Our overall goal is to define the roles that cadherins play during myelination of model neural structures derived from the CNS (optic nerve) and PNS (sciatic nerve). To this end our specific aims are: 1. TO COMPLETE THE CHARACTERIZATION OF A NOVEL OPTIC NERVE-ENRICHED CADHERIN WE HAVE IDENTIFIED, O-CADHERIN (OCAD), AND TO IDENTIFY THE FULL COMPLEMENT OF CADHERINS EXPRESSED IN OPTIC AND SCIATIC NERVES; (1) We will complete the sequence of OCAD by isolating cDNAs from an optic nerve cDNA library that we have prepared from mouse tissue, using as probes partial OCAD cDNAs, and/or polyclonal antibodies we have raised against an OCAD fusion protein. The complete OCAD primary sequence will be compared with those of other cadherins, an interpreted in light of the anticipated solution of the crystal structure of the first extracellular domain (ECI) of NCAD that we are completing in related experiments. (2) Using specific and degenerate oligonucleotide primers representing segments of the homologous calcium binding domains and cytoplasmic segments common to the cadherin family, we will identify the full complement of cadherins from mouse optic and sciatic nerves, which are heavily myelinated neural structures. We expect that several cadherins are expressed in these nerves, and for each novel cadherin, we will generate cDNA probes, and prokaryotic fusion polypeptides from which specific antisera will be raised. We will identify the cell(s) of origin in optic or sciatic nerve that express each cadherin, in particular for OCAD, and for any other novel cadherin we identify we will provide tissue distributions and developmental profiles using protein blotting, in situ hybridization, and immunocytochemical methods. II. TO EXPLORE CADHERIN FUNCTION IN NERVE MYELINATION: The capacity of individual cadherins identified in optic and/or sciatic nerves to interact with each other will be assessed (1) by confocal immunomicroscopy to precisely localize each cadherin in situ. In particular, high resolution imaging with specific antisera will be useful in revealing putative co- localization patterns for cadherins, their associated protein (e.g., catenins and actin) and other adhesion molecules at cell; cell interfaces; (2) and in transfection studies. Full length cadherin cDNA clones (O-, N-, E-and/or R-cadherin, for example) will be used singly and in various combinations for expression in host cells. The capacity of the expressed cadherins to associate with each other within a single cell, and induce homophilic and/or heterophilic adhesive contacts between plasma membranes at apposed cell-cell borders will be assessed by confocal immunomicroscopy. (3) Stable cadherin transfectants and co-transfectants will be used in assays for adhesion that measures the ability of single cells to form aggregates. The sensitivity of observed adhesive interactions to inhibition by antibodies and soluble cadherin fragments will be tested collaboratively in tissue culture systems that his laboratory has established.
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