Plan apo 1.4 na objective

Budding yeast strains EMS219 (Mat alpha, his5 leu2–3,212 ura3–50 CAN1 asp5 gal2 (form I1 rDNA::leu2 URA3+)), intact rDNA and EMS60-UVR-12 (LEU2+, URA3+ CANS form I1 rDNA), translocated rDNA (22 (link)), were transformed with CDC14-GFP:KAN to label the nucleolus to generate DCY1021.1 and DCY1017.2, respectively. DCY1021.1 was transformed using pS01 plasmid to introduce the brn1–9 allele into strain AY1009. DCY1021.1 was transformed to knockout Fob1 and Hmo1 in strains DCY1055.1 and DCY1056.1, respectively. Cells were grown in YPD (1% Yeast extract, 2% Bacto-peptone, 2% Dextrose) with excess adenine. Strains were grown until mid-log phase prior to imaging. Images were acquired at room temperature for wild-type (WT), fob1Δ and hmo1Δ mutant strains (25°C). brn1–9 strains were shifted to 37°C 3 h prior to image analysis. G1 cells were found in the population by visual inspection of bud size. Images were acquired using a Ti-Eclipse inverted microscope (Nikon) with a 100 × Plan Apo 1.4 NA objective (Nikon) and Clara CCD digital camera (Andor) using MetaMorph 7.7 imaging software (Molecular Devices). Single stacks contained 7 Z-planes sections with 300 nm step-size. Image stacks were cropped and maximum intensity projections were created using ImageJ.

To assay for mRNA export defects, FISH against poly(A)-RNA was performed as previously described (Cole et al., 2002 (link)). In brief, REF (BMY83) and mex67-5 (BMY135) strains were grown to mid-log phase at a permissive temperature (26°C) and then shifted to a nonpermissive (37°C) temperature for 30 min with prewarmed media. After fixation, poly(A)-RNA was detected using a fluorescein-labeled dT₅₀ probe, and DNA was visualized using DAPI. Imaging was performed on a microscope (DeltaVision Elite) equipped with a front-illuminated sCMOS camera driven by softWoRx 6 at 23°C using a 60× 1.4 NA oil objective. To localize Mex67p, haploid strains were generated (KWY5566 and KWY5567) expressing Ndc1p-tdTomato, GFA1-PP7, and GFP-tagged Mex67. To avoid cross talk from the PP7-CP tagged with YFP, we used strains that did not express the coat protein. Cells were grown in a synthetic complete medium at 26°C and then imaged in a 384-well plate coated with concanavalin A at 26°C using an inverted epifluorescence microscope (Ti; Nikon) equipped with a Spectra X LED light source and an sCMOS camera (Flash 4.0; Hamamatsu Photonics) using a 100× Plan-Apo 1.4 NA objective and the NIS Elements software (Nikon). All image processing was done using Fiji (Schindelin et al., 2012 (link)).