Microtubule-associated protein 1a: Analysis of its microtubule binding domain and its function in differentiating P19 neurons.
|Title:||Microtubule-associated protein 1a: Analysis of its microtubule binding domain and its function in differentiating P19 neurons.|
|Authors:||Vaillant, Andrew R.|
|Abstract:||The expression of several microtubule-associated proteins (MAPs), including MAP1a, was examined in RA-induced P19 embryonal carcinoma (EC) neurons. Immunofluorescence microscopy revealed that MAP1a and MAP1b were detected associated with microtubules (MTs) in undifferentiated cells while MAP2 was absent. At day 2, cells spontaneously formed aggregates and in these aggregates, MAP2 and increased amounts of MAP1b were detected in some cells. By day 4 of differentiation, neurite outgrowth was observed from these aggregates and MAP1a, MAP1b and MAP2 were colocalized with MTs in all neurites, including growth cones. HMW-MAP2 was restricted to the proximal regions of neurites and cell bodies. By day 8, substantial neurite outgrowth had occurred and MAP1a, MAP1b and MAP2 were seen in all processes. HMW-MAP2 was restricted to cell aggregates and largely absent from processes. At day 12, no further neurite outgrowth was evident and existing neurites were organized into fascicles, which were MAP1a, 1b and MAP2 positive but weaker than at day 4. Also, the restriction of HMW-MAP2 to aggregates was even more marked. Western blotting and ELISA showed that MAP1a, 1b and LMW-MAP2 protein levels increased during differentiation. Peak accumulation occurred no later than day 8, coinciding with the period of neurite outgrowth, and then decreased after day 8. HMW-MAP2 was absent in undifferentiated cells and increased steadily as development progressed. Juvenile tau appeared at day 4, and adult tau appeared at day 6. Both tau forms increased during development. These results show that MAP expression in serum-free differentiating P19 EC cells is similar to that seen in brain development, with the exception of MAP1a, which mirrors expression patterns seen only in axons. These observations suggest that MAP1a may modulate microtubule dynamics during neurite outgrowth. To determine how MAP1a interacts with MTs, several myc-tagged MAP1a fragments were expressed in P19 EC and HeLa cells. To investigate the role of LCs in MAP1a function during development, the expression of LC3 during the differentiation of P19 EC cells was characterized. The pattern of expression and effects on MT stability compared with MAP2 suggest that MAP1a is a weak stabilizer that functions in the growth of neurites and in the adult may function to mediate MT stabilization by other adult MAPs. LC3 may also play a role in the regulation of MAP1a function during neuronal differentiation. (Abstract shortened by UMI.)|
|Collection||Thèses, 1910 - 2010 // Theses, 1910 - 2010|