Diversity in Nuclear Pore Complexes Figure 8. Radial intensity distribution graphs of NUP62 and NUP214 immunofluorescence in cultured cell lines. Immunofluorescent images for fields of TOV112D, HEK293 (293), COS7, or S12 cells (similar to those shown in Fig. 5), and images from several different Hs 832(C).T cells, were analyzed. Radial intensity distributions for NUP62 or NUP214 were determined for each cell line and normalized to arc length, and a minimum of 24 sectors derived from at least six independent nuclei were used for each analysis. Radial distances were normalized from 0 (point of origin) to 1 (surface of nuclear envelope). Distribution points are colored according to residuals (blue: less than one standard error; green: less than two standard errors; red: greater than two standard errors). Plotted in red are 95% confidence intervals. doi:10.1371/journal.pone.0036137.g008 transcriptionally more active than genes associated with NPCs [30,31]. We have observed intranuclear accumulation of protein that binds NUP214 antibody, although this immunolabeling was relatively resistant to siRNA-mediated knockdown. It is possible that intranuclear NUP214 immunolabeling occurs in regions of active transcription, as the labeling appears to occur in areas of less intense DAPI staining (characteristic of euchromatin). Chromatin associated with NPCs has been found to be enriched in a subset of genes involved in development and cell cycle regulation, albeit in a transcriptionally less active state than genes associated with nucleoplasmic nucleoporins [30]. From those studies, activation of silent genes at the nuclear periphery by transcriptional regulators appears to result in inward migration either accompanied by or followed by association with nucleo- plasmic nucleoporins. Thus, while actively transcribing genes may be transferred to the inner nucleus, genes poised to receive positive regulators for activation may be retained near or moved to the nuclear envelope. One of the functions of the FG-repeat proteins is to direct specific cargo through the NPC, and NPCs bearing different FG-repeat proteins would be expected to transport different cargo. In addition, the specific functions of FG-repeat proteins in nuclear transport differ, and may even be antagonist [29]. The NUP214-NUP88 complex is required for NF-kB translocation into the nucleus [57,58], indicating that NUP214+ Figure 9. Optical sections of NUP62 and NUP214 immunolabel- ing in S12 cell nuclei using confocal and high resolution STED microscopy. HIgh resolution STED (NUP214; red) and standard confocal (NUP62; green) double immunofluorescence microscopy were performed on S12 neuroblastoma cells. Individual optical sections were selected from the ventral (culture plate side) nuclear surface (0 um) upward to the dorsal surface at intervals of 250 nm. Yellow, overlap bewteen green and red. Bar represents 5 um. doi:10.1371/journal.pone.0036137.g009 NPCs mediate this process. The characteristic distribution of NUP214+ NPCs suggests that NF-bkB is given access to specific subnuclear domains, and consequently to specific genes. Alterna- tively, the MUC1-C oncoprotein directly binds the central domain of NUP62, and this interaction is required both for nuclear import and for the oncogenic manifestations of MUC1-C over-expression in carcinoma cells [59]. The activity of MUC1-C would be directed at subnuclear domains accessed by NUP62+ NPCs. Similarly, nuclear delivery of the glucocorticoid receptor complex would be mediated by NUP62+ NPCs [60]. Targeting to specific subnuclear domains, and hence to specific genes, could be conferred by cell-type specific expression and sorting of FG-repeat proteins among NPC populations. Modulation of the distribution and composition of NPC populations may constitutes an epigenetic mechanism through which cell type-specific patterns of gene expression can be generated from more general external and intrinsic signaling streams. Recruitment of target genes to the nuclear periphery and to the domains of specific NPCs thus may program how the cell responds to future cues [61]. As nuclear exits, NPCs control the first stage of delivering nascent RNAs to specific cytoplasmic sectors, and thereby may play an important role in controlling subcellular organization. The identification and mapping of NPC populations divulges a novel parameter for illuminating nuclear polarity, which through the selective delivery of RNAs could contribute to or be required for the development and/or maintenance of cellular polarity. Transport of RNA from nuclear to cytoplasmic domains requires certain FG-repeat proteins [28], and some evidence has suggested that different RNAs may require specific FG-repeat proteins for nuclear export [62]. Variations in the distribution of NPC populations reported here suggest that transcripts may be selectively delivered to specific cytoplasmic sectors in certain cell types, thereby mediating differentiation and the acquisition of polarity. Understanding NPC diversity and distribution will help elucidate how the 3D structure of the nucleus contributes to cell asymmetry and differentiation, as well as to patterning and stereological control of gene expression. Materials and Methods Cell lines and antibodies Human ovarian carcinoma (TOV112D and TOV21G), benign cystadenoma cells (Hs 832(C).T), COS7, and HEK293 cell lines were either purchased from the American Type Culture Collection (ATCC) or acquired from long-term laboratory stocks. The S12 neuroblastoma cell line is a spontaneously differentiating clone derived from SH-SY5Y [63]. All cells were maintained Dulbecco’s modified Eagle medium (DMEM; Cellgro) supple- mented with 15% fetal bovine serum. Antibodies to NUP62 were purchased from BD Transduction Laboratories (product numbers 611962 and 610498 (Ab1)) and Santa Cruz Biotechnology (product number H-122 sc-25523 (Ab2)). Antibodies to NUP214 were purchased from Bethyl Laboratories (product numbers IHC- 00103 (Ab4) and A300–717A (Ab3)). Antibody to NUP133 was purchased from Abnova (product number H00055746-M01). Antibody to GAPDH (MAB374) was purchased from Chemicon (Millipore). Monoclonal anti-V5 epitope antibody was purchased PLoS ONE | www.plosone.org 11 April 2012 | Volume 7 | Issue 4 | e36137