different cell preparation protocols. The fluorescence assay was able to detect lower lectin binding ligand levels than the bead assay, while the bead assay, because it can so rapidly detect cells with large numbers of lectin binding ligands, is ideal for initial screening studies that seek to identify cells that are rich in surface binders for specific molecules. The direct use of frozen, thawed and fixed cells allows rapid mass screening for surface molecules, without the requirement for costly and time consuming cell culture.

A novel approach to study adhesion mechanisms by isolation of the interacting system.

For decades most investigations into mechanisms of adhesive interactions have examined whole organisms or single cells. Results using whole organisms are often unclear because it may not be known if a probe used in an experiment is directly affecting the cellular interaction under study or if it is an indirect effect resulting from action on some other structure or pathway. Here we develop a novel approach to isolate the structural components of a cellular interaction by dissecting them out of the organism to study them in a pristine environment away from all confounding factors. We used the adhesion between the archenteron and blastocoel roof of the sea urchin gastrula stage embryo as a model that can be replicated in many other developmental and pathological systems. The isolated components of the cellular interaction and those in the whole organism possessed identical cell surface receptors and adhesive affinities.

Carbohydrate involvement in cellular interactions in sea urchin gastrulation.

The sea urchin embryo is a model for studying cellular interactions that occur in higher organisms because of its availability, transparency, and accessibility to molecular probes. In previous studies, we found that the mannose/glucose-binding lectin Lens culinaris agglutinin entered living sea urchin embryos, bound to specific cell types and caused exogastrulation, when the developing gut (archenteron) falls out of the embryo proper. We have proposed that the lectin bound to sugar-containing ligands, thus preventing attachment of the archenteron to the blastocoel roof, resulting in exogastrulation. Here, we have continued our study of cellular interactions in this model using Lytechinus pictus sea urchin embryos, and have found that inhibitors of glycoprotein/proteoglycan synthesis, tunicamycin and sodium selenate, and the specific glycosidases, beta-amylase, alpha-glucosidase, and alpha-mannosidase, all inhibit archenteron organization, elongation, and attachment to the blastocoel roof in viable swimming embryos. We also show that single cells obtained by disaggregation of 32-h-old sea urchin embryos bind to L. culinaris agglutinin- and concanavalin A-derivatized beads; the binding is blocked by alpha-methyl mannose, but not l-fucose. These cells also bind to beads derivatized with mannan. These results provide evidence for a role of carbohydrate-containing molecules in cellular interactions in sea urchin gastrulation. In a second set of experiments, we found that the supernatant obtained by disaggregation of 24-32-h-old L. pictus embryos in calcium- and magnesium-free sea water contains molecules that cause exogastrulation, archenteron disorganization, inhibition of archenteron elongation and inhibition of archenteron attachment to the blastocoel roof in viable swimming embryos. We propose that the supernatant contains ligands and/or receptors that mediate archenteron development and attachment to the blastocoel roof and are released when embryos are disaggregated into single cells. These studies may lead to a better understanding of the molecular basis of mechanisms that control cellular interactions during development.