In the long term, we want to understand how CNS and PNS myelin sheaths are assembled. We anticipate that the assembly process can be productively investigated by observing living, actively myelinating cells, using modified myelin proteins as markers and high resolution microscopy to define how separate intracellular biosynthetic pathways ultimately converge to yield myelin. As a first aim, we will engineer and express in host cells a battery of cDNAs encoding the major myelin proteins, each fused to a fluorescent protein (FP) derivative from the jellyfish, Aequorea victoria. Green or Blue Fluorescent Protein (FP cDNA cassettes will be linked to cDNAs encoding the 14K or 21.5k myelin basic proteins (MBP), Po, the myelin associated glycoproteins (MAG), the proteolipid proteins (DM20/PLP), and N- and E-cadherin (NCAD, ECAD), all representative and characteristic myelinating cell components in PNS and/or CNS. The intracellular fates of the naturally fluorescent polypeptides encoded by the chimeric cDNAs will be followed over time in living host cells. In a second aim, the chimeric reagents will be utilized as completely novel, potentially highly informative probes for high resolution visualization of myelin sheath assembly in living Schwann cells and oligodendrocytes. We will deliver the in vitro synthesized mRNAs, singly and in combinations, by rapid intracellular injection (in a pulse) directly into myelinating cells in co-culture with neurons, where protein-protein and protein-membrane interactions can be observed, and the patterns of incorporation into living myelin studied. Traffic in living Schwann cells or oligodendrocytes of the de novo synthesized and fluorescently tagged proteins as they progressively move through intracellular compartments and arrive and distribute in myelin will be monitored over time. It is anticipated that the successful completion of the proposed experiments will reveal for the first time the precise intracellular loci at which proteins destined for myelin first associate with each other, and where newly synthesized proteins and membrane are incorporated into the forming myelin spiral.
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