5.11.1 Fluorescence microscopy
In general, formaldehyde-fixed yeast cells with fluorescent signals were examined with a Leica DMI6000B fluorescence microscope and pictures were obtained by using the Leica DFC360 FX camera and the LAS AF 1.6.2 software. To avoid movement of the cells, they were transferred onto microscope slides that were previously coated with 0.3 % (w/v) poly-L-lysine hydrobromide. For preparation of the
slides, the polylysine solution was dropped onto each well of the microscope slide, dried on a 65°C heating block and subsequently washed with water.
0.1 M phosphate-buffer (pH 6.5): 33 mM K2HPO4
67 mM KH2PO4
P-solution (pH 6.5): 0.1 M phosphate-buffer pH 6.5 1.2 M sorbitol
1x PBS (pH 7.4): 137 mM NaCl 2.7 mM KCl 10 mM Na2HPO4
1.8 mM KH2PO4
Mounting Solution (pH 8.0): 2 % (w/v) n-Propyl gallate 80 % (v/v) Glycerol
in PBS (pH 8.0)
5.11.2 GFP-microscopy
The cellular localization of GFP-tagged proteins was examined in formaldehyde-fixed cells, as described previously (Windgassen and Krebber, 2003). For that, GFP-fusion proteins expressing yeast cells were grown in selective media to log-phase, shifted for 1 h to their non-permissive temperatures and shortly fixed with 2.6 % (v/v) formaldehyde. To prevent destruction of the fluorescent GFP, the cells were immediately centrifuged for 5 min at 2 050 x g and 4°C and washed once with 0.1 M phosphate-buffer and once with P-solution. Depending on the size of the pellet, the cells were resuspended in 50-150 µl of P-solution and 20 µl of this suspension was transferred onto one well of the polylysine-coated microscope slide. After 20 min incubation, the cells were permeabilized by treatment with 0.5 % (v/v) TritonX-100 in P-solution for 5 min. Upon washing with 1x PBS, the DNA was stained with Hoechst 33342 (diluted 1:10 000 in PBS) for 5 min followed by two washing steps with 1x PBS. Finally, the slides were dried and sealed with mounting solution and a cover slip.
5.11.3 Fluorescence in situ hybridization (FISH)
Fluorescence in situ hybridization (FISH) is a method to localize specific nucleic acids such as DNAs and RNAs in tissues or cells by using fluorescent labeled probes that specifically hybridize with the nucleic acids of interest. To localize the different ribosomal RNAs in yeast cells, FISH experiments with specific DIG-labeled
RNA-probes (generation see section 5.10.4) were performed according to Amberg et al.
(1992) with some modifications. Furthermore, a Cy3-end labeled oligo(dT)50 probe was used for detection of poly(A)+RNAs.
All buffers and solutions were prepared with DEPC-treated water.
Hyb-Mix: see section 5.10.4 20x SSC: see section 5.10.5
Zymolyase-Solution: 10 mg/ml Zymolyase
2 mM Vanadyl ribonucleoside complexes 1 mg/ml Heparin
Antibody blocking buffer (ABB): 1x PBS
5 % (w/v) heat-inactivated FBS 0.3 % (v/v) Triton-X100
Liquid cultures with log-phase yeast cells were shifted to their restrictive tempe-ratures or induced by galactose addition as indicated. Subsequently, the cells were fixed by addition of 4 % (v/v) formaldehyde and incubation with agitation at RT for 1 h and then pelleted by centrifugation for 5 min at 2 050 x g and 4°C. Upon washing three times with 1 ml P-solution, the cells were resuspended in 100 µl of P-solution and treated with zymolyase to digest the cell wall and produce spheroplasts. For that, the cell suspension was incubated with 10 mM DTT for 10 min at RT and sub-sequently 50 µg zymolyase was added. The enzymatic reaction was performed at RT and its progress was followed by examination with a light microscope. When half of the cells appeared dark indicating a successful cell wall digestion, 1 ml of P-solution was added to stop the reaction. The spheroplasts were carefully pelleted by centrifugation for 3 min at 400 x g and 4°C and washed two times in P-solution.
Afterwards, the spheroplasts were resuspended in an appropriate volume of P-solution (~100 µl) and 25 µl of the suspension were transferred onto one well of the polylysine-coated microscope slides. Upon 15 min incubation, the adhered sphero-plasts were permeablized with 0.5 % (v/v) triton X-100 in P-solution for 10 min at RT and rinsed with P-solution. Subsequently, equilibration in 0.1 M triethanolamine pH 8.0 was performed for 2 min and the polar groups of proteins were blocked with 0.25 % (v/v) acetic anhydride in 0.1 M triethanolamine pH 8.0 for 10 min at RT. Upon rinsing with P-solution, the cells were pre-hybridized with Hyb-mix supplemented with 500 µg/ml of tRNAs and 500 µg/ml of denatured Salmon sperm carrier-DNAs for 1 h at 37°C. Afterwards, hybridization with specific probes was performed overnight at
37°C. For that, DIG-labeled RNA probes (0.1 µl per well) or Cy3-labeled oligo(dT)50
probes (0.5 µl per well) in tRNA and ssDNA supplemented Hyb-mix were added to each well. The next day, the following washing steps were performed for 30-60 min each: 2x SSC at RT, 1x SSC at RT, 0.5x SSC at 37°C and 0.5x SSC at RT. Upon blocking with ABB for 1 h at RT, a sheep anti-digoxigenin Fab-FITC antibody (Roche) was diluted 1:200 in ABB and added overnight at 4°C for DIG detection. Then, the following washing steps were performed at RT: twice with ABB for 15 min, once with ABB for 30 min and twice with 0.1 % (v/v) Tween-20 in PBS for 30 min. Afterwards, the DNA was stained with Hoechst 33342 (diluted 1:10 000 in PBS) for 5 min and three times washed with PBS. Finally, the slides were air dried and sealed with mounting-solution and cover slips.
5.11.4 Statistical analyses
The ratio of cells that showed a nuclear accumulation of the fluorescent signal was determined from several microscopy pictures and subsequently, the average was calculated for every strain.
To determine the average enrichment of the nuclear signal, the fluorescent signals of the whole cell and of its nucleus of at least 10 cells with phenotype per strain were quantified by using the Fiji software. Subsequently, the intensity of the nuclear signal was related to the complete cell and to the ratio of the wild type. The significance of the enrichment was calculated by performing an unpaired Student’s t-test (type 2) with the Microsoft Excel software.