Uapo40

Conjugating pairs expressing GFP-tagged protein were suspended in 3% low-melting point agarose (SeaPlaque Agarose; Takara) dissolved in 10 mM Tris-HCl (pH 7.5) at 37°C. Conjugating pair-suspended agarose solution (100 µl) was placed in the center of a glass-bottomed dish and followed by placing a cylinder shaped, flat-soled epoxy resin block (0.95 g weight and 0.44 cm² bottom area) gently on top of the agarose solution. Gelation of agarose was effected by floating the glass-bottomed dish on ice-cold water (within 5 sec) followed by gentle removal of the resin block. This step produced a thin, flat gel in which conjugating pairs were pushed down near to the bottom of the glass. Procedures from suspending the cells in agarose solution to agarose gelation were executed as quickly as possible. The medium used for cell conjugation (100 µl) was layered on top of the agarose gel to prevent the gel from drying out during observation. For microscopic observation, the DeltaVision system (GE Healthcare, Buckinghamshire, England), placed in a temperature-controlled room, was used (Haraguchi et al., 2008 (link)). Time-lapse images (16–41 stacks) along the z-axis at 0.5–1.5 µm intervals were taken using an oil-immersion objective lens UApo40 (NA = 1.35, Olympus, Tokyo Japan). The images were processed by iterative deconvolution and/or denoising (Boulanger et al., 2009 (link)) when necessary.

Intracellular localizations of GFP-tagged Nups were observed by performing fluorescence microscopy (IX-70; Olympus, Tokyo, Japan). Images were taken using the DeltaVision microscope system (GE Healthcare, Issaquah, WA) with oil-immersion objective lens UApo40 (NA=1.35) (Olympus). Line profiles of fluorescence intensity were obtained with a measurement tool included in the DeltaVision system. Background fluorescence was measured from the cytoplasm as an averaged value of 5×5 pixels and was subtracted from the peak values of fluorescence on the NE.