Differences in vimentin distribution in glial cells in culture revealed with an antibody against a phosphorylated epitope.
We have previously described that spatial and temporal changes in the organization of vimentin that are correlated with protein kinase C (PKC)-induced phosphorylation of vimentin can be detected with the mouse monoclonal antibody B3 in cultures of amoeboid microglia [Ciesielski-Treska et al. (1991) J. Neurosci. Res. 29, 362-378]. The antibodies were generated to native form of vimentin-containing filaments and antibody B3 reveals a restricted immunostaining of vimentin in glial cells from human, rat and mouse origin. In the present study we show the distribution of epitope B3 analyzed by immunofluorescence within defined populations of rat glial cells. Relatively high immunoreactivity was found in Type 1 astrocytes, Type 2 astrocytes and oligodendrocytes had low immunoreactivity. Although the results suggested that in Type 1 astrocytes the phosphorylated epitope is prominent, its phosphorylation was not found to be cell cycle-dependent, and appeared unrelated to the organizational changes of intermediate filaments associated with the morphological conversion of polygonal to stellate astrocytes. As expected, the immunofluorescence was increased by exposition of astrocyte cultures to an activator of PKC, confirming our previous conclusion that the immunoreactivity of the epitope B3 depends on PKC-mediated phosphorylation. In addition, the finding that the immunofluorescence of vimentin was more homogeneous in quiescent, serum-deprived astrocytes and also in astrocytes exposed to an inhibitor of protein synthesis, cycloheximide, may suggest that phosphorylation of the epitope B3 depends on a protein factor present in fetal calf serum. The immunofluorescence studies on cultures enriched in Type 2 astrocytes and in oligodendrocytes indicate that the epitope B3 is hypophosphorylated in glial cells of this lineage and becomes dephosphorylated after terminal differentiation of oligodendrocytes. These observations suggest that in Type 2 astrocytes and in oligodendrocytes the low level of phosphorylation of vimentin could be related to the down regulation in vimentin expression.
Ciesielski-Treska
J
J
Unité INSERM U-338 de Biologie de la Communication Cellulaire, Strasbourg, France.
Ulrich
G
G
Aunis
D
D
D000818:Animals; D001253:Astrocytes / Q000276:immunology / Q000378:metabolism; D015153:Blotting, Western; D002478:Cells, Cultured; D003513:Cycloheximide / Q000494:pharmacology; D004591:Electrophoresis, Polyacrylamide Gel; D000939:Epitopes / Q000276:immunology*; D005455:Fluorescent Antibody Technique; D007150:Immunohistochemistry; D008856:Microscopy, Fluorescence; D009457:Neuroglia / Q000276:immunology / Q000378:metabolism*; D009836:Oligodendroglia / Q000276:immunology / Q000378:metabolism; D010766:Phosphorylation; D011493:Protein Kinase C / Q000276:immunology / Q000378:metabolism; D011506:Proteins / Q000378:metabolism; D051381:Rats; D013755:Tetradecanoylphorbol Acetate / Q000494:pharmacology; D014746:Vimentin / Q000276:immunology / Q000378:metabolism*
D000939:Epitopes; D011506:Proteins; D014746:Vimentin; D003513:Cycloheximide; D011493:Protein Kinase C; D013755:Tetradecanoylphorbol Acetate