The goal is to understand how plasma membranes of myelinating cells in the central and peripheral nervous systems (CNS, PNS) become organized into mature myelin sheaths. Two major integral proteins unique to myelin are thought to be mediators of myelin compaction in the PNS (protein zero, P(o)) and CNS (the myelin proteolipid protein, PLP). These proteins are unrelated to each other biochemically, but in their respective nervous system divisions they are each believed to be responsible for the formation and maintenance of the intraperiod line (IPL), formed by the close apposition of the extracellular leaflets of the myelin bilayer. In non-glial cells transfected with a P(o) cDNA, P(o) expression at the plasma membranes of adjacent cells is accompanied by the formation of intercellular adhesive contacts, readily detectable by immunofluorescence, which ultrastructurally resemble an IPL. The formation of these contacts is mediated by a 'functional domain' in the extracellular segment of P(o) that will be identified (Aim I). By using site-directed mutagenesis methods, segments of the cDNA encoding the P(o) molecule will be mutated and expressed in non-glial cells. The capacity of each construction to induce adhesive zones at cell-cell borders will be assessed immunocytochemically by confocal microscopy, and by routine and immunoelectron microscopy of stable transfectants.Stable transfectants will be used in an assay for adhesion that measures the ability of single cells to form aggregates in suspension. Poly- and monospecific antibodies directed against peptide segments, and truncated P(o) molecules secreted by stable transfectants will be used to inhibit cell-cell adhesion, mediated by the intact P(o) molecule. Several models for the topology of PLP have been proposed. An accurate topological map (Aim II) is essential for functional studies on this molecule. intramembranous, cytoplasmic and extracellular domains of the PLP molecule will be defined using proteases, and poly- and monospecific antibodies as probes. The role of specific segments of the polypeptide in establishing the disposition of PLP in the bilayer will be assessed by site-directed mutagenesis studies. Mutagenized cDNAs will be expressed in a coupled transcription-translation system containing membranes derived from rough endoplasmic reticulum, that are the acceptor membranes for the nascent polypeptide in vivo. Cultured oligodendrocytes and non-glial transfectants expressing high levels of PLP will be used in conjunction with PLP antibodies of defined specificity in immunocytochemical experiments that will localize the extracellular and intracellular domains.
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