The major myelin-resident protein PLP is transported to myelin membranes via a transcytotic mechanism: involvement of sulfatide

Abstract

Myelin membranes are sheet-like extensions of oligodendrocytes that can be considered membrane domains distinct from the cell's plasma membrane. Consistent with the polarized nature of oligodendrocytes, we demonstrate that transcytotic transport of the major myelin-resident protein proteolipid protein (PLP) is a key element in the mechanism of myelin assembly. Upon biosynthesis, PLP traffics to myelin membranes via syntaxin 3-mediated docking at the apical-surface-like cell body plasma membrane, which is followed by subsequent internalization and transport to the basolateral-surface-like myelin sheet. Pulse-chase experiments, in conjunction with surface biotinylation and organelle fractionation, reveal that following biosynthesis, PLP is transported to the cell body surface in Triton X-100 (TX-100)-resistant microdomains. At the plasma membrane, PLP transiently resides within these microdomains and its lateral dissipation is followed by segregation into 3-[(3-cholamidopropyl)-dimethylammonio]-1-propanesulfonate (CHAPS)-resistant domains, internalization, and subsequent transport toward the myelin membrane. Sulfatide triggers PLP's reallocation from TX-100- into CHAPS-resistant membrane domains, while inhibition of sulfatide biosynthesis inhibits transcytotic PLP transport. Taking these findings together, we propose a model in which PLP transport to the myelin membrane proceeds via a transcytotic mechanism mediated by sulfatide and characterized by a conformational alteration and dynamic, i.e., transient, partitioning of PLP into distinct membrane microdomains involved in biosynthetic and transcytotic transport.

FIG 1

PLP undergoes a conformational alteration during transport to myelin membranes. (A) Localization of PLP in developing OLGs analyzed by immunostaining (4C2). Note that in imOLGs, PLP is localized at the cell body plasma membrane (arrows), whereas in mOLGs, PLP-containing vesicular structures in the perinuclear region and myelin sheet were observed. (B, C) Surface staining of mOLGs with anti-PLP antibodies that recognize an extracellular epitope (ET3, O10). Single labeling is shown in panel B, and double labeling is shown in panel C. Note that ET3 binds primarily to the cell body plasma membrane, whereas O10 staining is more prominent in the myelin sheet. Representative pictures of four independent experiments are shown. The scale bars are 10 μm.

FIG 2

Overexpression of syntaxin 3 inhibits surface transport of PLP in OLGs concomitant with TX-100 resistance. (A, B) OPCs were transduced with the vector only (mock), syntaxin 3 (S3↑), or syntaxin 4 (S4↑). (A) Surface and intracellular localization of PLP in mOLGs as determined by immunostaining with anti-PLP antibodies directed against extracellular (O10) and intracellular PLP epitopes (2D2). Syntaxin 3 and 4 overexpression was confirmed by Western blotting. Note the lack of PLP surface expression upon the overexpression of syntaxin 3. (B) TX-100 and CHAPS membrane microdomain association of PLP in mOLGs. PLP (4C2) and caveolin were visualized by Western blotting. Detergent-resistant membrane microdomains are present in fractions 3 and 4. Note that PLP is TX-100 resistant in syntaxin 3-overexpressing OLGs but largely TX-100 soluble in mock-transduced OLGs. (C) ImOLGs were pulse-labeled with Tran³⁵S-label and chased for the times indicated. PLP was immunoprecipitated from the TX-100-soluble (black bars) and -insoluble (white bars) fractions, and the amounts were analyzed by radioactivity counting. The statistical significance of differences between 0-min values and values obtained at the time points indicated is shown (n = 3; **, P < 0.01; ***, P < 0.001; one-way ANOVA with Tukey's posttest). Note that following biosynthesis, PLP is transiently TX-100 resistant. (D) Cell surface proteins of mOLGs were biotinylated, lysed, and subjected to TX-100 extraction and OptiPrep density gradient centrifugation. Biotinylated, surface-localized proteins and nonbiotinylated, i.e., intracellularly localized, proteins were separated by SA immunoprecipitation. PLP was visualized by Western blotting (4C2 antibody). Note that TX-100-resistant PLP is present at the surface. (E) imOLGs were subjected to in situ detergent extraction with either TX-100 or CHAPS prior to fixation. Note that TX-100-resistant PLP localized to the cell body plasma membrane (arrow), whereas CHAPS-resistant PLP resided mainly intracellularly. Pictures and blots representative of at least three independent experiments are shown. The scale bars are 10 μm.

FIG 3

PLP-eGFP is present in the apical and basolateral membrane domains of polarized HepG2 cells. (A) Schematic model of polarized HepG2 cells. The apical (BC, red) and basolateral (blue) membrane domains are separated by tight junctions (green). (B) Localization of PLP-eGFP upon stable expression in HepG2 cells. Note that PLP-eGFP is present in the apical (arrow) and basolateral (arrowhead) membrane domains. (C) Localization of extracellular PLP (ET3) and total PLP-eGFP (inset) at the basolateral surface of HepG2 cells. Nuclei were visualized with DAPI. (D) TX-100 and CHAPS membrane microdomain association of PLP-eGFP in HepG2 cells. PLP-eGFP was visualized by Western blotting (anti-GFP antibody). Detergent-resistant membrane microdomains are present in fractions 3 and 4. (E, F) HepG2-PLP-eGFP cells were left untreated (ctrl) or subjected to in situ detergent extraction with either TX-100 or CHAPS prior to fixation. Apical membranes were visualized by immunostaining for MRP2 (E, insets, red). Note that TX-100-resistant PLP-eGFP localized to apical membranes, whereas CHAPS-resistant PLP-eGFP resided at both membrane domains. The number of PLP-positive BCs among the total MRP2-positive BCs was determined under the conditions indicated (F). Each bar represents the mean result + the SD of four independent experiments. (G) Basolateral-to-apical transcytosis of PLP-eGFP and DPPIV as analyzed by an antibody (Ab) trafficking assay. Antibodies (red) were visualized by immunostaining, and nuclei were stained with DAPI. Note that DPPIV, but not PLP-eGFP (ET3), was transcytosed to the apical membrane domain. The localization of total PLP-eGFP is shown in the insets (green) (H) TLC analysis of cellular glycosphingolipids in HepG2 and HepG2-PLP-eGFP cells. Pictures and blots representative of at least three independent experiments are shown. Scale bars are 10 μm.

FIG 4

Sulfatide is not essential for the transport of PLP to the cell body plasma membrane. OPCs were differentiated for 3 (A, B) or 7 (C) days in the absence (ctrl) or presence of sodium chlorate, an inhibitor of sulfatide synthesis. (A) The surface localization of PLP was determined by immunostaining with anti-PLP antibodies directed against extracellular epitopes (ET3, O10); this was followed by fixation, permeabilization, and staining with the 4C2 anti-PLP antibody. The percentage of PLP surface-positive cells is shown in panel B. Statistically significant differences from the results obtained with untreated cells (ctrl) were assessed with the Student t test (n = 4; **, P < 0.001). Note the prominent PLP surface expression (ET3 antibody) at the cell body plasma membrane upon inhibition of sulfatide biosynthesis. (C) TX-100 and CHAPS membrane microdomain association of surface PLP. Cell surface proteins were biotinylated and subjected to TX1-100 or CHAPS extraction, density gradient fractionation, and immunoprecipitation with SA-agarose. PLP was visualized by Western blotting (4C2 antibody). Note that surface PLP is TX-100 and CHAPS resistant in untreated cells but CHAPS soluble upon inhibition of sulfatide synthesis (chlorate). Pictures and blots representative of at least three independent experiments are shown. Scale bars are 10 μm.

FIG 5

Sulfatide is essential for segregation to TX-100-soluble membrane microdomains. (A) TLC analysis of cellular glycosphingolipids in OLN-PLP (mock), OLN-PLP-G (GalC), and OLN-PLP-GS (GalC and sulfatide) cells. (B) Western blot analysis of PLP-eGFP expression in OLN-PLP cells (mock), OLN-PLP-G cells, OLN-PLP-GS cells, OPCs, and OLGs (10 days of differentiation). PLP was visualized with anti-PLP antibody 2D2. (C) Cell surface proteins were biotinylated, lysed, and immunoprecipitated with SA-agarose to separate surface-localized proteins (lanes S) and nonbiotinylated, i.e., intracellularly localized proteins (lanes I). PLP-eGFP was visualized by Western blotting (anti-GFP antibody). Note that PLP-eGFP is present at the surface of OLN-PLP cells (mock), i.e., in the absence of GalC (G) and sulfatide (S). (D) The surface localization of PLP-eGFP (ET3) was determined in mock-transduced (vector only) and syntaxin 3-overexpressing cells (S3↑). Note the decreased PLP-eGFP surface expression upon the overexpression of syntaxin 3 (arrow). (E) TX-100 and CHAPS membrane microdomain association of PLP-eGFP in OLN-PLP, OLN-PLP-G, and OLN-PLP-GS cells. PLP-eGFP was visualized by Western blotting (anti-GFP antibody). Detergent-resistant membrane microdomains are present in fractions 3 and 4. Note that PLP-eGFP is TX-100 resistant in the absence of sulfatide. PLP (4C2) and caveolin were visualized by Western blotting. Pictures and blots representative of at least three independent experiments are shown. The scale bar is 10 μm.

FIG 6

PLP is conveyed from TX-100- to CHAPS-resistant membrane microdomains at the plasma membrane. (A) Colocalization of PLP-eGFP with GalC and/or sulfatide in OLN-PLP-GS cells. Immunostaining with anti-GalC (O1) and antisulfatide (O4) antibodies was performed with live or fixed and permeabilized cells. Note that PLP-eGFP colocalized with sulfatide but not GalC at the surface of OLN-PLP-GS cells, whereas intracellular PLP-eGFP partially colocalized with GalC but not sulfatide (insets). (B, C) Endosomes and lysosomes were separated by organelle fractionation and centrifugation on a discontinuous sucrose gradient. The distribution of EEA1, LAMP1, and PLP-eGFP (anti-GFP antibody) was visualized by Western blotting. The distribution of sulfatide was visualized with a dot blotting assay using antisulfatide antibody O4 for detection. The boxes mark the pooled fractions of the gradient used for TX-100 and CHAPS extraction of the enriched endosomal and lysosomal fractions shown in panel C. Detergent-resistant membrane microdomains are present in fractions 3 and 4. Note that PLP-eGFP is TX-100 soluble and CHAPS insoluble in both endosomes and lysosomes in OLN-PLP-GS cells but still CHAPS soluble in endosomes in galactolipid-deficient OLN-PLP cells. The scale bar is 10 μm.

FIG 7

Sulfatide modulates the conformation of PLP at the plasma membrane. Surface expression of PLP-eGFP in differentiated OLN-PLP, OLN-PLP-G, and OLN-PLP-GS cells with three different anti-PLP antibodies against extracellular epitopes, i.e., the first (4C2) and second (O10, ET3) extracellular loops. Note that O10 recognizes surface PLP-eGFP only when sulfatide is present (A). Quantification of the number of O10-positive cells among the total number of cells is shown in panel B. Each bar represents the mean value plus the SD. Statistically significant differences from the results obtained with OLN-PLP cells were assessed by one-way ANOVA with Tukey's posttest (n = 3; ***, P < 0.001). The scale bar is 10 μm.

FIG 8

Schematic overview of the trafficking of de novo-synthesized PLP to myelin sheets via transcytosis. (Part 1) Transport of PLP from the trans-Golgi network to the apical-surface-like (red) cell body plasma membrane is syntaxin 3 dependent and sulfatide independent and occurs as part of a TX-100-insoluble, CHAPS-soluble membrane microdomain (part 2). At the plasma membrane, PLP is redistributed to TX-100-soluble, CHAPS-insoluble microdomains, a process that is sulfatide mediated and likely involves a conformational alteration and/or oligomerization (part 3). Transport from the plasma membrane of the cell body to the basolateral-surface-like (blue) myelin-like membranes occurs by means of TX-100-soluble, CHAPS-insoluble microdomains. Along this route, PLP likely accumulates temporally in a late endosomal compartment (11, 12). See the text for further details.