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Fig. 4. The 13 amino acid N-terminus of PLP is sorted into MLMs in a palmitoylation-dependent way. Primary oligodendrocyte cultures were infected with recombinant SFV producing 1-13PLP-EYFP or its palmitoylation-deficient mutant 1-13PLP(C5,6,9S)-EYFP, and processed for immunofluorescence 5 hours after infection. Cells were stained for PLP (O10) to visualize MLMs (arrows). 1-13PLP(C5,6,9S)-EYFP showed a diffuse staining of the oligodendroglial soma and some processes (A), whereas 1-13PLP-EYFP was distributed over the entire oligodendrocyte membrane and was detectable in MLMs (B). 1-13PLP-EYFP was also found on intracellular membranes in the Golgi region. Confocal microscopy analysis showed colocalization of 1-13PLP-EYFP with GM130, a marker for the Golgi apparatus (C). Treatment with BFA (10 µg ml–1) for the last 4 hours during post-infection time resulted in a redistribution of 1-13PLP-EYFP from the distal membrane extensions into the soma and proximal part of some processes (D). Primary oligodendrocytes were infected with SFV-1-13PLP-EYFP, SFV-1-13PLP(C5,6,9S)-EYFP or SFV-EYFP, and light-membrane fractions were isolated according to the myelin-isolation protocol to enrich MLMs. The amounts of virus-derived 1-13PLP-EYFP, 1-13PLP(C5,6,9S)-EYFP and EYFP in the myelin preparations were determined by immunoblotting with an anti-GFP antibody (E). The sequence of the N-terminal 13 amino acids is shown for 1-13PLP-EYFP and 1-13PLP(C5,6,9S)-EYFP (F). Bars, 20 µm.
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