hydrolysis of RanGTP to RanGDP, and dissociation of the complex. RanGDP returns to the nucleus through its interaction with nuclear transport factor 2 (NTF2), and the nucleus guanine nucleotide exchange factor (RanGEF or RCC1) mediates recharging to RanGTP (reviewed in [26]). A shared structural feature of many nucleoporins (referred to as FG-repeat containing) are hydrophobic core amino acid repeats, such as GLFG or FXFG, which mediate selective interactions with carrier proteins involved in nuclear/cytoplasmic translocation. For example, NTF- 2 binds primarily FXFG nucleoporins, whereas importin-b and the mRNA export factor TAP bind both FXFG and GLFG nucleoporins [27,28]. In addition, the functional roles of different FG-repeat proteins are probably not equivalent, and interactions between FG-repeat proteins may influence their activities [29]. Finally, recent studies have shown that some FG-repeat nucleo- porins accumulate in the nucleoplasm, and independently of the NPC associate with and function in transcriptional regulation of specific segments of chromatin [30,31]. The NUP62 complex assembles from O-glycosylated proteins of molecular masses 62, 58, 54, and 45 kDa [15,16]. The 62 kDa component of the complex, NUP62, contains three domains: N- terminal FG-repeat, central threonine/alanine-rich linker, and C- terminal alpha helical coiled-coil [15]. Similarly, NUP214 contains an N-terminal FG-repeat domain, while the C-terminal region is rich in serine, threonine and proline [17]. Although bound to the ring structure on the cytoplasmic face of the NPC, the FG-repeat domain of NUP214 is flexible and can reside on either side of the pore, and its topology is influenced by the transport state of the NPC or the local concentration of chemical mediators [32,33]. Nucleoporin 214 has been reported to interact with NUP88/ NUP82p [34], NUP358 [35], NUP62 [36], and NUP98 [13]. Although global alterations in the architecture and stoichiometry of the NPCs have been observed after knock-down of NPC components either by genetic or other experimental methods [19,37,38,39], architectural and distributional diversity among native NPC populations in cells are largely unexplored. Here we report that NPCs display architectural and distributional hetero- geneity in adherent cultured cells. Populations of NPCs are distinguished by NUP62 and NUP214 immunolabeling and by their spatial distribution on the nuclear envelope. Results Populations of NPCs distinguished by NUP62 and NUP214 immunolabeling The three dimensional shape of the nucleus of most adherent cultured cells approximates an oblate ellipsoid, which is essentially a moderately flattened sphere consisting of contralateral surfaces facing either the culture plate or medium connected by an intervening, steeply-arcked rim structure. Several of the micro- scopic images presented in this report represent stacks of serial optical sections taken from the medium side through the entire nucleus to the culture plate side, and are visualized as a projections of the stack to the x,y, x,z, and z,y planes. By viewing signals from two perspectives, it is possible to distinguish co-localized and disparate immunolabeling. Nucleoporin immunofluorescent sig- nals emanating from the nuclear envelope (contralateral surfaces and rim) are likely to represent NPCs, whereas intranuclear signals, by definition, would not arise from NPCs and may represent nucleoporins involved in functions other than nucleocy- toplasmic transport [40]. Western blot analyses revealed that knockdown of NUP62 or NUP214 to ,25% of control levels resulted from treatment of TOV112D cells (derived from a high grade ovarian carcinoma Diversity in Nuclear Pore Complexes [41]) with combinations of targeted siRNAs (Fig. 1A). Knockdown of NUP62 was accomplished in 48 hours with a single treatment of three specific siRNAs; knockdown of NUP214 required 72 hours and two treatments with six specific siRNAs. Although knockdown of NUP62 is accompanied by growth inhibition of TOV112D cells, this resulted from mitogenic arrest rather than loss of cell viability [42]. In contrast, NUP214 siRNA treatment of TOV112D cells for extended periods (longer than one week) resulted in death of TOV112D ovarian tumor cells, followed by colonial growth of some remaining cells. For the NUP214 knockdown studies presented below, original NUP214 siRNA treated TOV112D cells were employed and not cells recovered from remaining colonies. Immunofluorescence microscopy re- vealed that knockdown of NUP62 results in regional loss of immunolabeling among NPCs rather than a general loss of signal from all NPCs (Fig. 1B). In particular, signal is lost from groups of NPCs on the rim of the nucleus (readily apparent when viewing projections on the z,y and x,y planes). Knockdown of NUP214 resulted in loss of signal from the contralateral surfaces of the nucleus, with the remaining signal localized to intranuclear accumulations (Fig. 1B). Intranuclear immunolabeling by the NUP214 antibody is further addressed below, but as this labeling lies distal to the nuclear envelope it would, by definition, not be attributable to NPC-bound NUP214. Double immunofluores- cence microscopy of TOV112D cells with NUP62 and NUP133 (a component of the conserved NPC inner core ring complex [38]) revealed co-localization of NUP62 with NUP133 at the nuclear rim; in contrast, double immunofluorescence with NUP214 and NUP133 antibodies revealed preferential co-localization of NUP214 with NUP133 on the contralateral surfaces the nucleus (Fig. 1C). Employing a recently reported method that is rapid, gentle, and preserves protein-protein interactions [43], nuclear and cytosolic fractions were isolated from Hs 832(C).T (slowly proliferating fibroblastoid cells derived from an ovarian benign cystadenoma), TOV112D, HEK293(adenovirus transformed cells derived from human embryonic kidney), S12 (a clone derived from SH-SY5Y neuroblastoma cells), and COS7 (green monkey SV40 trans- formed kidney cells). Samples were immunoblotted with the NUP62 and NUP214 antibodies used for immunofluorescence (Ab1 and Ab4, below). As shown in Fig. 2, binding of the NUP214 antibody was predominantly at the NUP214 band and another band of ,35 kDa. While the latter band is detected in the nuclear fraction, it does not appear to contribute a signal in immunoflu- orescence microscopy. For example, while the 35 kDa band is absent from immunoblots of COS7 cell extracts, the NUP214 immunofluorescent patterns of TOV112D (which display the 35 kDa band) and COS7 cells are quite similar (see below). In addition, immunoblots of both HEK293 and TOV112D cells display the 35 kDa band, but their NUP214 immunofluorescent patterns are different. It is likely that the 35 kDa band contains an epitope that is revealed only in immunoblots after denaturation of the proteins in SDS. The NUP62 antibody bound exclusively to ,62 kDa bands (Fig. 2). The NUP214 and NUP62 antibodies detected varying levels of nucleoporins in the cytosolic fractions, which is consistent with other reports of cytoplasmic stores of nucleoporins in annulate lamellae and other compartments [44], and with immunofluorescence analyses shown below. Architecturally distinct NPC populations are positioned on the nuclear envelope and can be distinguished from intranuclear nucleoporins To distinguish intranuclear signals from those originating at the nuclear envelope, single optical sections from the nuclear surfaces PLoS ONE | www.plosone.org 2 April 2012 | Volume 7 | Issue 4 | e36137