High resolution immunogold analysis reveals distinct subcellular compartmentation of protein kinase C γ and δ in rat Purkinje cells

Abstract

High resolution immunogold cytochemistry was used to investigate the subcellular distribution of protein kinase C γ and δ in Purkinje cells of the rat cerebellum. Postembedding incubation with an antibody raised to a peptide sequence near the C-terminus of protein kinase C γ resulted in strong labelling along the dendrosomatic plasma membrane. A quantitative analysis indicated that this labelling reflected the existence of two pools of PKC γ; one membrane associated pool and one cytoplasmic pool located within 50 nm of the plasma membrane. The labelling along the plasma membrane showed a pronounced and abrupt increase when moving from the cell body into the axon initial segment. Gold particles signalling protein kinase C γ were also enriched in putative Purkinje axon terminals in the dentate nucleus. The only organelle showing a consistent immunolabelling for protein kinase C γ was the Golgi apparatus where the gold particles were restricted to the trans face. Protein kinase C γ immunoreactivity also occurred in the Purkinje cell spines, with an enrichment in or near the postsynaptic density. Antibodies to protein kinase C δ produced a very different labelling pattern in the Purkinje cells. Most of the gold particles were associated with rough endoplasmic reticulum, particularly with those cisternae that were located close to the nucleus or in the nuclear indentations. No significant protein kinase C δ immunolabelling was detected at the plasma membrane or in Purkinje cell spines. The present data point to a highly specific compartmentation of the two major protein kinase C isozymes in Purkinje cells and suggest that these isozymes act on different substrates and hence have different regulatory functions within these neurons.