Microtubule-associated Protein 2 (MAP2) Expression in Transiently and Stably Transfected P19 Embryonal Carcinoma Cells

Résumé du livre

In developing neurons, microtubule-associated protein 2 (MAP2) occurs as high molecular weight MAP2 (HMW-MAP2) and low molecular weight MAP2 (MAP2c) isoforms. Though both HMW-MAP2 and MAP2c have been shown to stabilize microtubules (MTs) in vitro and in vivo, their developmental regulation has suggested that HMW-MAP2 may stabilize MTs to a greater extent than MAP2c. To test this hypothesis, MAP2c and HMW-MAP2 were independently transfected into undifferentiated P19 embryonal carcinoma (EC) cells using the constitutive phosphoglycerate kinase (PGK) promoter to drive expression. MT stability was assayed by treating transfected cells with the MT depolymerizing drug colchicine for various lengths of time and measuring the number of transfected cells possessing stable MT bundles. MAP2c and HMW-MAP2 stabilized MTs to similar extents suggesting that, in developing neurons, the increase in MT stability can not be attributed directly to the developmental exchange in MAP2c and HMW-MAP2 expression. Both MAP2c and HMW-MAP2 have been shown to promote the in vitro and in vivo assembly of MTs and inhibition of MAP2 expression in cultured neurons affects neuronal morphogenesis as indicated by a reduction in MT mass accumulation and neuritic growth. It is hypothesized that MAP2c and HMW-MAP2 are both required for neuronal morphogenesis. Comparison of relative MAP2 protein levels from untransfected and stable, MAP2cmyc-transfected neuronal cultures by Western blotting and ELISA showed higher MAP2c expression in the transfected cell line. MAP2cmyc expression was restricted to neurons and was first detected three days after neuronal induction at the time of neuronal outgrowth. In many neurons, MAP2cmyc was localized to all neurites and the morphology of these neurons appeared indistinguishable from untransfected, control neurons. (Abstract shortened by UMI.).