Domain topology of nucleoporin Nup98 within the nuclear pore complex.

Antibodies

The following antibodies were used in this study: a rabbit polyclonal anti-xNup98 peptide3 ( Powers et al., 1995 ) against the C terminus of Xenopus Nup98 (residues 843-855; QGAQFVDRPESG); a rat monoclonal anti-Nup98 antibody against the N terminus of human Nup98 (residues 1-466; clone 2H10, Sigma-Aldrich, St. Louis, MO; ( Fukuhara et al., 2005 )); a mouse monoclonal anti-myc antibody (clone 9E10, supernatant of hybridoma cell line). For immunofluorescence, the following antibodies were used: rabbit anti-huNup98 C-terminal domain, residues 506-863 ( Griffis et al., 2002 ); rat monoclonal anti-huNup98 GLFG domain ( Fukuhara et al, 2005 ; Sigma); rabbit anti-huNup153 Zn Finger domain, (gift from Katie Ullman); mouse monoclonal SA1 anti-Nup153 C-terminal domain (gift from Brian Burke); rabbit anti-Nup358 IR domain (gift from Mary Dasso); mouse monoclonal 414 (Abcam, Cambridge, MA); Alexa-labeled secondary antibodies (Invitrogen, Carlsbad, CA). DNA constructs pcDNA3-myc-Nup98 was generated by digestion of pCS2-MT-Nup98 with BamHI and XhoI. This excises Nup98 along with 6 copies of the myc epitope derived from pCS-MT. The Nup98 fragment was then ligated into pcDNA3.0 (Invitrogen, Corporation, Carlsbad, CA). pEGFP-Nup358 was a kind gift of Dr. Joachim Köser (Biozentrum, University of Basel, Switzerland). Western blotting For HeLa protein extracts, approximately 1×10 6 cells were resuspended in lysis buffer (50 mM Tris-HCl pH 8, 150 mM NaCl, 1% Nonidet P-40, containing protease inhibitor cocktail tablets from Roche) and then cleared by centrifugation. After protein quantification, 20 μg of protein were mixed with Laemmli loading buffer for gel electrophoresis. For Xenopus protein extracts, 15 nuclei isolated from oocytes were resuspended in 60μl of low salt buffer followed by addition of 30 μl of 3X Laemmli loading buffer. For gel electrophoresis, 20 μl of Xenopus extract were loaded. Proteins were separated on 7% SDS-PAGE before being transferred to PVDF membrane which was subsequently blocked with 5% non-fat milk. For immuno-blotting the following primary antibodies were used: anti-GLFG Nup98 (clone 2H10, 1/2000) and anti-C Nup98 (1/1000). All secondary antibodies were alkaline phosphatase-coupled anti-IgG antibodies (1/20.000; Sigma). All dilutions were carried out in 5% non-fat milk. Blots were developed using CDP-Star (Applied Biosystems).

Immuno-EM of isolated nuclei from Xenopus oocytes

Mature (stage 6) oocytes were surgically removed from female Xenopus laevis, and their nuclei were isolated as described ( Fahrenkrog et al., 2002 ). Colloidal gold particles, ~8-nm in diameter, were prepared by reduction of tetrachloroauric acid with sodium citrate in the presence of tannic acid and antibodies were conjugated to colloidal gold particles as described ( Slot and Geuze, 1985 ). Isolated nuclei were labeled as described previously ( Fahrenkrog et al., 2002 ). Thin sections were cut on a Reichert Ultracut microtome (Reichert-Jung Optische Werke, Vienna, Austria) using a diamond knife (Diatome, Biel, Switzerland). The sections were collected on parlodion coated copper grids and stained with 6% uranyl acetate for 1 h followed by 2% lead citrate for 2 min. Electron micrographs were recorded with a Philips CM-100 transmission electron microscope (FEI Company, Hillsboro, OR) operated at an acceleration voltage of 80 kV equipped with a CCD camera. Microinjection and immuno-EM of tagged human Nup98 in Xenopus nuclei For microinjection of myc-Nup98 into nuclei, freshly isolated oocytes from Xenopus laevis were prepared and processed for microinjection as described ( Fahrenkrog et al., 2002 ). 5 ng per microliter of pcDNA-myc-Nup98 was injected into each oocyte nucleus. The localization of the fusion proteins within the NPC were determined by using a monoclonal anti-myc directly conjugated to 8-nm colloidal gold.

Immunofluorescence

For immunofluorescence staining, HeLa cells were grown on glass #1.5 coverslips in DMEM with 10% FBS, 1% Glutamax (Invitrogen) and 1% each penicillin/streptomycin. Cells were simultaneously fixed and permeabilized with 2% formaldehyde (Ted Pella Inc, Redding, CA), 0.2% Triton-X100 in PBS for 15 minutes at RT. Cells were blocked for 30 minutes at RT in 5% BSA, 5% normal goat serum, 0.02% Triton-X100 in PBS and then incubated for 1 hr in primary antibody diluted in block solution. After washing, cells were incubated for 1 hr with the appropriate Alexa fluor-labeled secondary antibody diluted in block solution. Cells were then washed, stained with Hoechst and mounted with Vectashield (Vector Laboratories, Burlingame, CA). For transfection of GFP-Nup358 1.5 μg of plasmid was used per well of 6 well culture dishes. Cells were transfected with HeLa-Monster (Mirus Bio, Madison, WI) 48 hr before fixation and staining.

Microscopy and Image analysis

Fluorescence microscopy was carried out using a Nikon N-SIM microscopy system on an Eclipse Ti inverted microscope run with Nikon Elements software (Nikon Instruments Inc., Melville, NY). The samples were imaged with a 100x 1.49 NA objective and an iXon DU897 EM-CCD camera (Andor Technology PLC, Northern Ireland). Widefield images were acquired with a Hg lamp and the appropriate filters: 480/30 ex 535/40 em (Alexa488 and GFP) or 540/25 ex 692/68 em (Alexa555). Widefield images were deconvolved with Huygens software (Scientific Volume Imaging, Netherlands). SIM images were acquired with laser excitation and emission filters 488 nm ex 520/40 em (Alexa488 and GFP) and 561 nm ex and 640/40 em (Alexa 555). Images were acquired in 3D SIM mode (for each SIM image 15 images with 5 different phases of 3 different angular orientations of illumination were collected) and z-stacks were collected for each image. SIM images were processed with the Nikon Elements software. The reconstruction parameters were optimized to be: Structured illumination contrast = 1.5; Apodization Filter = 1.0; Width of 3D-SIM filter = 0.18. SIM images of 5-10 cells were acquired for each condition. Images were analyzed in ImageJ (NIH, Bethesda, MD). Lines with a width of 3 pixels were drawn perpendicular to and intersecting the nuclear envelope at a point where both labels were present. The fluorescence intensity along the lines was recorded in each channel. Multiple line scans were assessed for each condition. In each case, a representative line scan is shown. For some antibody pairs, not every NPC showed separation of labels, but, when separated the relative position was never observed to be opposite of that illustrated.