RhoB expression is induced after the transient upregulation of RhoA and Cdc42 during neuronal differentiation and influenced by culture substratum and microtubule integrity

RhoGTPases are important intracellular signalling switches in the regulation of cytoskeleton organization. They likely have an important role in ontogenesis because cytoskeletal rearrangements accompany cell differentiation and specialization. Western blotting showed that protein expression of RhoA, RhoB and Cdc42 RhoGTPases dramatically increased, in a programmed manner, during neuronal differentiation of P19 mouse embryonal carcinoma cells with retinoic acid. RhoA and Cdc42 expression were sequentially upregulated and peaked during the commitment period while that of RhoB was induced in post-mitotic neurons. Although RhoB had a higher expression on matrices allowing cell spreading and neurite elongation, it was distributed throughout cell volume by immunocytofluorescence and associated with various cell compartments by centrifugal subfractionation, suggesting a role not restricted at neurites at this stage of differentiation. RhoA and Cdc42 were mainly cytosolic and RhoB particulate in the P19 cell model. Treatment of cells with cytoskeleton disruptors showed that poisons of microtubules but not of actin filaments or neurofilaments increased the cytosolic level of RhoB. The results indicate that RhoA, Cdc42 and RhoB must intervene at specific stages of neuronal development and there exists a relationship between RhoB expression/distribution, the microtubule network and the extracellular matrix during this process. (C) 2001 Elsevier Science B.V. All rights reserved.