Computational protocol: Expression, stability, and replacement of glucan-remodeling enzymes during developmental transitions in Saccharomyces cerevisiae

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[…] Cells were observed by phase-contrast microscopy, and sporulation was scored by counting at least 200 cells after a mild sonication. For Gas1p-GFP visualization, 1 ml sporulating culture was centrifuged at 8000 rpm for 2 min at 4°C, washed twice with cold PBS, and incubated for 15 min on ice. If required, 8.3 μg/ml 4,6-diamidine-phenylindole (DAPI) was added to the cells. Samples were then incubated for 15 min at room temperature in the dark before microscopy observation. Cells were observed as wet mounts using an Eclipse 90i (Nikon, Tokyo, Japan) or a DMRXA (Leica, Wetzlar, Germany) microscope equipped with epifluorescence, Nomarski optics, and a Hamamatsu ORCA-ER camera (Nuhsbaum, McHenry, IL). The setup, including the microscope and camera, was controlled by MetaMorph software (Molecular Devices, Sunnyvale, CA). Alternatively, cells were examined with an Olympus BX60 microscope (Olympus Optical, Tokyo, Japan) connected to a DC290 Kodak digital camera. The images were analyzed using ImageJ-BMF software (McMaster Biophotonics Facility, Hamilton, ON, Canada) and Adobe Photoshop (San Jose, CA). The observation of the natural fluorescence of dityrosine was performed as described previously (Briza et al., ).For indirect immunofluorescence, sporulating cells (20 OD450) were fixed in 3.7% formaldehyde–0.1 M KPO4 buffer for 30 min. After a 3-min centrifugation at 1500 rpm, the cells were resuspended in the same volume of fixing solution (0.1 M KPO4, 3.7% formaldehyde) for 2–4 h. Fixed cells were washed and resuspended in SHA buffer (1 M sorbitol, 0.1 M HEPES-KOH, pH 7.5, and 5 mM NAN3) at a concentration of 108 cells/ml. A small aliquot of fixed cells (150 μl) was centrifuged and resuspended in SHA supplemented with 25 μg/ml Zymolyase 20T (ICN Biomedicals) and 0.2% β-mercaptoethanol. After a 30-min incubation at 37ºC, removal of the ascus sac was checked before proceeding. Spheroplasts were then permeabilized by incubating in SHA and 0.1% Triton X-100 for 5 min. After attaching to glass slides, cells were plunged into −20°C methanol for 6 min, followed by −20°C acetone for 30 s. After a blocking step in PBS + block (1% milk, 0.5% bovine serum albumin [BSA] in PBS) for 10 min, slides were incubated in primary antibody diluted in PBS + block for 2 h. Primary antibodies were anti-Gas1p rabbit serum (diluted 1:500), anti-HA monoclonal antibody (mAb) (diluted 1:1000; Covance, Berkeley, CA), and anti-Gas4p serum. Rabbit immunoglobulin (Ig) G was purified from anti-Gas4p serum using a protein A microspin column and diluted 1:50. Slides were washed 12 times with PBS + 0.5 mg/ml BSA and then incubated in Alexa Fluor 594 goat anti–mouse IgG (1:1000 dilution) or Alexa Fluor 488 goat anti–rabbit IgG (1:1500 dilution) for 1 h. After 12 washes with PBS, or with PBS–0.5% BSA–0.1% Triton X-100 for Gas4p, a mounting media (Gel Mount; Biomeda Corporation, Foster City, CA) was added to the slides, which were kept at 4ºC for 1 h and observed under an epifluorescence microscope. […]

Pipeline specifications

Software tools MetaMorph, ImageJ
Applications Phase-contrast microscopy, Microscopic phenotype analysis
Organisms Saccharomyces cerevisiae
Chemicals Glycosylphosphatidylinositols