Nestin and Steroid Receptor Figure 7. GR redistributes with shortened IFs composed of a tailless vimentin protein. (A–D) Confocal images of cytoskeletons prepared from B8 VimMT3B4 cells, which stably express a tailless vimentin protein; tailless vimentin forms a fragmented IF network (A, B); the filaments contain also nestin (C); GR colocalizes with vimentin/nestin filament fragments (A, C); Bars in A and C = 1 mm; B = 5 mm. doi:10.1371/journal.pone.0006084.g007 treatment with nestin shRNA alone. The data substantiate the hypothesis that anchoring of GR by nestin compromises its growth suppressive functions. Discussion Our data indicate that nestin in conjunction with vimentin determines cytoplasmic accumulation of the glucocorticoid receptor, a major sensor of stress response at the cellular level. Neither nestin nor vimentin were sufficient on its own, but anchoring required their copolymerization into an IF system. Nestin, due to its short N-terminal domain, cannot self-assemble into filaments, but forms heteromeric filaments with other class III IF-proteins, such as vimentin, desmin, or GFAP, with nestin prosumably locating at the filament periphery [47,48,49,50,51]. Therefore, a certain structural configuration of the IFs resulting from copolymerization of nestin and vimentin seems to be required for an efficient binding of a GR containing complex. Our data at least indicate that the tail domain of vimentin is not essential for anchoring of GR. All of the cell types we have tested so far for cytoplasmic anchoring of GR expressed the class III IF protein vimentin. In differentiation, vimentin expression often precedes that of the tissue specific IF protein, suggesting that vimentin could have a specific role in conjunction with nestin. Further studies must show whether other class III IF proteins can substitute for vimentin in this function. The differential reaction of phosphospecific GR antibodies gives evidence that specifically the non-activated GR form is anchored to the vimentin/nestin filament system. This form is in a hypophosphorylated state, as deduced from its increased mobility in SDS-PAGE. Any reaction with this phosphor site specific antibody was abolished in the presence of dexamethasone which shifted GR into the nucleus. The data indicate that GR anchored to the IF system is not irreversibly withdrawn from the activation cycle but awaits binding of a ligand. It is well known that the activation status of the receptor in the cytoplasm is also defined by its association with chaperone molecules. Binding of chaperone molecules is necessary for the receptor to gain the high affinity, steroid binding form. GR- PLoS ONE | www.plosone.org 9 June 2009 | Volume 4 | Issue 6 | e6084