Computational protocol: Phosphoregulation of the budding yeast EB1 homologue Bim1p by Aurora/Ipl1p

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Protocol publication

[…] Binding assays were performed essentially as previously described (). Precleared Bim1WT, Bim1WT phosphorylated preparatively by Ipl1, and Bim16D (at 1 µM) were incubated with increasing concentrations of taxol-stabilized MTs (0–10 µM) in 25 mM Hepes, pH 7.5, 150 mM KCl, 4 mM MgCl2, 1 mM EGTA, and 10% glycerol. Bim1 bound to MTs was separated from unbound fraction by ultracentrifugation. The amount of Bim1p in supernatant and pellet was quantified by densitometry of Coomassie-stained SDS-PAGE gels using ImageJ software (National Institutes of Health). To determine apparent Kd of Bim1 for MTs, data points from three independent experiments were fitted into the equation Y = 0.5 × {(Kd + Bmax + X) − [(Kd + Bmax + X)2 − (4BmaxX)]1/2} using Prism 4.0 (GraphPad Software, Inc.), where Y is the concentration of Bim1 partitioning to the pellet with the MTs, Bmax is the maximal fractional Bim1–MTs complex, Kd is the dissociation constant, and X is the concentration of polymeric tubulin.In the fluorescent-based MT-binding assay, 1 µM of purified Bim1 constructs was subjected to MT binding as described in the previous paragraph and centrifuged. The amount of Bim1 bound to MTs was measured fluorometrically using a plate reader (Synergy 2; BioTek), and averaged data from three individual experiments were similarly plotted as binding affinity curves. [...] To quantify spindle elongation kinetics, cells were arrested with α factor, released into synthetic medium, and imaged on concanavalin A–coated culture dishes (Matek) at 30°C. z stacks (eight stacks 0.4 µm apart) were acquired at 1-min intervals on a microscope (Axiovert 200M; Carl Zeiss, Inc.) equipped with a 100× α Plan-Apochromat 1.46 NA objective and a CoolSNAP HQ camera (Photometrics) controlled by MetaMorph software (MDS Analytical Technologies). Spindle length measurements were performed using ImageJ. Strains expressing Bim1–3× GFP and mCherry-Tub1 were visualized by live cell Deltavision deconvolution microscopy (Applied Precision, LLC) on a microscope (IX-71; Olympus) controlled by SoftWoRx (Applied Precision, LLC) and equipped with a 100× Plan-Apochromat 1.4 NA objective (Olympus) and a CoolSNAP HQ camera at 25°C. z stacks (eight stacks 0.35 µm apart) were acquired at 15-s intervals, deconvoluted, and projected into 2D images. […]

Pipeline specifications

Software tools ImageJ, MetaMorph
Applications SPIM, Microscopic phenotype analysis
Organisms Saccharomyces cerevisiae
Diseases Graft vs Host Disease