CADHERINS EXPRESSED DURING NERVE MYELINATION

我们的总体目标是确定钙粘蛋白在细胞凋亡过程中所起的作用。 源自CNS（视神经）的模型神经结构的髓鞘形成 PNS（坐骨神经） 为此，我们的具体目标是：1。 到 一种新的富含视神经的钙粘蛋白的性质研究 O-cadherin（OCAD），并确定 视神经和坐骨神经表达的钙粘蛋白;（1）我们将完成 通过从视神经cDNA文库中分离cDNA， 我们已经从小鼠组织中制备，使用部分OCAD cDNA作为探针， 和/或针对OCAD融合蛋白的多克隆抗体。 将完整的OCAD一级序列与其他OCAD一级序列进行比较。 钙粘蛋白，根据预期的解决方案进行解释， NCAD的第一胞外结构域（ECI）的晶体结构， 在相关实验中完成。 (2)使用特定和退化 寡核苷酸引物代表同源钙离子的片段， 结合域和细胞质片段共同的钙粘蛋白家族，我们 将从小鼠的视神经和坐骨神经中鉴定钙粘蛋白的完整补体， 神经，其是有大量髓鞘的神经结构。 我们预计 几种钙粘蛋白在这些神经中表达，并且对于每一种新的 钙粘蛋白，我们将产生cDNA探针，和原核融合多肽 从中产生特异性抗血清。 我们将识别细胞（S） 来源于表达每种钙粘蛋白的视神经或坐骨神经， 特别是对于OCAD，以及对于我们鉴定的任何其他新型钙粘蛋白，我们将 利用蛋白质提供组织分布和发育概况 印迹、原位杂交和免疫细胞化学方法。 二. 探讨钙粘蛋白在神经髓鞘形成中的作用： 在视神经和/或坐骨神经中鉴定的单独的钙粘蛋白相互作用 （1）通过共聚焦免疫显微镜进行评估， 在原位精确定位每个钙粘蛋白。 特别是高分辨率 用特异性抗血清成像将有助于揭示推定的共- 钙粘蛋白，其相关蛋白（例如， 连环蛋白和肌动蛋白）和其它粘附分子;细胞界面; (2)和转染研究中。 全长钙粘蛋白cDNA克隆（O-，N-， 例如，E-和/或R-钙粘蛋白）将单独使用和以各种形式使用。 用于在宿主细胞中表达的组合。 表达的能力 钙粘蛋白在单个细胞内相互结合，并诱导 质膜之间的嗜同性和/或嗜异性粘附接触 将通过共聚焦免疫显微镜来评估并置的细胞-细胞边界。 (3)将使用稳定的钙粘蛋白转染子和共转染子。 粘附测定法，其测量单细胞形成 集料. 观察到的粘合剂相互作用对 将测试抗体和可溶性钙粘蛋白片段的抑制作用 在他的实验室拥有的组织培养系统中 确立了习

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.