Planapon60xosc na1

Conjugating pairs of cells expressing GFP–Nup93 were embedded in an agarose gel on a glass-bottomed dish with an addressing grid (grid size, 175 µm) on the coverslip (AGC Techno Glass). Live cells were monitored on the DeltaVision system; to fix cells, 2.5% glutaraldehyde solution was layered over the agarose gel and left for 2 hrs. 3D images (50–60 z-stacks×0.2 µm intervals) were obtained using an oil immersion objective lens (PlanApoN60xOSC/NA1.4; Olympus) and processed by deconvolution. EM observation of the same cells was carried out as described previously (Haraguchi et al., 2008 (link)): Briefly, the cells in the agarose gel were post-fixed with 1% OsO₄ (Nisshin EM, Tokyo, Japan) for 1 hr. After sequential dehydration with ethanol, the samples were stained en bloc with 2% uranyl acetate (Wako, Osaka, Japan) for 30 min. The epoxy block containing the cells of interest was trimmed according to the coordinates of the grid and sliced into 80-nm sections. Image data were collected by JEM-2000EX II (JEOL, Tokyo, Japan) with an acceleration voltage of 80 kV.

After overnight incubation on ME plates, cells were re-suspended in EMM-N+5S medium. To disperse sporulating cells, suspensions were subjected by brief sonication (Handy Sonic; Tomy Seiko, Tokyo, Japan); 20 µl of the cell suspension was then dropped onto lectin (0.2 mg/ml; Sigma-Aldrich, Tokyo, Japan)-coated 35-mm glass-bottomed culture dishes (MatTek, Ashland, MA, USA) to immobilize cells (Asakawa and Hiraoka, 2009 (link)). For imaging Latrunculin A-treated cells, Latrunculin A (Thermo Fisher Scientific, Tokyo, Japan) was added at a final concentration of 1 µM prior to cell immobilization. Cells undergoing sporulation were selected for live-cell imaging.
A DeltaVision microscope equipped with a CoolSNAP HQ² charge-coupled device (GE Healthcare, Tokyo, Japan) was used for image acquisition. Optical section images were acquired at 0.5-µm focus intervals using an oil-immersion 60× objective lens (PlanApoN60x OSC, NA1.4; Olympus, Tokyo, Japan). Images were processed using the de-noising algorithm (Boulanger et al., 2009 (link)) and by constrained iterative deconvolution (Agard et al., 1989 (link)).