Chronological expression of microtubule-associated proteins (MAPs) in EC cell P19 after neuronal induction by retinoic acid

Abstract

Pluripotent murine embryonal carcinoma (EC) P19 cells are induced at a high rate into neural cells using retinoic acid and serum-free medium. EM observation revealed great increase of microtubules (MTs) after neuronal induction. To study the expression of microtubule-associated proteins (MAPs), immunoblotting and immunocytochemistry were performed with phosphorylated MAP1B (pMAP1B)-, MAP2-, and MAP1A-specific monoclonal antibodies. They did not stain undifferentiated cells. Early MAPs (pMAP1B and MAP2C) appeared 12 h after the neuronal induction, changing to late MAPs (MAP1A and MAP2A/B) at 3-5 days. These expression patterns are quite similar to those of neural cells in vivo. Anti-pMAP1B stained not only neurites but also the cell body and varicosities. But after extraction of the soluble component by permeabilization, pMAP1B was found in only MT-domains of the neurites at LM and EM levels, indicating that some part of pMAP1B is a structural component of neurite MTs and others exist in a soluble form. After culturing for more than 5 days, pMAP1B disappeared from the soma, but still remained in the distal ends of neurites. Here we showed that P19 is a good model system for studying the expression of MAPs on the continuous course of neuronal differentiation.