Computational protocol: Yeast Silent Mating Type Loci Form Heterochromatic Clusters through Silencer Protein-Dependent Long-Range Interactions

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Protocol publication

[…] For live and intact cell imaging, cultures carrying two color tagged integration sites were grown exponentially in YPD to OD600 nm< = 0.4 (∼1×107 cells/ml), and rinsed in complete synthetic medium before imaging. Microscopy was performed at 25°C. Cells were spread on synthetic complete 3% agarose patches for acquisition.3D images were captured on a Metamorph driven Olympus IX81 wide-field microscope equipped with a Coolsnap HQ camera and a Polychrome V (Till Photonics). Stacks of 21 images were acquired with a step size of 0.2 µm either at 490 nm for 200 ms for GFP or alternating the wavelength between 435 nm (CFP) and 512 nm (YFP) at every image plane, and exposures of 400 ms per wavelength. A 100×/1.4 Oil Plan-Apochromat objective (Olympus) was used. Position of GFP tagged loci relative to the nuclear rim identified by the Nup49-GFP signal were determined as a percentage of fluorescent spots in one of three concentric nuclear zones of equal surface in the plane bearing the brightest GFP-lacI or tetR-GFP focus using the Pointpicker plug-in for Image J ,. Only the 10 core focal planes were scored. For 3D distance determination, CFP and YFP images were automatically analyzed using SpotDistance implemented as a plug-in for ImageJ, freely available at: http://bigwww.epfl.ch/spotdistance/ . The measured distances were loaded into R software (www.r-project.org) and the measured distance distributions translated into a box plot. Outliers are defined as 1.5 times the Inter Quartile Range (IQR) and are represented as open circles. Different distance distributions (determined as non-normal using a Kolmogorov-Smirnov test) were scored for significant difference in R using the Wilcox test.2D time-lapse imaging () was performed on a Zeiss LSM510 confocal microscope using a 100× Plan-Apochromat objective (NA = 1.4) partly at the RIO Imaging facility in Toulouse, France, and partly in Susan Gasser's laboratory at the University of Geneva, Switzerland. Live imaging was performed as described . Nine independent 2D time-lapse series of 50 to 150 confocal images were acquired at 1.5 s intervals of G1-phase nuclei, following the tagged foci by adjusting the focal plane. 2D distances were measured manually using Metamorph. […]

Pipeline specifications

Software tools MetaMorph, ImageJ
Applications Laser scanning microscopy, Microscopic phenotype analysis
Organisms Saccharomyces cerevisiae