Computational protocol: Phosphorylation by Cdk1 Increases the Binding of Eg5 to Microtubules In Vitro and in Xenopus Egg Extract Spindles

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Protocol publication

[…] Alexa-568 (Invitrogen) labeled microtubules were generated by polymerizing 10 mg/ml purified tubulin with 2 mg/ml Alexa-568-labeled tubulin and 1 mM GTP in BRB80 (80 mM PIPES pH 6.8, 1 mM MgCl2, 1 mM EGTA). After a tenfold dilution in BRB80 containing 20 µM paclitaxel (Sigma), microtubules were pelleted for 5 min in a tabletop centrifuge at 14,000 rpm (Eppendorf 5417C). The microtubule pellet was resuspended in BRB80/paclitaxel.A flow chamber with a volume of about 5 µl was built by assembling two glass coverslips on top of each other using double sticky tape (Tesa). The flow chamber was first washed with assay buffer (72 mM Pipes, 0.9 mM MgCl2, 0.9 mM EGTA, 50 mM KCl, 20 µM paclitaxel, 14 mM 2-mercaptoethanol, 1% Glucose, 2 mM Mg-ATP), followed by 300 nM motor in kinase assay buffer (see above). The motor was incubated 10 min on ice and 2 min at room temperature, and then the flow chamber was washed with 4 flow chamber volumes of assay buffer. Finally, motility buffer (assay buffer supplemented with 0.1 mg/ml β-casein, 0.4 mg/ml glucose oxidase (Serva), 0.2 mg/ml catalase (Sigma) and microtubules diluted 1∶2000) was flowed in.For imaging, an inverted microscope (as described above) was used. Usually sequences of 2.5 min time lapse were recorded with 1 frame taken every 5 s and 500 ms exposure time per frame. The experiment was performed at 21°C. 4 time-lapse movies per condition were analyzed with the Kymograph plugin of ImageJ . For statistical analysis a t-test was performed. [...] To quantify the amount of Eg5 on spindle microtubules, fluorescence images of fixed spindles formed in the presence of Eg5-GFP fusions were taken as described above. Using ImageJ, the mean intensity per area was measured in a region of interest drawn around randomly selected structures. After subtraction of background measured in areas not containing microtubules, the GFP and TAMRA intensities of at least 10 structures were averaged (except for the addition of 46 nM Eg5-GFP for which 6 structures were analyzed). From the averaged GFP intensities an averaged background signal of spindles in mock depleted extract not containing Eg5-GFP was then subtracted. Typically, 2 or 3 different conditions corresponding to different concentrations of added Eg5-GFP were analyzed per extract, and in total 6 different extracts were used. As the measured TAMRA fluorescence intensity of spindles varied from extract to extract, we used the total average of TAMRA intensities from spindle structures in all extracts divided by the average TAMRA intensity of spindle structures per extract to correct the measured TAMRA values of each spindle in order to be able to compare experiments performed in different extracts. Finally, the ratios of GFP intensities to corrected TAMRA intensities were calculated and then normalized so that the ratio was set to 1 at the Eg5-GFP concentration corresponding to 460 nM, which is roughly the endogenous concentration . Fits to the data of were performed using gnuplot. […]

Pipeline specifications

Software tools ImageJ, Gnuplot
Applications Miscellaneous, Microscopic phenotype analysis
Organisms Xenopus laevis