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Classic 18.5- and 21.5-kDa Myelin Basic Protein Isoforms Associate with Cytoskeletal and SH3-Domain Proteins in the Immortalized N19-Oligodendroglial Cell Line Stimulated by Phorbol Ester and IGF-1

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Abstract

The 18.5-kDa classic myelin basic protein (MBP) is an intrinsically disordered protein arising from the Golli (Genes of Oligodendrocyte Lineage) gene complex and is responsible for compaction of the myelin sheath in the central nervous system. This MBP splice isoform also has a plethora of post-translational modifications including phosphorylation, deimination, methylation, and deamidation, that reduce its overall net charge and alter its protein and lipid associations within oligodendrocytes (OLGs). It was originally thought that MBP was simply a structural component of myelin; however, additional investigations have demonstrated that MBP is multi-functional, having numerous protein-protein interactions with Ca2+-calmodulin, actin, tubulin, and proteins with SH3-domains, and it can tether these proteins to a lipid membrane in vitro. Here, we have examined cytoskeletal interactions of classic 18.5-kDa MBP, in vivo, using early developmental N19-OLGs transfected with fluorescently-tagged MBP, actin, tubulin, and zonula occludens 1 (ZO-1). We show that MBP redistributes to distinct ‘membrane-ruffled’ regions of the plasma membrane where it co-localizes with actin and tubulin, and with the SH3-domain-containing proteins cortactin and ZO-1, when stimulated with PMA, a potent activator of the protein kinase C pathway. Moreover, using phospho-specific antibody staining, we show an increase in phosphorylated Thr98 MBP (human sequence numbering) in membrane-ruffled OLGs. Previously, Thr98 phosphorylation of MBP has been shown to affect its conformation, interactions with other proteins, and tethering of other proteins to the membrane in vitro. Here, MBP and actin were also co-localized in new focal adhesion contacts induced by IGF-1 stimulation in cells grown on laminin-2. This study supports a role for classic MBP isoforms in cytoskeletal and other protein-protein interactions during membrane and cytoskeletal remodeling in OLGs.

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Abbreviations

CNS:

Central nervous system

DAPI:

4′,6-diamidino-2-phenylindole

DMEM:

Dulbecco’s Modified Eagle Medium

GFP:

Green fluorescent protein

Golli:

Gene of oligodendrocyte lineage

IGF-1:

Insulin-like growth factor-1

MBP:

Myelin basic protein

MAGUK:

Membrane-associated guanylate kinase

NGS:

Normal goat serum

OLG:

Oligodendrocyte

OPC:

Oligodendroglial progenitor cell

PBS:

Phosphate-buffered saline

PKC:

Protein kinase C

PMA:

Phorbol-12-myristate-13-acetate

PNS:

Peripheral nervous system

PSD-95:

Post-synaptic density protein of 95 kDa

RFP:

Red fluorescent protein

TIRF:

Total internal reflection fluorescence

UTR:

Untranslated region

ZO-1:

Zona occludens 1

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Acknowledgments

This work was supported by the Canadian Institutes of Health Research (MOP #86483, J.M.B. and G.H.), and the Natural Sciences and Engineering Research Council of Canada (RG121541 to G.H.). G.H. is a Tier I Canada Research Chair. G.S.T.S. is a recipient of a Doctoral Studentship from the Multiple Sclerosis Society of Canada. The pGEX2T-ZO-1-SH3 plasmid was a kind gift from Dr. Maria Balda (University College London, UK). The pGFP-ZO-1 plasmid was a kind gift from Dr. Heidi Wunderli-Allenspach (Institute of Pharmaceutical Sciences, ETH-Zürich). The authors are grateful to Dr. Vladimir Bamm for many helpful discussions, Ms. Sara Gagnon and Ms. Lindsay Petley-Ragan for their technical assistance in the early stages of this project, and to Dr. Reihua (Ray) Lu for generous use of his epifluorescence microscope.

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Authors and Affiliations

  1. Department of Molecular and Cellular Biology, University of Guelph, 50 Stone Road East, Guelph, ON, N1G 2W1, Canada

    Graham S. T. Smith & George Harauz

  2. Molecular Structure and Function Program, Hospital for Sick Children, Toronto, ON, Canada

    Lopamudra Homchaudhuri & Joan M. Boggs

  3. Department of Laboratory Medicine and Pathobiology, University of Toronto, Toronto, ON, Canada

    Lopamudra Homchaudhuri & Joan M. Boggs

Authors
  1. Graham S. T. Smith
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  2. Lopamudra Homchaudhuri
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  3. Joan M. Boggs
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  4. George Harauz
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Corresponding author

Correspondence to George Harauz.

Additional information

Special issue: In honor of Dr. Bob Ledeen.

Electronic supplementary material

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11064_2011_700_MOESM1_ESM.tif

Supplementary Figure 1. Fluorescent intensity profile analyses of RFP-MBP-C1-UTR (red) within membrane-ruffled regions of N19-OLGs when compared to β-actin, γ-actin, α-tubulin, and the SH3-domain-containing proteins cortactin and ZO-1 following 5 min stimulation with PMA. Areas in dashed squares are shown magnified on the right. The yellow lines in each image indicate the area within the ruffled regions that was examined for intensity. Increases in co-localization of MBP with cytoskeletal proteins can be observed for each treatment as indicated by the intensity profiles. Identical observations were observed with RFP-MBP-C8-UTR and RFP-MBP-21.5-UTR (not shown). Scale bar = 20 μm (TIFF 18829 kb)

11064_2011_700_MOESM2_ESM.tif

Supplementary Figure 2. Three-dimensional reconstructions of 0.3 μm serial images of N19-OLGs treated with PMA, demonstrating an enrichment of RFP-MBP-C1-UTR (red) in membrane-ruffled regions when compared to β-actin, γ-actin, α-tubulin, and the SH3-domain-containing proteins cortactin and ZO-1, following a 5 min stimulation using PMA. Increases in co-localization of MBP and these cytoskeletal proteins can be observed along ruffled membrane edges (yellow, white arrowheads) of OLGs compared to GFP alone. Images for each PMA-stimulation of MBP are provided from both a vertical and angular perspective to help distinguish structural changes of the ruffled plasma membrane. Scale bar = 20 μm (TIFF 18403 kb)

Video 1. Transfected N19-OLGs responding to PMA treatment showing dynamics of RFP-MBP-C1-UTR (red) and GFP-β-actin (green) over a 10 min time course. MBP and actin show co-localization within membrane-ruffled regions at the periphery of the cell and throughout the cell body. Images for each channel were acquired every 10 s, and the frame rate of the video was set at 10 frames/s. Scale bar = 10 μm (Video 1) or 5 μm (Video 2) (MP4 238 kb)

Video 2. Transfected N19-OLGs responding to PMA treatment showing dynamics of RFP-MBP-C1-UTR (red) and GFP-β-actin (green) over a 10 min time course. MBP and actin show co-localization within membrane-ruffled regions at the periphery of the cell and throughout the cell body. Images for each channel were acquired every 10 s, and the frame rate of the video was set at 10 frames/s. Scale bar = 10 μm (Video 1) or 5 μm (Video 2) (MP4 283 kb)

Video 3. Transfected N19-OLGs responding to PMA treatment showing dynamics of RFP-MBP-C1-UTR (red) and GFP-α-tubulin (green) over a 6.5 min time course. The MBP and α-tubulin show co-localization within membrane-ruffled regions at the periphery of the cell and throughout the cell body. Images for each channel were acquired every 10 s, and the frame rate of the video was set at 10 frames/s. Scale bar = 10 μm (Video 3) or 5 μm (Video 4) (MP4 81 kb)

Video 4. Transfected N19-OLGs responding to PMA treatment showing dynamics of RFP-MBP-C1-UTR (red) and GFP-α-tubulin (green) over a 6.5 min time course. The MBP and α-tubulin show co-localization within membrane-ruffled regions at the periphery of the cell and throughout the cell body. Images for each channel were acquired every 10 s, and the frame rate of the video was set at 10 frames/s. Scale bar = 10 μm (Video 3) or 5 μm (Video 4) (MP4 82 kb)

Video 5. Transfected N19-OLGs responding to PMA treatment showing dynamics of RFP-MBP-21.5-UTR (red) and GFP-β-actin (green) over a 10 min time course. The MBP and actin show co-localization within membrane-ruffled regions at the periphery of the cell and throughout the cell body. The white arrows show membrane-ruffled regions, whereas the blue arrows show active areas of actin polymerization and co-localization with MBP. Images for each channel were acquired every 10 s, and the frame rate of the video was set at 10 frames/s. Scale bar = 20 μm. (MP4 741 kb)

Video 6. Transfected N19-OLGs responding to PMA treatment showing dynamics of RFP-MBP-C1-UTR (red) and GFP-ZO-1 (green) over a 8 min time course. The MBP and ZO-1 show co-localization within membrane-ruffled regions throughout the plasma membrane and cell body. Images for each channel were acquired every 10 s, and the frame rate of the video was set at 10 frames/s; highlighted pixels (white) show areas of co-localization of MBP and ZO-1. Scale bar = 20 μm (MP4 131 kb)

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Smith, G.S.T., Homchaudhuri, L., Boggs, J.M. et al. Classic 18.5- and 21.5-kDa Myelin Basic Protein Isoforms Associate with Cytoskeletal and SH3-Domain Proteins in the Immortalized N19-Oligodendroglial Cell Line Stimulated by Phorbol Ester and IGF-1. Neurochem Res 37, 1277–1295 (2012). https://doi.org/10.1007/s11064-011-0700-2

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  • DOI: https://doi.org/10.1007/s11064-011-0700-2

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