Computational protocol: Pheromone-induced morphogenesis and gradient tracking are dependent on the MAPK Fus3 binding to Gα

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Protocol publication

[…] Strains C699-5 (FUS3), C699-192 (fus3A180F182), and C699-200 (fus3Δ) were grown in synthetic complete medium. When cultures reached a cell density of ∼1 × 107 cells/ml, a 10-ml aliquot was removed to provide an uninduced (t = 0) reference sample. α-Factor was added to a final concentration of 100 nM in the remaining portion of the cultures, and 10-ml aliquots of cells were removed after 30 and 90 min of incubation. Cell lysis, extract preparation, and trichloroacetic acid precipitation were done according to the procedures described by ). The precipitated protein was suspended in buffer (0. 1 M Tris-HCl, pH 11.0, 3% SDS), boiled for 5 min, and centrifuged for 10 min to remove insoluble aggregates. A 40-μl amount of each sample was mixed with 8 μl of 6× SDS–PAGE loading buffer and boiled for 2 min before being loaded onto 10% SDS–PAGE gels for protein fractionation. Proteins were transferred from the gels to polyvinylidene fluoride membranes for detection of dual-phosphorylated Fus3 and Kss1 by using anti–phospho-p44/42 MAPK rabbit polyclonal antibodies (1:500; Cell Signaling Technologies, Danvers, MA) as primary antibody with horseradish peroxidase (HRP)–conjugated goat anti-rabbit immunoglobulin G (IgG; 1:10,000; Jackson ImmunoResearch, West Grove, PA) as secondary antibody. Antibodies were washed from the membranes by treatment with stripping buffer (Tris-HCl, pH 6.0, SDS, β-mercaptoethanol) and reprobed for glucose-6-phosphate dehydrogenase (G6PDH) as the internal loading control by using rabbit monoclonal anti-G6PDH (1:20,000; Sigma-Aldrich, St. Louis, MO) primary antibody with HRP-conjugated goat anti-rabbit IgG (1:10,000; Jackson ImmunoResearch) as the secondary antibody. Immunoreactive species were detected using ECLplus (GE Healthcare, Amersham, Pittsburgh, PA) and exposing the developed blots to x-ray film. Bands on the scanned x-ray films were quantified using ImageJ software (National Institutes of Health, Bethesda, MD). [...] These analyses were done in 5–50 nM pheromone gradients using a microfluidic device and methods for cell culturing, cell loading, and gradient generation as described in the supplement to ). Differential interference contrast (DIC) images of cells from four to six different fields that span the entire gradient were captured at 5-min intervals for a 5-h time course.Microscopy was performed with a Nikon Ti-E inverted microscope using a Plan Fluor ELWD 40×/0.6 Nikon objective and a Photometrics CoolSNAP HQ2 Monochrome camera. Acquisition was performed with MetaMorph software (Molecular Devices, Sunnyvale, CA). Image processing and cell scoring were aided by use of ImageJ software.The morphological categories scored after 5 h of growth in the pheromone gradient are illustrated by micrographs in . Unbudded cells (whether vegetative, shmoo, or hyperelongated) in each of the fields were also scored to assess the percentage of G1 cells in the population. The distinction between mothers with large buds (G2/M) versus unbudded mother–daughter pairs (G1) was aided by time-lapse imaging. Cells were scored as G1 if a bud emerged from either within the next two 5-min intervals.Gradient tracking was quantified in cells 4.5 h after growth in the pheromone gradient. The angle between the cell center and the furthest point of the leading edge was measured using the straight-line tool in FIJI (fiji.sc/Fiji; ). The measured angles were then normalized in relation to the direction of the gradient and the chamber orientation. A histogram of the angles was generated using Matlab (MathWorks, Natick, MA). The histogram was normalized to represent the percentage of cells in each bin and plotted as a figure using the Polar function in Matlab. […]

Pipeline specifications

Software tools ImageJ, MetaMorph
Application Microscopic phenotype analysis
Organisms Saccharomyces cerevisiae