Nestin and Steroid Receptor Figure 8. Visualization of the cytoskeleton architecture by EM replica technique. C6D8 cells were grown on glass coverslips, extracted with a non-ionic detergent, fixed with aldehyde, critical point dried, rotary shadowed with platinum/carbon (A) respectively carbon (B), detached from the glass, and transferred onto EM grids. The micrographs reveal maintenance of the 3D organization of the networks with this technique. doi:10.1371/journal.pone.0006084.g008 chaperone complexes are highly unstable and undergo constant cycles of dissociation and association. As we show here, GR containing immunecomplexes obtained with nestin antibodies from the cytoskeleton fraction of anchoring competent cells contained the chaperone HSC70, but not HSP90. The immuno- precipitation assay was limited by the low solubility of nestin/ vimentin IFs under the mild conditions, applied to generate fractions for IP. However, HSP90 was also not observed on the cytoskeleton by immunocytochemical staining in situ staining, whereas HSC70 coincided with GR present on the cytoskeleton. In contrast nestin was isolated at significant quantities by immunoprecipitation from vimentin deficient cells. But this sample showed no coprecipitation of GR. GR complexes isolated from the detergent soluble fraction contained HSP90 in addition to HSC70. According to the current model, which largely results from in vitro studies with purified proteins, the presence of HSC70 in a GR containing complex indicates that GR associated with the IF system is in a certain advanced stage in the activation cycle. HSC70 and the subsequently bound HSP90 induce the formation of a deep hydrophobic cleft in the ligand binding domain of GR, required for hormone binding [52,53,54]. After ligand binding the chaperone molecules are displaced from the receptor molecules, the receptor dimerizes and translocates into the nucleus [55,56,57]. The data raise the question whether chaperone molecules themselves may mediate cytoplasmic retention. But previous studies of others showed that GR and chaperone molecules can move together into the nucleus suggesting that association with chaperones is not sufficient for cytoplasmic retention [58,21]. In addition, overexpression of the glucocorticoid receptor raised the nuclear level of na¨ıve, chaperone associated receptor molecules [59,60]. These data contradict a direct role of chaperone molecules in cytoplasmic retention of GR. Our data give evidence that anchoring of GR to nestin/ vimentin is related to the maintenance of a high proliferation rate. A first, indirect hint was obtained with mouse embryo fibroblasts. Anchoring of GR in these primary cells was typical of early passages, when the proliferation rate was high. In advanced passages the cells ceased expression of nestin, contained GR in the nucleus, and concomitantly halted proliferation. It suggested a link between nestin expression, anchoring of GR and maintenance of proliferative capacity. Our RNAi experiments corroborate the suggestion. First, suppression of nestin abolished cytoplasmic localization of GR and induced a sustained growth arrest. Secondly, the growth suppressive effect could be rescued by blocking GR expression prior to that of nestin. The data extend a recent report, showing that suppression of nestin in repopulating mesangial cells lowered the number of BrdU incorporating cells [61]. Thus nestin/vimentin dependent anchoring can modulate growth suppression by GR. The coincident accumulation of nestin and GR in embryonic mouse tissue let us assume that nestin/vimentin dependent PLoS ONE | www.plosone.org 10 June 2009 | Volume 4 | Issue 6 | e6084