Abstract
Abstract: :
Purpose: The formation of retina tissue during embryonic development requires interactions between differentiating and migrating cells and a dynamic extracellular matrix. These changing cell to cell and cell to substratum interactions are modulated by changes in the conformation of specific receptors at the cell surface. In early embryonic retina some conformational changes are mediated by disulfide exchanges carried out by a cell surface protein disulfide isomerase, retina cognin. The question this work begins to ask is which receptors are the substrates for disulfide exchange at the cell surface. Previous protein interaction cloning had identified three protein sequences as potential targets: an avidin–like sequence, an EGF–repeat sequence and a tetraspanin sequence. Using a simple retina cell aggregation assay, we asked if known ligands for these molecules interfered with retina cell interactions. Methods: Embryonic chicken retina and forebrain (from embryonic days E6–11) were dissected and separated into suspensions of single cells using either trypsin or trypsin–free procedures. Biotin and recombinant inactivated diphtheria toxin (CRM–197) were used to probe for avidin–like proteins or the heparin–binding EGF–like growth factor (HB–EGF), respectively. Control and experimentally–treated cell suspensions were allowed to reaggregate for 1 to 2 hours during which the cells not in aggregates were counted. Under these conditions untreated cells rapidly reaggregate, for retina in a cognin–dependent manner. Results: Previous work indicated that the substrate for, at least, cognin binding was a membrane protein that was not sensitive to trypsin. The HB–EGF growth factor is a membrane protein but the location of most members of the vertebrate avidin family is not known. Thus, we tested retina cells whose surfaces had been exposed to trypsin and those who had been removed from the tissue by calcium and magnesium removal only. Neither biotin nor CRM–197 perturbed the cognin–mediated reaggregation of the retina cells or the reaggregation of forebrain cells prepared in these ways. Conclusions: The results rule out the simplest hypothesis that either HB–EGFR or an avidin–like protein forms direct cell to cell contacts in the embryonic retina because they are modified by cognin–mediated disulfide exchange. We are currently investigating whether these proteins participate in a more complicated multi–protein adhesion complex, perhaps containing a tetraspanin, at the embryonic retina cell surface.
Keywords: cell adhesions/cell junctions • retinal development • cell membrane/membrane specializations