Fluorescent myelin proteins provide new tools to study the myelination process.
We present here a new approach which permits us to follow myelin proteins within living, actively myelinating cells. We have developed probes to study the spatial and temporal incorporation of proteins into the myelin sheath by expressing myelin proteins fused to the green fluorescent protein (GFP). GFP from the jellyfish Aequorea victoria and its derivatives, e.g., blue fluorescent protein (BFP) were used as molecular reporters to monitor the intracellular distribution of myelin proteins. Fusion proteins (14 kD myelin basic protein [MBP]-GFP, 21 kD MBP-GFP) were expressed in primary Schwann cells (SCs) and their distribution was monitored by confocal microscopy. The autofluorescent chimeric proteins were readily visualized and their subcellular localization was unaffected by the GFP reporter. However, because of the length of culturing time necessary to establish permanent cell lines, we found that it was not possible to obtain MBP-GFP stable SCs that also were capable of myelinating neuronal axons. We therefore devised a way of introducing vectors under conditions where cells are dividing in response to endogenous stimuli, and therefore are still capable of myelinating. We designed a protocol in which SCs cocultured with dorsal root ganglion (DRG) neurons are transfected while they are actively dividing. SCs transfected in this way exhibit a good level of protein expression and retain their myelinating phenotype. The fusion protein expression lasts long enough to observe "green myelin. " These fluorescently tagged myelin proteins will allow high-resolution examination of the protein and membrane traffic in normal myelinating cells.
Pedraza
L
L
Program in Cell Adhesion, Department of Biochemistry and Molecular Biology, Mount Sinai School of Medicine, New York, New York 10029, USA.
Colman
D R
DR
D001369:Axons / Q000378:metabolism; D002462:Cell Membrane / Q000378:metabolism; D002467:Cell Nucleus / Q000378:metabolism; D018920:Coculture Techniques; D005727:Ganglia, Spinal / Q000166:cytology / Q000502:physiology; D049452:Green Fluorescent Proteins; D006367:HeLa Cells; D006801:Humans; D007202:Indicators and Reagents / Q000378:metabolism; D008164:Luminescent Proteins / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism; D008970:Molecular Weight; D009185:Myelin Proteins / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism / Q000502:physiology*; D009186:Myelin Sheath / Q000502:physiology*; D011993:Recombinant Fusion Proteins / Q000737:chemistry / Q000378:metabolism; D012583:Schwann Cells / Q000378:metabolism / Q000502:physiology
D007202:Indicators and Reagents; D008164:Luminescent Proteins; D009185:Myelin Proteins; D011993:Recombinant Fusion Proteins; D049452:Green Fluorescent Proteins