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Exogenous hyalin and sea urchin gastrulation. Part IV: a direct adhesion assay – progress in identifying hyalin's active sites

Published online by Cambridge University Press:  08 June 2009

Haike Ghazarian
Affiliation:
Department of Biology and Center for Cancer and Developmental Biology, California State University, Northridge, 18111 Nordhoff Street, Northridge, CA 91330–8303, USA.
Catherine Coyle-Thompson
Affiliation:
Department of Biology and Center for Cancer and Developmental Biology, California State University, Northridge, 18111 Nordhoff Street, Northridge, CA 91330–8303, USA.
William Dalrymple
Affiliation:
Department of Biology and Center for Cancer and Developmental Biology, California State University, Northridge, 18111 Nordhoff Street, Northridge, CA 91330–8303, USA.
Virginia Hutchins-Carroll
Affiliation:
Department of Biology and Center for Cancer and Developmental Biology, California State University, Northridge, 18111 Nordhoff Street, Northridge, CA 91330–8303, USA. Department of Chemistry and Biochemistry, California State University, Northridge, 18111 Nordhoff Street, Northridge, CA 91330–8262, USA.
Stan Metzenberg
Affiliation:
Department of Biology and Center for Cancer and Developmental Biology, California State University, Northridge, 18111 Nordhoff Street, Northridge, CA 91330–8303, USA.
Ziba Razinia
Affiliation:
Department of Cell Biology, Yale University School of Medicine, 333 Cedar Street, New Haven, CT 06510, USA.
Edward J. Carroll Jr
Affiliation:
Department of Chemistry and Biochemistry, California State University, Northridge, 18111 Nordhoff Street, Northridge, CA 91330–8262, USA.
Steven B. Oppenheimer*
Affiliation:
Department of Biology and Center for Cancer and Developmental Biology, California State University, Northridge, 18111 Nordhoff Street, Northridge, CA 91330–8303, USA. Department of Biology and Center for Cancer and Developmental Biology, California State University, Northridge, 18111 Nordhoff Street, Northridge, CA 91330–8303, USA.
*
All correspondence to: Steven B. Oppenheimer. Department of Biology and Center for Cancer and Developmental Biology, California State University, Northridge, 18111 Nordhoff Street, Northridge, CA 91330–8303, USA. Tel: +818 677 3336. Fax: +818 677 2034. e-mail: [email protected]

Summary

In Strongylocentrotus purpuratus the hyalins are a set of three to four rather large glycoproteins (hereafter referred to as ‘hyalin’), which are the major constituents of the hyaline layer, the developing sea urchin embryo's extracellular matrix. Recent research from our laboratories has shown that hyalin is a cell adhesion molecule involved in sea urchin embryo-specific cellular interactions. Other laboratories have shown it to consist of 2–3% carbohydrate and a cloned, sequenced fragment demonstrated repeat domains (HYR) and non-repeat regions. Interest in this molecule has increased because HYR has been identified in organisms as diverse as bacteria, flies, worms, mice and humans, as well as sea urchins. Our laboratories have shown that hyalin appears to mediate a specific cellular interaction that has interested investigators for over a century, archenteron elongation/attachment to the blastocoel roof. We have shown this finding by localizing hyalin on the two components of the cellular interaction and by showing that hyalin and anti-hyalin antibody block the cellular interaction using a quantitative microplate assay. The microplate assay, however, has limitations because it does not directly assess hyalin's effects on the adhesion of the two components of the interaction. Here we have used an elegant direct assay that avoids the limitations, in which we microdissected the two components of the adhesive interaction and tested their re-adhesion to each other, thereby avoiding possible factors in the whole embryos that could confound or confuse results. Using both assays, we found that mild periodate treatment (6 h to 24 h in sodium acetate buffer with 0.2 M sodium periodate at 4 °C in the dark) of hyalin eliminates its ability to block the cellular interaction, suggesting that the carbohydrate component(s) may be involved in hyalin's specific adhesive function. This first step is important in identifying the molecular mechanisms of a well known cellular interaction in the NIH-designated sea urchin embryo model, a system that has led to the discovery of scores of physiological mechanisms, including those involved in human health and disease.

Type
Research Article
Copyright
Copyright © Cambridge University Press 2009

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References

Alvarez, M., Nnoli, J., Carroll, E.J. Jr., Hutchins-Carroll, V., Razinia, Z. & Oppenheimer, S.B. (2008). Exogenous hyalin and sea urchin gastrulation. Part II: hyalin, an interspecies cell adhesion molecule. Zygote 16, 73–8.CrossRefGoogle ScholarPubMed
Bidwell, J.P. & Spotte, S. (1985). Artificial Seawaters, Formulas and Methods. Boston: Jones & Barlett Publishers, Inc.Google Scholar
Callebout, I., Gilges, D., Vignon, I. & Mornon, J.P. (2000). HYR, an extracellular module involved in cellular adhesion and related to the immunoglobulin-like fold. Protein Sci. 9, 1382–90.CrossRefGoogle Scholar
Carroll, E.J. Jr, Hutchins-Carroll, V., Coyle-Thompson, C. & Oppenheimer, S.B. (2008). Hyalin is a cell adhesion molecule involved in mediating archenteron-blastocoel roof attachment. Acta Histochem. 110, 265–75.CrossRefGoogle ScholarPubMed
Citkowitz, E. (1971). The hyaline layer: its isolation and role in echinoderm development. Dev. Biol. 24, 348–62.CrossRefGoogle ScholarPubMed
Contreras, A., Vitale, J., Hutchins-Carroll, V., Carroll, E.J. & Oppenheimer, S.B. (2008). Exogenous hyalin and sea urchin gastrulation. Part III: Biological activity of hyalin isolated from Lytechinus pictus embryos. Zygote 16, 355–61.CrossRefGoogle ScholarPubMed
Coyle-Thompson, C. & Oppenheimer, S.B. (2005). A novel approach to study adhesion mechanisms by isolation of the interacting system. Acta Histochem. 107, 243–51.CrossRefGoogle ScholarPubMed
Davidson, E.H. (2006). The sea urchin genome: where will it lead us? Science 314, 939–40.CrossRefGoogle ScholarPubMed
Davidson, E.H. & Cameron, R.A. (2002). Arguments for sequencing the genome of the sea urchin Strongylocentrotus purpuratus. <www.genome.gov/pages/research/sequencing/SegProposals/SeaUrchin_Genome.prob.2002>..>Google Scholar
Gray, J., Justice, R., Nagel, G.M. & Carroll, E.J. Jr (1986). Resolution and characterization of a major protein of the sea urchin hyaline layer. J. Biol. Chem. 261 (20), 9282–8.CrossRefGoogle Scholar
Herbst, C. (1900). Ueber das auseinanderegene im furchungs- und gewebe-zellen in kalkfreiem medium. Arch. F. Entwick 9, 424–63.CrossRefGoogle Scholar
Khurrum, M., Hernandez, A., Eskalaei, M., Badali, O., Coyle-Thompson, C. & Oppenheimer, S.B. (2004). Carbohydrate involvement in cellular interactions in sea urchin gastrulation. Acta Histochem. 106, 97106.CrossRefGoogle ScholarPubMed
Kondo, K. (1973). Cell-binding substances in sea urchin embryos. Dev. Growth Differ. 15, 201–16.CrossRefGoogle ScholarPubMed
Latham, V.H., Tully, M.J. & Oppenheimer, S.B. (1999). A putative role for carbohydrates in sea urchin gastrulation. Acta Histochem. 101, 293303.CrossRefGoogle ScholarPubMed
Quinn, G.P. & Kough, M.J. (2006). Experimental Design and Data Analysis for Biologists. Cambridge: Cambridge University Press.Google Scholar
Razinia, Z., Carroll, E.J. Jr & Oppenheimer, S.B. (2007). Microplate assay for quantifying developmental morphologies: effects of exogenous hyalin on sea urchin gastrulation. Zygote 15, 159–64.Google Scholar
Sajadi, S., Rojas, P. & Oppenheimer, S.B. (2007). Cyclodextrin, a probe for studying adhesive interactions. Acta Histochem. 109, 338–42.Google Scholar
Spiro, R.G. (1964). Periodate oxidation of the glycoprotein fetuin. J. Biol. Chem. 239, 567–73.Google Scholar
Spiro, R.G. (1966a). Analysis of sugars in glycoproteins. In Methods in Enzymology vol. 8 (eds Neufeld, E.F. & Ginsburg, V.), pp. 326. New York: Academic Press.Google Scholar
Spiro, R.G. (1966b). Characterization of carbohydrate units of glycoproteins. In Methods in Enzymology vol. 8 (eds Neufeld, E.F. & Ginsburg, V.), pp. 2652. New York: Academic Press.Google Scholar
Stephens, R.E. & Kane, R.E. (1970). Some properties of hyalin: the calcium-insoluble protein of the hyaline layer of the sea urchin egg. J. Cell Biol. 44, 611–7.Google Scholar
Vater, C.A. & Jackson, R.C. (1990). Immunolocalization of hyalin in sea urchin eggs and embryos using an antihyalin-specific monoclonal antibody. Mol. Reprod. Dev. 25, 215–26.Google Scholar
Wessel, G.M., Berg, L., Adelson, D.L., Cannon, G. & Mcclay, D.R. (1998). A molecular analysis of hyalin—a substrate for cell adhesion in the hyaline layer of the sea urchin embryo. Dev. Biol. 193, 115–26.CrossRefGoogle ScholarPubMed