isolated from the cytosol of mouse L-cells also contained the IF protein vimentin in addition to actin and tubulin [24,25]. Here we show, that intermediate filaments composed of nestin and vimentin mediate cytoplasmic anchoring specifically of the unliganded receptor in undifferentiated cells. Anchoring of GR to these filaments is of functional relevance as suppression of nestin is sufficient to activate an irreversible growth arrest, a function which is overcome by concomitant suppression of GR. The finding is consistent with the hypothesis that nestin acts as a survival factor, impeding negative regulation of growth by GR in undifferentiated cells. Results Nestin colocalizes with GR in embryonic mouse tissue Immunoperoxidase labelling of nestin or GR on consecutive sections of mouse embryo heads (E14.5) demonstrated strong nestin and GR signals in neural cells lining the third ventricle (Figure 1, A, B, and C) and in muscle cells adjacent to a hair follicle (Figure 1, A, D, and E). Confocal microscopy revealed a strong nestin signal (red color) in the neuroepithelium and in radial glia (Figure 1F). This is in accordance with previous findings of others showing selective presence of nestin in areas of continuous neurogenesis, namely the lateral wall of the lateral ventricle and in ependymal and subventricular cell layers [26,27,1]. Nestin staining clearly coincided with the staining of GR (green color). In addition, a coincident staining of both proteins was found in muscle cells adjacent to a hair follicle (Figure 1G). The data suggest that nestin positive cells in developing tissue accumulate significant amounts of the glucocorticoid receptor. Nestin and GR costain in undifferentiated human blood cells Individual steps in blood cell differentiation are well defined by specific markers, making it an ideal system to determine the relationship between nestin/GR colocalization and the status of cell differentiation. Nestin expression is known to identify blood precursor or stem cells [28]. Here we assayed peripheral blood mononuclear cells (PBMCs) isolated from human cord blood for this phenotype. CD34 and CD133 were used as markers of early stages of blood cell differentiation. Only a small proportion (less than 1%) of human cord blood cells was nestin positive (Figure 2, A1–A3). Nestin positive cord blood cells were negative for CD34, an early marker of the hematopoietic lineage and vice versa (Figure 2B), but they expressed the stem cell marker CD133 (Figure 2C). When cord blood cells were extracted with a nonionic detergent, only those cells which were positive for nestin and CD133 retained GR in the cytoplasm (Figure 2D). Cells which lacked nestin showed a nuclear GR signal (Figure 2D, arrow). The data indicate that cytoplasmic retention of GR in nestin expressing PBMCs defines adult stem or progenitor cells. Nestin dependent retention of GR in mouse embryo fibroblasts is a marker of early passages We next asked if the coincident presence of nestin and GR is a stable phenotypic marker that is maintained after explantation of primary cells in tissue culture. Mouse embryo fibroblasts [29] were isolated from BALB/c embryos (E 14.5). At the 2nd passage after explantation about 5 to 20% of the cells in a culture showed a coincident staining of nestin and GR, and both colocalized with the vimentin filament system. (Figure 3, A and B). In nestin negative cells the receptor localized to the nucleus (Figure 3, B1, arrows). Addition of the GR ligand dexamethasone abolished the GR signal on the nestin/vimentin filament system and induced an Nestin and Steroid Receptor accumulation of GR in the nucleus (Figure 3C). The proportion of nestin expressing cells declined gradually during passaging, decreasing to less than 1% nestin positive cells at the 6th passage, whereas vimentin expression was maintained. Concomitant with downregulation of nestin, the cells accumulated GR in the nucleus, adopted a senescent phenotype characterized by a flattened cell shape, positive staining for senescence associated b-galactosidase and entry into a sustained growth arrest (data not shown). Similarily, primary cell cultures derived from the hippocampus of embryonic rats (E 14.5 ), a proliferative region of the embryonic brain with significant nestin-immunoreactivity [11,30] contained cells which expressed nestin in addition to vimentin and showed a cytoplasmic accumulation of GR (data not shown). Nestin negative cells exhibited a nuclear GR signal only. The data indicate that cytoplasmic retention of GR in embryonic cells coincides with expression of nestin and vimentin and is a feature of early passages. It is lost concomitantly with adoption of a senescent phenotype. Anchoring of GR to IFs requires the coincident presence of vimentin and nestin To determine the need of nestin and vimentin for GR anchoring we analyzed cell lines varying in IF-protein composi- tion. Rat C6 subclones which either express vimentin or completely lack cytoplasmic IFs [31,32] were used to determine this relationship in detail. GR was present in the cytoplasm of C6D8 cells, a subclone which contains an IF network composed of vimentin and nestin (Figure 4, A1). In contrast, GR was strictly nuclear in C6D10 cells, which lack cytoplasmic IFs (Figure 4, B1). About 40% of the cells in a C6D10 culture displayed a diffuse nestin staining in the cytoplasm (Figure 4, B2). C6D10 cells contained significantly lower amounts of nestin than vimentin positive cells (Figure 5A). To determine an association of GR with the cytoskeleton the cells were lysed in situ by use of the detergent Triton-X100. GR staining was retained on cytoskeletons of extracted C6D8 cells (Figure 4, A2). Confocal microscopy and colocalization analysis pointed to coincident staining patterns of GR and vimentin in these cells (Figure S1, A1–A3). In contrast, neither tubulin (Figure S1, B1–B2) nor actin (Figure S1, C1–C2) colocalized with GR, as indicated by the broad scatter diagrams. We subjected C6D8 cells to treatment with cytochalasin B, a drug which disrupts the actin filament system, but leaves the vimentin filament system intact. In these cells the GR label still colocalized with vimentin filaments and did not redistribute with actin into patches (Figure S2). To substantiate the role of the IF-system in GR anchoring we restored the vimentin filament network in C6D10 cells by stably expressing an exogenous mouse vimentin protein. The exogenous protein contained an epitope in the rod domain which facilitated its detection by the human vimentin specific monoclonal antibody VIM3B4 [33]. Endogenous rat vimentin protein was identified by reaction with the monoclonal antibody V9, which binds to rat but not to mouse vimentin [33]. Cells (C6K23) with the reconstituted vimentin filament system showed a filamentous co-staining of GR, vimentin and nestin (Figure 4C). In addition, vimentin and GR could be co-immunoprecipitated from the cytoskeleton fraction of C6D8 cells (Figure 5C, lane 7). The precipitate was negative for lamin B, indicating that the presence of GR did not result from co- immunoprecipitation of residual nuclei. Tubulin and GR could not be co-immunoprecipitated (Figure 5D), which is consistent with the confocal data, showing no colocalization of GR and tubulin (Figure S1, B1–B2). Thus, the data substantiate a role of vimentin in cytoplasmic anchoring of GR. PLoS ONE | www.plosone.org 2 June 2009 | Volume 4 | Issue 6 | e6084