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The Journal of Cell Biology, Vol 103, 545-557, Copyright © 1986 by The Rockefeller University Press
ARTICLES |
MM Black, JM Aletta and LA Greene
We have used the nerve growth factor (NGF)-responsive line of PC12 pheochromocytoma cells as a model system to study microtubule specializations associated with neurite outgrowth. PC12 cells treated with NGF cease proliferating and extend neurites. Long-term NGF treatment results in a two- to threefold increase in the proportion of total cellular tubulin that is polymerized in PC12 cells. The increase in this parameter first becomes apparent at 2-4 d with NGF and increases steadily thereafter. Several changes in microtubule- associated proteins (MAPs) of PC12 cells also occur after exposure to NGF. In immunoprecipitation assays, we observed the levels of MAP-2 to increase by at least several-fold after treatment with NGF. We also found that the compositions of three MAP classes with apparent Mr of 64K, 67K, and 80K are altered by NGF treatment. These MAPs, recently designated "chartins," are biochemically and immunologically distinct from the similarly-sized tau MAPs (Peng et al., 1985 Brain Res. 361: 200; Magendantz and Solomon, 1985 Proc. Natl. Acad. Sci. 82: 6581). In two-dimensional isoelectric focusing x SDS polyacrylamide gels, each chartin MAP class resolves into a set of proteins of similar apparent Mr but distinct pI. Peptide mapping analyses confirm that the isoelectric variants comprising each chartin MAP class are closely related in primary structure. Several striking differences in the composition of the chartin MAPs of PC12 cells grown with or without NGF were consistently observed. In particular, following longterm NGF treatment, the abundances of the more acidic variants of each chartin MAP class were markedly enhanced relative to the more basic members. This occurs without substantial changes in the abundance of each MAP class as a whole relative to total cell protein. The combined results of in vivo phosphorylation and peptide mapping experiments indicate that the NGF-inducible chartin MAP species are not primary translation products, but are generated posttranslationally, apparently by differential phosphorylation of other chartin MAPs. These observations suggest that NGF treatment of PC12 cells leads to changes in the posttranslational processing of the chartin MAPs. The time course of these changes closely resembles that for the increase in the proportion of cellular tubulin that is polymerized and for neurite outgrowth. One of the important events in the growth and stabilization of neurites appears to be the formation of microtubule bundles that extend from the cell body to the tips of the neurites.(ABSTRACT TRUNCATED AT 400 WORDS)
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