Lack of stabilized microtubules due to the absence of major MAPs in CAD cells does not preclude neurite formation

BISIG C .G.; CHESTA M. E.; ZAMPAR G. G.; PURRO S. A.; SANTANDER V. S.; ARCE C. A.

FEBS JOURNAL

WILEY-BLACKWELL PUBLISHING, INC

Año: 2009 vol. 276 p. 7110 - 7123

1742-464X

In many laboratories, the requirement of microtubule-associated proteins(MAPs) and the stabilization of microtubules for the elongation of neuriteshas been intensively investigated, with controversial results being obtained.We have observed that the neurite microtubules of Cath.a-differentiated(CAD) cells, a mouse brain derived cell, are highly dynamic structures, andso we analyzed several aspects of the cytoskeleton to investigate the molecularcauses of this phenomenon. Microtubules and microfilaments were presentin proportions similar to those found in brain tissue and weredistributed similarly to those in normal neurons in culture. Neurofilamentswere also present. Analysis of tubulin isospecies originating from post-translationalmodifications revealed an increased amount of tyrosinated tubulin,a diminished amount of the detyrosinated form and a lack of the Delta2form. This tyrosination pattern is in agreement with highly dynamic microtubules.Using western blot analyses with specific antibodies, we found thatCAD cells do not express several MAPs such as MAP1b, MAP2, Tau, doublecortin,and stable-tubule-only-peptide. The presence of the genes correspondingto these MAPs was verified. The absence of the correspondingmRNAs confirmed the lack of expression of these proteins. The exceptionwas Tau, whose mRNA was present. Among the several MAPs investigated,LIS1 was the only one to be expressed in CAD cells. In addition, wedetermined that neurites of CAD cells form and elongate at the same rateas processes in a primary culture of hippocampal neurons. Treatment withnocodazol precluded the formation of neurites, and induced the retractionof previously formed neurites. We conclude that the formation and elongationof neurites, at least in CAD cells, are dependent on microtubuleintegrity but not on their stabilization or the presence of MAPs.