Fig 2: Nup133 is able to recruit an extensive array of nucleoporins to the LacO array. Immunofluorescence microscopy was performed on U2OS 2–6–3 cells transiently transfected with LacI-CFP-Nup133. The location of LacI-CFP-Nup133 was visualized by CFP fluorescence (left panel). To assay for recruitment of endogenous nucleoporins, the transfected cells were stained with antibody against: (A) Nup85, (B) Nup160, (C) ELYS, (D) Nup53, (E) Nup93, (F) Nup214, and (G) Nup98, followed by a Texas Red-labeled secondary antibody (middle panel). DNA was visualized by DAPI staining. The smaller panels show a magnified image of the LacO array. The right hand panels are a merge of the previous 2 images together with a DAPI-stained image. We note that when Nup133 was used as the LacI-CFP-tagged fusion protein, a large percentage of the transfected cells showed rim localization of the LacO array. Quantitation is shown in Table 1.

Fig 3: (A) A cut-away schematic representation of the massive nuclear pore complex, which has 8 radial spokes, is shown (inset). The subcomplexes that comprise one radial spoke and their predicted localization within that spoke are shown in the enlarged portion of (A). The nucleoporins used as LacI-CFP fusions in this study are highlighted in yellow. Each separate shape indicates a different subcomplex that must assemble with others to form a nuclear pore. The different colors are used to demarcate distinct regions of the formed nuclear pore, such as the large Y complex subunits (blue), ELYS (lavender), the central scaffold (green), the central transporter regions (gold), the cytoplasmic ring (purple), the cytoplasmic filaments (deep purple), and the nuclear basket (light brown). The three transmembrane Nups are shown in gray. (A few nucleoporins such as Aladin and hCG1 have been omitted for clarity). (B) Details of the Nup107–160 or “Y” complex, with the nucleoporins tested as LacI-CFP fusions underlined. (C) Diagram of the LacI-LacO system used in this study. A diagram of a U2OS 2–6–3 cell is shown, with the LacO array integrated into one location in the genome. The enlargement shows the expected binding of a fusion LacI-CFP-NupX (green) to each of the multiple copies of the LacO array (blue). (D–F) U2OS 2–6–3 cells were mock transfected (D), transfected with the negative control fusion LacI-CFP (E), or transfected with LacI-CFP-Nup fusion such as LacI-CFP Nup214 (F). To test for LacI-CFP-NupX binding to the LacO array, cells were visualized with anti-LacI to detect the fusion protein (left panel in each set), or a specific anti-nucleoporin antibody (middle panel in E and F). DNA was detected with DAPI stain. The right-hand panel in each set shows a merge of all 3 stains. The arrows point to the position of the LacO array.

Fig 4: Widespread recruitment of nucleoporins to the LacO array by the LacI-CFP-Nup153 fusion protein. Immunofluorescence microscopy of U2OS 2–6–3 cells transiently transfected with a LacI-CFP-Nup153 construct. The location of LacI-CFP-Nup153 was visualized by CFP fluorescence or staining with anti-LacI antibody (left panel). Endogenous nucleoporin recruitment was assessed with antibody against: (A) Nup133, (B) ELYS, (C) Nup62, or (D) Nup93 (middle panel). Chromatin was visualized by DAPI staining. The smaller panel shows a magnified image of the LacO array for each image; the right hand panel is a merge of the previous 2 images with the DAPI DNA stain. Data for additional nucleoporins tested as well as the quantitation for colocalization and rim localization for all tested nucleoporins are shown in Table 1.