Laser-based spinning disk confocal microscope (Andor Technology). Filtered images (Semrock emission filters in a Sutter filter wheel) were captured with a D-977 iXon EMCCD+ camera (Andor Technology) after twofold magnification (Andor Technology) by using a 1006TIRFM/1.45 objective (Olympus). Z-Stacks were recorded with a spacing of 0.2 mm over the Dinaciclib web entire cell (10?5 planes). Images were processed with ImageJ software (http://rsbweb.nih. gov/ij/) and the MBF ImageJ for Microscopy collection of plugins (http://www.macbiophotonics.ca/imagej/). For quantification of the Glc7GFP signal, single Z-slices of confocal images that had been recorded under identical conditions were used. The average GFP fluorescence intensity was measured in an area of equal size in the nucleus and cytoplasm using ImageJ software, and the ratio was calculated. Fluorescence microscopy of Glc7GFP localization 23727046 upon additional expression of untagged GLC7 (Fig. S3) was carried out using a Zeiss Axiovert 200 M microscope equipped with an Axio Apochrom (Zeiss) 1006/1.4 oil objective and the filter set #10 (FITC). Images were captured using an AxioCam MRm TV2/30 0.636 (Zeiss) camera and AxioVision LE software. For the analysis of sister chromatid separation (Fig. 5cd), cultures were grown to log-phase in SC medium+/22 mM methionine, harvested, and resuspended in sterile filtered medium. 1.4 low-melting agarose was added in equal volume to mount the samples on cover slips. Microscopy was carried out on a Nikon TiE inverted live cell system with a motorized Prior Z-stage and Perfect Focus System using a 1006 1.45 NA objective (Nikon). Eleven Z-Stacks (spacing 0.3 mm) were recorded with a Photometrics HQ2 camera and analyzed using Nikon NIS Elements software. For differential interference contrast (DIC) microscopy, a single snap-shot was taken. All images were recorded using identical exposure times. Medium- to large-budded cells in eachYIplac128-PMET25-glc8T-118A3HA YIplac211-SHP1 YIplac211-shp1-7 YIplac211-shp1-b1 YIplac211-shp1-a1 YIplac211-shp1-a3 YIplac211-shp1-a4 YIplac211-shp1-a5 YIplac211-shpDUBAYIplac211-shp1DUBX YIplac128-256xlacO YIplac211-GFPLacIdoi:10.1371/journal.pone.0056486.tmutated region. Double mutants were constructed by crossing the respective conditional allele with the shp1-7 mutant carrying YCplac33-SHP1. Yeast was cultured in DMXAA standard YPD and SC media [119]. For the induction of the PMET25 promoter, cells were first grown in SC media supplemented with 2 mM methionine, washed twice with H2O, and then transferred to SC medium 1317923 lacking methionine.a-factor arrest/releaseOvernight cultures of wild-type and mutant strains were diluted to an OD600 nm of 0.1 (0.15 for shp1 mutants) in 50 ml YPD. The cultures were then grown at 25uC for approximately four hours until reaching an OD600 nm of 0.3?.35. 10 mM a-factor (central core facility, Max Planck Institute of Biochemistry, Martinsried, Germany) in DMSO were added, and the cells were allowed to arrest for three hours at 25uC. Directly before addition of a-factor, a control sample from the asynchronous culture was collected, and the pellet was frozen in liquid nitrogen. The efficiency of the arrest was determined by FACS analysis and/or Western blot for Clb2 levels after three hours of arrest. The cultures were then washed two times with equal volumes of YPD and resuspended to a finalRegulation of Glc7 by Cdc48ShpTable 2. Yeast strains used in this study.Strain DF5a YAB589 YAB1729 YAB1568 YAB1564 YAB1288 YAB171.Laser-based spinning disk confocal microscope (Andor Technology). Filtered images (Semrock emission filters in a Sutter filter wheel) were captured with a D-977 iXon EMCCD+ camera (Andor Technology) after twofold magnification (Andor Technology) by using a 1006TIRFM/1.45 objective (Olympus). Z-Stacks were recorded with a spacing of 0.2 mm over the entire cell (10?5 planes). Images were processed with ImageJ software (http://rsbweb.nih. gov/ij/) and the MBF ImageJ for Microscopy collection of plugins (http://www.macbiophotonics.ca/imagej/). For quantification of the Glc7GFP signal, single Z-slices of confocal images that had been recorded under identical conditions were used. The average GFP fluorescence intensity was measured in an area of equal size in the nucleus and cytoplasm using ImageJ software, and the ratio was calculated. Fluorescence microscopy of Glc7GFP localization 23727046 upon additional expression of untagged GLC7 (Fig. S3) was carried out using a Zeiss Axiovert 200 M microscope equipped with an Axio Apochrom (Zeiss) 1006/1.4 oil objective and the filter set #10 (FITC). Images were captured using an AxioCam MRm TV2/30 0.636 (Zeiss) camera and AxioVision LE software. For the analysis of sister chromatid separation (Fig. 5cd), cultures were grown to log-phase in SC medium+/22 mM methionine, harvested, and resuspended in sterile filtered medium. 1.4 low-melting agarose was added in equal volume to mount the samples on cover slips. Microscopy was carried out on a Nikon TiE inverted live cell system with a motorized Prior Z-stage and Perfect Focus System using a 1006 1.45 NA objective (Nikon). Eleven Z-Stacks (spacing 0.3 mm) were recorded with a Photometrics HQ2 camera and analyzed using Nikon NIS Elements software. For differential interference contrast (DIC) microscopy, a single snap-shot was taken. All images were recorded using identical exposure times. Medium- to large-budded cells in eachYIplac128-PMET25-glc8T-118A3HA YIplac211-SHP1 YIplac211-shp1-7 YIplac211-shp1-b1 YIplac211-shp1-a1 YIplac211-shp1-a3 YIplac211-shp1-a4 YIplac211-shp1-a5 YIplac211-shpDUBAYIplac211-shp1DUBX YIplac128-256xlacO YIplac211-GFPLacIdoi:10.1371/journal.pone.0056486.tmutated region. Double mutants were constructed by crossing the respective conditional allele with the shp1-7 mutant carrying YCplac33-SHP1. Yeast was cultured in standard YPD and SC media [119]. For the induction of the PMET25 promoter, cells were first grown in SC media supplemented with 2 mM methionine, washed twice with H2O, and then transferred to SC medium 1317923 lacking methionine.a-factor arrest/releaseOvernight cultures of wild-type and mutant strains were diluted to an OD600 nm of 0.1 (0.15 for shp1 mutants) in 50 ml YPD. The cultures were then grown at 25uC for approximately four hours until reaching an OD600 nm of 0.3?.35. 10 mM a-factor (central core facility, Max Planck Institute of Biochemistry, Martinsried, Germany) in DMSO were added, and the cells were allowed to arrest for three hours at 25uC. Directly before addition of a-factor, a control sample from the asynchronous culture was collected, and the pellet was frozen in liquid nitrogen. The efficiency of the arrest was determined by FACS analysis and/or Western blot for Clb2 levels after three hours of arrest. The cultures were then washed two times with equal volumes of YPD and resuspended to a finalRegulation of Glc7 by Cdc48ShpTable 2. Yeast strains used in this study.Strain DF5a YAB589 YAB1729 YAB1568 YAB1564 YAB1288 YAB171.
