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 (PK C)-induced phosphorylation of vimentin can be detected with the mouse monoclonal antibody B3 in cultures of amoeboid microglia [Ciesielski-T reska et al. (1991) J. Neurosci. Res. 29,362-378]. The antibodies were generated to native form of vimentin-containing filaments and antibod y B3 reveals a restricted immunostaining of vimentin in glial cells fr om human, rat and mouse origin. In the present study we show the distr ibution of epitope B3 analyzed by immunofluorescence within defined po pulations of rat glial cells. Relatively high immunoreactivity was fou nd in Type 1 astrocytes, Type 2 astrocytes and oligodendrocytes had lo w immunoreactivity. Although the results suggested that in Type 1 astr ocytes the phosphorylated epitope is prominent, its phosphorylation wa s not found to be cell cycle-dependent, and appeared unrelated to the organizational changes of intermediate filaments associated with the m orphological conversion of polygonal to stellate astrocytes. As expect ed, the immunofluorescence was increased by exposition of astrocyte cu ltures to an activator of PKC, confirming our previous conclusion that the immunoreactivity of the epitope B3 depends on PKC-mediated phosph orylation. In addition, the finding that the immunofluorescence of vim entin was more homogeneous in quiescent, serum-deprived astrocytes and also in astrocytes exposed to an inhibitor of protein synthesis, cycl oheximide, may suggest that phosphorylation of the epitope B3 depends on a protein factor present in fetal calf serum. The immunofluorescenc e studies on cultures enriched in Type 2 astrocytes and in oligodendro cytes indicate that the epitope B3 is hypophosphorylated in glial cell s of this lineage and becomes dephosphorylated after terminal differen tiation of oligodendrocytes. These observations suggest that in Type 2 astrocytes and in oligodendrocytes the low level of phosphorylation o f vimentin could be related to the down regulation in vimentin express ion.