Quantitation of pore-related nuclear membrane structures in gp210-siRNA HeLa cells. We identified (A) and quantified (B and C) three distinct morphological features including mature NPCs (m), fusion-arrested structures (f), and twinned membranes (t) on TEM micrographs of gp210-siRNA cells and control GL2-siRNA HeLa cells. The number of counted structures for each category is shown in B, and graphed in C as the percentage of total structures counted in either gp210-siRNA or control GL2-siRNA cells. Bars in A, 50 nm.

Gp210 in C. elegans. (A) A schematic diagram of full-length Ce-Gp210 (residues 1–1847), drawn approximately to scale, showing the exons (numbered boxes), a single predicted transmembrane domain (TM), and the bacterial Ig-like type 2 (Big2) domain. Regions used as antigen are marked by orange lines and labeled by serum number (3586 and 3783). (B) Western blot of crude protein extract of mixed stage C. elegans probed with preimmune (P) or immune (I) serum 3586. (C) Confocal images of an adult C. elegans gonad double-stained by indirect immunofluorescence using Ce-Gp210 serum 3783 (left panel), FG-repeat nucleoporins (mAb414; NUPs; middle panel), and the merged images (right panel).

Phenotypes of HeLa cells with reduced gp210. (A) Transfection of HeLa cells with dsRNA specific for human gp210 gene (gp210-siRNA) caused significantly reduced expression of gp210 relative to control cells transfected with nonspecific dsRNA (GL2-siRNA). Cells were double-stained for gp210 (Courvalin et al., 1990) by indirect immunofluorescence and DAPI to visualize DNA and imaged by fluorescence microscopy. (B) The growth rates of gp210-siRNA and untreated HeLa cells were quantified by a colorimetric assay (see MATERIALS AND METHODS). The graph shows the ratio of OD₍₆₃₀₎ measurements for gp210-siRNA cells vs. untreated cells at different time points. The exponential regression is monotonously descending (with a negative exponential coefficient) showing that the growth rate of gp210-siRNA cells was lower than that of untreated cells at all times. (C) Downregulation of gp210 disrupts FG-repeat nucleoporins. Gp210-siRNA cells and control GL2-siRNA cells were double-stained for human gp210 and FG-repeat nucleoporins (mAb414) and visualized by laser confocal microscopy. The exposure times shown for immunostaining are identical within each set of panels in A and C. (D) Thin-section transmission electron micrographs of GL2-siRNA control cells (a and b) and gp210-siRNA cells (c–f), 3 d after siRNA treatment. Cells with reduced gp210 had condensed chromatin (c), clustering of mature NPCs (d), enlarged nuclear envelope lumenal space (e), and clusters of pore-related membrane structures (f). Arrows in D depict mature NPCs (b and d), membrane blebs (e), and various arrested pore structures (f), which were named “twinned membranes” (t) and “fusion-arrested” membranes (f), as described in text. Bars: 30 μm in A, 20 μm in C, 200 nm in D (b, d–f), and 2000 nm in D (a and c).

Localization of nuclear lamins and emerin in gp210-siRNA cells. (A) Immunofluorescence of GL2-siRNA and gp210-siRNA cells double-stained for gp210 plus either lamin B (a), lamin A/C (b), or emerin (c). Images were obtained by laser confocal microscopy. (B) Assay for in vitro import of rhodamine-labeled NLS-BSA into nuclei of digitonin-permeabilized gp210-siRNA cells (left), GL2-siRNA cells (middle), or unpermeabilized cells (right). Each arrow indicates one nucleus. Bars: 20 μm in A and 10 μm in B.

RNAi-induced downregulation phenotype for Ce-Gp210. (A) Double immunostaining of control embryos (left) or Cegp210(RNAi) embryos (right; exons 8–15 were targeted), imaged by fluorescence microscopy. Embryos were stained by indirect immunofluorescence using antibodies against Ce-Gp210 (serum 3783), Ce-lamin (Liu et al., 2000), or FG-repeat nucleoporins (Nups; mAb414). Arrows indicate NPC clusters. (B) Thin-section TEMs of control untreated embryos (a) and Ce-gp210(RNAi) embryos in which exons 3–5 were targeted (b and c). Cegp210(RNAi) embryos had more condensed chromatin (b), an enlarged nuclear envelope lumen (b), and clustered NPCs (c). NU, nucleus; Cyt, cytoplasm. Bars: 10 μm (A), 200 nm (B, c), 750 nm (B, b), and 1500 nm (B, a).