AN ANALYSIS OF AN AXONAL GRADIENT OF PHOSPHORYLATED MAP 1B IN CULTURED RAT SENSORY NEURONS

The present study investigated the cellular distribution of a developm entally regulated phosphorylated form of MAP 1B recognized by monoclon al antibody (mAb) 150 in cultures of dorsal root ganglia. The cell som a and the whole axon, when it first appears, are labelled, but longer axons label with a proximodistal gradient, such that the cell soma and proximal axon become unlabelled, whilst the distal axon and growth co ne label strongly. Double-labelling experiments with mAb 150 and a pol yclonal antibody (N1-15) that recognizes all forms of MAP 1B demonstra ted that MAP 1B is distributed along the entire length of axons with g radients, so the gradient of phosphorylated MAP 1B is not due to a los s or absence of MAP 1B from the proximal axon. The proportion of axons from 20 h cultures that were labelled with a mAb 150 gradient was at least 80% and this proportion was independent of the nerve growth fact or concentration of the culture medium. Analysis of axons ranging in l ength from 100 to 700 mu m and labelled with a gradient showed that th e unlabelled proximal portions of axons increased in length more slowl y than the labelled distal axon. Axons labelled along their entire len gth accounted for no more than 19% of the axonal population and analys is of these showed them to be frequently <400 mu m long. After simulta neously fixing and detergent-extracting cultures this proportion rose significantly to 93%, suggesting that in the proximal axon the mAb 150 epitope is masked by some factor(s) that is removed by detergent extr action. The possibility that mAb 150 could not access the epitope in t he proximal axon was discounted because another IgM, mAb 125, which re cognizes a different phosphorylation epitope on MAP 1B, labelled the p roximal axon of conventionally fixed cultures. In growth cones of fixe d and extracted neurons examined by immunofluorescence, the mAb 150 la belling strongly colocalized to bundled microtubules in the distal axo n shaft and the C-domain. In the P-domain, mAb 150 staining was weaker and more widely distributed than the microtubules. Immunogold electro n microscopy confirmed that antibody N1-15 and mAb 150 strongly labell ed the bundled microtubules in the C-domain and also showed that indiv idual microtubules in the P-domain, some of which lie alongside actin filament bundles of filopodia, were labelled lightly and discontinuous ly with both antibodies. This suggests that the phosphorylated isoform of MAP 1B recognized by mAb 150 may be involved in bundling microtubu les in the proximal region of the growth cone and in the interaction b etween microtubules and actin filaments in the P-domain.