Computational protocol: Gβ Regulates Coupling between Actin Oscillators for Cell Polarity and Directional Migration

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[…] A spinning disc Nikon Eclipse Ti fitted with a spinning disc head, 405 nm, 488 nm, and a 561 nm laser line and appropriate emission filters were used to record CFP, RFP, and GFP (or YFP) double- or triple-labeled cells at room temperature. Images were routinely recorded using a 60x (1.45 NA) objective, a Clara Interline CCD camera (Andor Technologies), and NIS Elements software. After analysis, when necessary for presentation, contrast was adjusted uniformly using ImageJ or Photoshop, and to image sets of some experiments a uniform Gaussian Blur was applied. To quantify oscillations, a single two- or three-channel image was taken to assess Gβ sequestration, followed by a 2-min movie (1 frame/second) to record behavior in the reporter channel at the lowest laser intensity necessary for reasonable signal-to-noise. Longer imaging periods (10 min) and/or adjustment of the focal plane close to the coverslip were used when necessary (e.g., to record individual oscillating foci or alternating polar and apolar states). [...] The electric fields were applied as previously described for vegetative Dictyostelium cells [] by using μ-Slides (Ibidi). These tissue-culture-treated slides with small cross-sectional area provide high resistance to current flow and minimized Joule heating during experiments. To eliminate toxic products from the electrodes that might be harmful to cells, agar salt bridges made with 1% agar gel in Steinberg’s salt solution were used to connect silver/silver chloride electrodes in beakers of Steinberg’s salt solution to pools of excess developing buffer (5 mM Na2HPO4, 5 mM KH2PO4, 1 mM CaCl2, and 2 mM MgCl2, pH 6.5) [] at either side of the chamber slide. EF strength is empirically chosen (~10V/cm) based on our previous experience [] and measured by a voltmeter before and after each experiment. Fields of HO547 cells were chosen based on the presence of Gβ and anchor expressing cells, which were distinguished by fluorescence imaging (see Microscopy section for details). High-definition DIC movies (1 frame/30 s) were recorded at room temperature for at least 30 min after the electric field was switched on. To quantify directionality and speed, time-lapse images were imported into ImageJ (http://rsbweb.nih.gov/ij). Tracks were marked by using the MtrackJ tool and plotted by using the Chemotaxis tool described []. All experiments were repeated and produced similar results. Data are combined and presented as means +/- SEM (standard error). To compare group differences, unpaired, two-tailed Student’s t test was used. A p-value of less than 0.05 is considered significant. […]

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