Abstract
BackgroundThe classic myelin basic protein (MBP) isoforms are intrinsically-disordered proteins of 14–21.5 kDa in size arising from the Golli (Gene in the Oligodendrocyte Lineage) gene complex, and are responsible for formation of the multilayered myelin sheath in the central nervous system. The predominant membrane-associated isoform of MBP is not simply a structural component of compact myelin but is highly post-translationally modified and multi-functional, having interactions with numerous proteins such as Ca2+-calmodulin, and with actin, tubulin, and proteins with SH3-domains, which it can tether to a lipid membrane in vitro. It co-localizes with such proteins in primary oligodendrocytes (OLGs) and in early developmental N19-OLGs transfected with fluorescently-tagged MBP.ResultsTo provide further evidence for MBP associations with these proteins in vivo, we show here that MBP isoforms are co-immunoprecipitated from detergent extracts of primary OLGs together with actin, tubulin, zonula occludens 1 (ZO-1), cortactin, and Fyn kinase. We also carry out live-cell imaging of N19-OLGs co-transfected with fluorescent MBP and actin, and show that when actin filaments re-assemble after recovery from cytochalasin D treatment, MBP and actin are rapidly enriched and co-localized at certain sites at the plasma membrane and in newly-formed membrane ruffles. The MBP and actin distributions change similarly with time, suggesting a specific and dynamic association.ConclusionsThese results provide more direct evidence for association of the predominant 18.5-kDa MBP isoform with these proteins in primary OLGs and in live cells than previously could be inferred from co-localization observations. This study supports further a role for classic MBP isoforms in protein-protein interactions during membrane and cytoskeletal extension and remodeling in OLGs.
Highlights
The classic myelin basic protein (MBP) isoforms are intrinsically-disordered proteins of 14–21.5 kDa in size arising from the Golli (Gene in the Oligodendrocyte Lineage) gene complex, and are responsible for formation of the multilayered myelin sheath in the central nervous system
Day [47]); mouse monoclonal anti-MBP, purified IgG2b, was from Covance (Emeryville, CA); mouse monoclonal anti-axotrophin (B2), purified IgG2, and rat monoclonal anti-zonula occludens 1 (ZO-1) antibody (R40.76), IgG2a, were from Santa Cruz Biotechnology (Santa Cruz, CA); rabbit polyclonal anti-α/β-tubulin antibody and rabbit polyclonal anti-cortactin antibody were purchased from Cell Signaling Technology (Beverly, MA); and goat antirabbit IgG conjugated to Horseradish peroxidase (HRP) was purchased from Jackson ImmunoResearch Labs (West Grove, PA)
Immunoprecipitation of the Triton X-100 (TX-100)-insoluble pellet with monoclonal anti-MBP (SMI 99) antibody followed by immunoblotting showed specific enrichment of MBP, tubulin, and actin (Figure 1, lane 5) compared to the control immunoprecipitate (Figure 1, lane 6)
Summary
The classic myelin basic protein (MBP) isoforms are intrinsically-disordered proteins of 14–21.5 kDa in size arising from the Golli (Gene in the Oligodendrocyte Lineage) gene complex, and are responsible for formation of the multilayered myelin sheath in the central nervous system. Myelin basic protein (MBP, the classic membrane-targeted 18.5-kDa isoform) is responsible for adhesion of the cytoplasmic surfaces of the multilayered myelin sheath [1,2], and may form a molecular sieve restricting many oligodendroglial proteins from access them to each other, and tether them and SH3-domain proteins (as demonstrated for the SH3-domain of Fyn-kinase) to a lipid surface [24,25,26] These varied interactions may allow MBP to participate as a signaling hub in myelin formation and remodeling, and to have many other functions in addition to membrane adhesion in compact multilamellar myelin [11,16,27]. Pull-down assays have revealed a potential interaction of MBP with β-tubulin and the cytoplasmic loop of the gap junction protein connexin-43 [46]
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