Diauxic shift-dependent relocalization of decapping activators Dhh1 and Pat1 to polysomal complexes

Drummond, Sheona P.; Hildyard, John; Firczuk, Helena; Reamtong, Onrapak; Li, Ning; Kannambath, Shichina; Claydon, Amy J.; Beynon, Robert J.; Eyers, Claire E.; McCarthy, John E. G.

Cells for high-resolution analysis were grown from an initial liquid culture to an OD 600 = 0.4 (for final OD 600 upon visualization of $0.5) or to an OD 600 = 2.0 and then 200 ml of culture was incubated with 2 mM ReAsH-EDT2 biarsenical dye (Invitrogen) and 1 mM 1, 2-ethanedithiol (EDT) for 1 h at 30 C in the dark, with shaking and aeration. Then cells were washed in appropriate growth media with glucose for exponentially growing cells or without glucose for diauxic shift cells supplemented with 25 mM EDT for 10 min in the dark, briefly centrifuged at 4000 rpm and then washed as before with 1 mM EDT then resuspended in 200 ml of appropriate media (to analyse ReAsH labelled Dhh1/Pat1 migration into P-bodies, cells were resuspended in media minus glucose) then mounted on slides treated with 2% polylysine. Images for Supplementary Figure S4 were acquired on a Confocal Laser Scanning microscope (Zeiss) with a 100Â oilimmersion objective lens (Zeiss) and LSM 5 software. All other images were acquired with a Deltavision Core Imaging System (Applied Precision) as 512 Â 512-pixel files with an EMCCD camera and Softworx software. Images in Figure 6 and Supplementary Figures S2, S6 and S10 are 3D projections of z-series compilations of multiple images at 0.1-micron intervals assembled using the Softworx software. All other images are single section images, deconvolved with the Huygens Deconvolution Software (Scientific Volume Imaging B.V.; SVI). For quantitation, cells were processed for microscopy and images comprising 3D projections of a z-series compilation of 50 images at 0.1-micron intervals were deconvolved and analysed using Huygens Co-localization Analyser software (SVI). Measurements for each tagging combination were performed on at least 10 cells, yielding the averages shown in Figure 2 . In order to quantify the extent of co-localization of the red channel (Dhh1/Pat1-TCM[ReAsH]) and green channel (RpL25-GFP, Rps2-GFP or translation factor-GFP) the Manders Intersection coefficients (i) were calculated. In this study the i 1 coefficient indicates the portion of voxels of signal 1 (red = TCM) that are intersecting with signal 2 (green = GFP) from the total volume occupied by signal 1. It is of note that as the red and green channels exhibit different signal intensities, calculation of the intersection co-efficient (i) will give more reliable quantitation of co-localization. In Figure 2 , i 1 (the degree to which the red(TCM-ReAsH) signal overlaps with the green (GFP) signal) was converted into 'percentage of total'.