Interaction of myelin basic protein with cytoskeletal and signaling proteins in cultured primary oligodendrocytes and N19 oligodendroglial cells

Abstract

Background: 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. 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.

Results: To 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.

Conclusions: These 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.

Figure 1

First level of stringency – extraction with Triton X-100. MBP, tubulin, and actin co-immunoprecipitate from the OLG Triton X-100 insoluble pellet. Primary OLGs were extracted with buffer containing 1% TX-100, and the soluble and resuspended insoluble fractions were immunoprecipitated in the lysis buffer with monoclonal anti-MBP SMI 99 antibody and a control antibody. Western blots were immunostained with rabbit polyclonal anti-MBP (E13) antibody, mouse monoclonal anti-MBP antibody (SMI 99), rabbit polyclonal anti-tubulin antibody, and mouse monoclonal anti-actin antibody. Representative results of 4 experiments are shown. Lane 1, supernatant fraction; lane 2, anti-MBP (SMI 99) immunoprecipitate of supernatant; lane 3, control antibody immunoprecipitate of supernatant; lane 4, pellet; lane 5, anti-MBP (SMI 99) immunoprecipitate of pellet; lane 6, control antibody immunoprecipitate of pellet; lane 7, anti-MBP SMI 99 IgG; lane 8, control antibody IgG. Supernatant fraction was not loaded for the gel used for the actin blot, and the immunoprecipitated supernatant fractions shown in lanes 2 and 3 for the actin blot were from a different gel than the remaining samples, due to overloading and overexposure of the immunoprecipitated supernatant samples on the gel/blot used for the remaining samples. Standards for MBP, tubulin, and actin were not loaded on the gels used for these samples, but can be seen in Figure 2a. The 3 bands for MBP at M_r values of approximately 16–27 kDa, indicated by arrows, represent the classic 14-, 18.5-, and 21.5-kDa isoforms of MBP. The 14-kDa isoform was not detected with the monoclonal anti-MBP (SMI 99) antibody but was immunoprecipitated by it, since it was detected by E13 antibody in the anti-MBP (SMI 99) immunoprecipitate (lanes 2,5). Lower-exposure blots show the MBP bands in the immunoprecipitates clearly but did not show those in the pellet or supernatant as well. Therefore, a more highly-exposed blot was chosen for this figure.

Figure 2

Second level of stringency – extraction with Triton X-100 and NP40. MBP, tubulin, actin, ZO-1, and Fyn co-immunoprecipitate from the OLG TX-100 plus NP40-insoluble pellet and supernatant fractions. Primary OLGs were extracted with buffer containing 1% TX-100 plus 1% NP40, and the supernatant and resuspended pellet were immunoprecipitated with mouse monoclonal antibody anti-MBP SMI 99 and a control antibody. Western blots were immunostained with (a) rabbit polyclonal anti-MBP (E13), rabbit polyclonal anti-tubulin, mouse monoclonal anti-actin, rat monoclonal anti-ZO-1, and (b) mouse monoclonal anti-Fyn. Representative results are shown from 3 experiments for MBP, tubulin, and actin, and from 2 experiments for ZO-1 and Fyn. (a, b) lane 1, standard purified protein sample (18.5-kDa isoform for MBP), except for ZO-1 and Fyn (the tubulin sample was run on the same gel as the other lanes, but separated from them by a M_r marker lane and, therefore, was cut out and shown separately to preserve lane alignment); lane 2, supernatant fraction; lane 3, anti-MBP (SMI 99) immunoprecipitate of supernatant; lane 4, control antibody immunoprecipitate of supernatant; lane 5, pellet; lane 6, anti-MBP (SMI 99) immunoprecipitate of pellet; lane 7, control antibody immunoprecipitate of pellet; lane 8, anti-MBP SMI 99 IgG; lane 9, control antibody IgG. Arrows for the MBP blot indicate the 21.5-, 18.5-, 17-, and 14-kDa isoforms of MBP. Lower-exposure blots showed the MBP bands in the immunoprecipitates clearly, but less well for the pellet or supernatant. Therefore, a more highly-exposed blot was chosen for this figure. The M_r of ZO-1 is about 220 kDa, but this antibody also detects several unidentified bands down to 110 kDa in rat liver, according to the manufacturer. (b) Fyn was detected in the pellet and supernatant at M_r of about 60 kDa (lanes 2,5).

Figure 3

Second level of stringency – extraction with Triton X-100 and NP40 – cortactin blot. MBP and cortactin co-immunoprecipitate from the OLG Triton X-100 plus NP40-insoluble pellet and supernatant fractions. Lane 1, supernatant fraction; lane 2, blank; lane 3, pellet; lane 4, anti-MBP (SMI 99) immunoprecipitate of supernatant; lane 5, control antibody immunoprecipitate of supernatant; lane 6, blank; lane 7, anti-MBP (SMI 99) immunoprecipitate of pellet; lane 8, control antibody immunoprecipitate of pellet; lane 9, anti-MBP SMI 99 IgG; lane 10, control antibody IgG. The supernatant and pellet fractions were run on a different gel from the immunoprecipitated samples. The blots were stained with rabbit polyclonal anti-cortactin antibody. Two bands at about 78 kDa and 82 kDa were detected for cortactin. Representative results are shown from 2 experiments.

Figure 4

Third level of stringency – extraction with Triton X-100 and NP40 and DOC. MBP, tubulin, actin, and ZO-1 do not co-immunoprecipitate in 1% TX-100 plus 1% NP40 plus 1% DOC; addition of DOC disrupts the protein complexes. Primary OLGs were extracted with buffer containing 1% TX-100 plus 1% NP40 plus 1% DOC, the soluble and resuspended insoluble fractions were immunoprecipitated with mouse monoclonal antibody anti-MBP SMI 99 and a control antibody. Western blots were immunostained with rabbit polyclonal anti-MBP (E13), rabbit polyclonal anti-tubulin, mouse monoclonal anti-actin, and rat monoclonal anti-ZO-1 antibodies. Lane 1, standard purified protein sample (18.5-kDa isoform for MBP), except for actin and ZO-1; lane 2, supernatant fraction; lane 3, anti-MBP (SMI 99) immunoprecipitate of supernatant; lane 4, control antibody immunoprecipitate of supernatant; lane 5, pellet; lane 6, anti-MBP (SMI 99) immunoprecipitate of pellet; lane 7, control antibody immunoprecipitate of pellet; lane 8, anti-MBP SMI 99 IgG; lane 9, control antibody IgG.

Figure 5

Live cell images of N19-OLGs treated with CytD for 2 hours, and then allowed to recover in its absence. The N19-OLGs after incubation with CytD for 2 hours (a-e), after 20 min recovery (f-j), after 40 min recovery (k-o), and after 60 min recovery (p-t). Panels a, f, k, and p show GFP-actin signal; panels b, g, l, and q show RFP-MBP signal; panels c, h, m, and r show the merge of GFP-actin and MBP-RFP signals; panels d, i, n, and s correspond to intensity plots along line 1, and panels e, j, o, and t represent intensity plots along line 2 for GFP-actin (green) and RFP-MBP (red). The intensity plots are in arbitrary units and do not quantitatively reflect the MBP and actin enrichment at the sites analyzed, but show changes in relative intensity of MBP and actin, their location and their degree of co-localization with time. The arrowheads in white indicate regions where ruffles were observed after removing CytD. The regions with ruffles are shown at larger size in the inset in panels f, g, h, k, l, and m, and the ruffles are indicated in the insets by arrows. Scale bar represents 30 μm.

Figure 6

Live-cell images of another N19-OLG treated with CytD for 2 hours (a-e), after 20 min recovery (f-j), after 40 min recovery (k-o), and after 60 min recovery (p-t). Panels a, f, k, and p show GFP-actin signal; panels b, g, l, and q show RFP-MBP signal; panels c, h, m, and r show the merge of GFP-actin and MBP-RFP signals; panels d, i, n, and s correspond to intensity plots along line 1, and panels e, j, o, and t represent intensity plots along line 2 for GFP-actin (green) and RFP-MBP (red). The intensity plots are in arbitrary units and do not quantitatively reflect the MBP and actin enrichment at the sites analyzed, but show changes in relative intensity of MBP and actin, their location and their degree of co-localization with time. The arrowheads in white indicate regions where ruffles were observed after removing CytD. Scale bar represents 30 μm.