Nestin and Steroid Receptor Figure 4. Retention of GR on the cytoskeleton requires vimentin and nestin. (A1–A2) The Vim expressing subclone C6D8 accumulates GR in the cytoplasm. Cytoplasmic GR colocalizes with Nes. (B1) GR in the Vim deficient subclone C6D10 is only nuclear. (B2) A proportion of the Vim negative C6D10 cells expresses Nes. (C) Expression of an exogenous mouse Vim protein in Vim deficient cells restores cytoplasmic accumulation of GR and colocalization of GR, Nes, and Vim. Bars in A–C = 5 mm. doi:10.1371/journal.pone.0006084.g004 Localization of IF associated GR complexes at the EM level To proof unequivocally that GR is bound to individual IF filaments we assayed its localization at an ultrastructural level by electron microscopy. We did an immunogold labelling analysis on cultured C6D8 cells by replica EM. The cells were extracted with detergent under controlled conditions, fixed, labelled with antibodies, critical point dried and shadowed with platinum carbon or only carbon[37,38]. The technique fills the gap between low resolution light microscopy and high resolution electron microscopy and is highly suited to understand changes in cytoskeleton architecture in relation to dynamic processes in the cell [39]. As shown in Figure 8 the procedure well preserved the 3D organization of the filament network. Immunogold labelling combined with carbon shadowing enabled us to distinguish vimentin filaments (10 nm immunogold particles) from other filament types, such as actin filaments (5 nm immunogold particles) (Figure 9A). Double labelling of GR (5 nm immunogold particles) and vimentin (10 nm immunogold particles) revealed an association of the GR label specifically with the vimentin containing filaments (Figure 9, B1–B3). The results suggest that GR is part of a larger complex bound to distinct sites at the vimentin/nestin filaments. The non-activated receptor form is bound to the cytoskeleton To define the functional status of the IF associated GR we asked whether nestin/vimentin associated GR complexes colocalize with HSC70 and HSP90, the major chaperone molecules stabilizing the conformation of GR in the activation cycle. Confocal microscopy indicated colocalization of GR and HSC70 (Figure S3), whereas HSP90 could not be detected on cytoskeletons. PLoS ONE | www.plosone.org 6 June 2009 | Volume 4 | Issue 6 | e6084