Neurochemical Research

, Volume 37, Issue 6, pp 1277–1295

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

  • Graham S. T. Smith
  • Lopamudra Homchaudhuri
  • Joan M. Boggs
  • George Harauz
Original Paper

DOI: 10.1007/s11064-011-0700-2

Cite this article as:
Smith, G.S.T., Homchaudhuri, L., Boggs, J.M. et al. Neurochem Res (2012) 37: 1277. doi:10.1007/s11064-011-0700-2

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.

Keywords

Membrane rufflingFocal adhesion contactsPhorbol esterIGF-1Laminin-2CytoskeletonMyelinationLive-cell imagingTIRF microscopyConfocal microscopy

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

Supplementary material

11064_2011_700_MOESM1_ESM.tif (18.4 mb)
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 (18 mb)
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)
View video

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)

View video

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)

View video

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)

View video

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)

View video

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)

View video

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)

Copyright information

© Springer Science+Business Media, LLC 2012

Authors and Affiliations

  • Graham S. T. Smith
    • 1
  • Lopamudra Homchaudhuri
    • 2
    • 3
  • Joan M. Boggs
    • 2
    • 3
  • George Harauz
    • 1
  1. 1.Department of Molecular and Cellular BiologyUniversity of GuelphGuelphCanada
  2. 2.Molecular Structure and Function Program, Hospital for Sick ChildrenTorontoCanada
  3. 3.Department of Laboratory Medicine and PathobiologyUniversity of TorontoTorontoCanada