An apical-type trafficking pathway is present in cultured oligodendrocytes but the sphingolipid-enriched myelin membrane is the target of a basolateral-type pathway

Abstract

Myelin sheets originate from distinct areas at the oligodendrocyte (OLG) plasma membrane and, as opposed to the latter, myelin membranes are relatively enriched in glycosphingolipids and cholesterol. The OLG plasma membrane can therefore be considered to consist of different membrane domains, as in polarized cells; the myelin sheet is reminiscent of an apical membrane domain and the OLG plasma membrane resembles the basolateral membrane. To reveal the potentially polarized membrane nature of OLG, the trafficking and sorting of two typical markers for apical and basolateral membranes, the viral proteins influenza virus-hemagglutinin (HA) and vesicular stomatitis virus-G protein (VSVG), respectively, were examined. We demonstrate that in OLG, HA and VSVG are differently sorted, which presumably occurs upon their trafficking through the Golgi. HA can be recovered in a Triton X-100-insoluble fraction, indicating an apical raft type of trafficking, whereas VSVG was only present in a Triton X-100-soluble fraction, consistent with its basolateral sorting. Hence, both an apical and a basolateral sorting mechanism appear to operate in OLG. Surprisingly, however, VSVG was found within the myelin sheets surrounding the cells, whereas HA was excluded from this domain. Therefore, despite its raft-like transport, HA does not reach a membrane that shows features typical of an apical membrane. This finding indicates either the uniqueness of the myelin membrane or the requirement of additional regulatory factors, absent in OLG, for apical delivery. These remarkable results emphasize that polarity and regulation of membrane transport in cultured OLG display features that are quite different from those in polarized cells.

Figure 1

Intracellular localization of HA and VSVG in virus-infected OLGs. After infection with either influenza (A and B) or VSV (C and D), the intracellular distribution of HA and VSVG was determined by immunostaining as described in MATERIALS AND METHODS. Their distribution was compared with that of medial Golgi-specific MG-160. (A and B) Costaining of HA and MG-160. (C and D) Costaining of VSVG and MG-160. Both HA and VSVG colocalize with MG-160 in the perinuclear region (arrows). Bar, 20 μm.

Figure 2

(E through H facing page). Effect of monensin on intracellular localization of HA and VSVG in infected OLGs. Two hours before fixation of infected OLGs, monensin was added to a final concentration of 10 μM and the distribution of HA (A and C) was compared with that of MG-160 (B) and the trans-Golgi-network–specific TGN-38 (D). Similarly, the distribution of VSVG (E and G) was compared with that of MG-160 (F) and TGN-38 (H). Monensin treatment induces fragmentation of the Golgi apparatus, as is demonstrated by a marked redistribution of the Golgi markers (B, D, F, and H; compare Figure 1D). This redistribution is matched by a similar redistribution of HA (A and C, arrows) and VSVG (E and G, arrows). Bar, 20 μm.

Figure 2

(E through H facing page). Effect of monensin on intracellular localization of HA and VSVG in infected OLGs. Two hours before fixation of infected OLGs, monensin was added to a final concentration of 10 μM and the distribution of HA (A and C) was compared with that of MG-160 (B) and the trans-Golgi-network–specific TGN-38 (D). Similarly, the distribution of VSVG (E and G) was compared with that of MG-160 (F) and TGN-38 (H). Monensin treatment induces fragmentation of the Golgi apparatus, as is demonstrated by a marked redistribution of the Golgi markers (B, D, F, and H; compare Figure 1D). This redistribution is matched by a similar redistribution of HA (A and C, arrows) and VSVG (E and G, arrows). Bar, 20 μm.

Figure 3

Immunoblots of TX-100-soluble and -insoluble fractions obtained at 4°C from OLGs infected with influenza virus or VSV. Monoclonal antibodies against HA (A) or VSVG (B) were used. Molecular weight markers were run alongside the extracts, as marked by horizontal bars. Samples are from the isolated viruses (v) or from cells (c), either infected (+) or noninfected (−). TX-100-soluble and -insoluble fractions are indicated by “s” and “i,” respectively. (A) HA. Lane 1 (v), solubilized influenza virus. The antibody marks HA at about 80 kDa (arrow) as well as a cluster of smaller proteins (accolade). Lane 2, TX-100-soluble fraction from uninfected OLGs; lane 3, TX-100-soluble fraction from influenza virus-infected OLGs; lane 4, TX-100-insoluble fraction from influenza virus-infected OLGs; and lane 5, TX-100-insoluble fraction from uninfected OLGs. Taking into account some nonspecific staining of the antibody (lanes 2 and 5), it is evident that HA (80-kDa band, arrow) primarily resides in the detergent-insoluble fraction (lane 3 versus lane 4). (B) VSVG. Lane 1 (v), solubilized VSV. The antibody demonstrates the presence of VSVG at about 60 kDa; lane 2, TX-100-soluble fraction from uninfected OLGs; lane 3, TX-100-soluble fraction from VSV-infected OLGs; lane 4, TX-100-insoluble fraction from VSV-infected OLG; and lane 5, TX-100-insoluble fraction from uninfected OLGs. Note that virtually all VSVG is present in the soluble fraction (lane 3).

Figure 4

Differential localization of HA and VSVG in sheet-forming OLGs. OLGs were infected and costained for MBP and the appropriate viral glycoprotein. Note that HA (B) is excluded from the MBP-stained sheets (A), whereas VSVG (D) colocalizes with MBP (C) in the sheets (C and D). Bar, 20 μm.

Figure 5

Polarized localization of HA and VSVG in neurons. Neurons, present in mixed brain cultures, were infected with influenza virus (A and B) and VSV (C and D), fixed, and costained with the neuron-specific TuJ1 antibody (A and C) and with antibodies against the viral glycoproteins. HA localizes exclusively to axon bundles (B, arrows), whereas G is only present in dendrites (D, arrowheads). Bar, 20 μm.

Figure 6

Effect of BFA on intracellular localization of HA in influenza-infected OLGs. Two hours after infection BFA was added to a final concentration of 18 μM. After another 2 h the OLGs were fixed and costained for MBP (A) and HA (B). No shift in intracellular localization of HA to the myelin sheet was detected. Arrow indicates a densely MBP-labeled part of the sheet. Bar, 20 μm.