Diversity in Nuclear Pore Complexes Figure 5. Effects of transfection reagents and cell density on nucleoporin distribution. A. TOV112D cells were treated in the absence of exogenous DNA with Lipo293D (SignaGen Laboratories), Lipofectamine (Invitrogen), or FuGENE HD (Promega), according to the instructions provided by the manufacturers. The cells were processed for immunofluorescence using NUP62 (green) and NUP214 (red) antibodies 48 hours after treatment. Immunofluorescent images represent 3D deconvolved projections of 250 nm optical sections through 10 to 15 um of the z axis. Projections are shown on x,y, x,z, and z,y planes, as indicated. Bar represents 5 um. Blue, DAPI stain; yellow, overlap between green and red. B. TOV112D were cultured at confluence for three days, and analyzed by immunofluorescence microscopy as described above. C. TOV21G cells were cultured at confluence for three days, and analyzed by immunofluorescence microscopy as described above. D. TOV21G cell cultured at low density and analyzed by immunofluorescence microscopy as described above. doi:10.1371/journal.pone.0036137.g005 COS7 cells. In addition, the analyses contrasts the NUP62 nuclear immunolabeling patterns among the cell lines. Preferential accumulation of NUP62+ NPCs at the nuclear rim appears to vary in degree, from a highly polarized distribution at the nuclear rim in TOV112D cells to a relatively even distribution in S12 and Hs 832(C).T cells (Fig. 8). In HEK293 cells, polarized distribution of both NUP62 and NUP214 is observed. High resolution immunofluorescence microscopy reveals architectural microheterogeneity in NPC populations Stimulated emission depletion (STED, Leica) combined with scanning confocal microscopy resolved immunofluorescent images of individual NPCs. The S12 neuroblastoma cell line was chosen for this analysis as it displays each of the three permutations of NUP62/NUP214 immunolabeled NPC types. Individual optical sections from the surfaces facing the culture plate or the medium and the central plane of the S12 nucleus revealed both common and disparate immunolabeled patterns for NUP62 and NUP214 (Fig. 9). Punctate immunolabeling for NUP62 and NUP214 was revealed on the surfaces of the S12 nucleus, with partial overlap. The central plane revealed partial overlap of NUP62 and NUP214 immunolabeling on the nuclear rim. Intranuclear filiform accumulations of NUP214 also were detected (Fig. 9). The resolving power of STED microscopy allowed groups of individual NPCs to be distinguished from what appeared in standard confocal images to be large indefinite clusters (Fig. 10A). Microheterogeneity in NPC populations was revealed by STED microscopy: in addition to NUP62+/NUP2142 and NUP622/ NUP214+ NPC populations, NUP62.NUP214 and NUP62,NUP214 populations also were detected (Fig. 10A and 10B). Remarkably, STED microscopy revealed NUP214 as a single dot, consistent with a position on the cytoplasmic annular ring, whereas NUP62 was revealed as pairs of dots (the dots are inclined consistently, and the distance between the dots appears greater when viewed more laterally though NPCs nearer the rim), consistent with the NUP62 complex forming two rings in each pore (Fig. 10C and 10E). Finally, sufficient resolution was provided by STED to roughly compare the diameters of the NPC populations (derived from the STED images of NPCs shown in Fig. 10C, 10D, and 10E). Measured from each outer edge across the pore, NUP62+/NUP2142 NPCs measured 158619 nm, NUP622/NUP214+ NPCs measured 186611 nm, and NUP62+/NUP214+ NPCs measured by the NUP62 antibody immunofluorescence as 178611 nm. These values are similar, and suggest the absence of profound gross structural differences between the NPC populations. The size of isolated NPCs has PLoS ONE | www.plosone.org 7 April 2012 | Volume 7 | Issue 4 | e36137