Computational protocol: Interphase adhesion geometry is transmitted to an internal regulator for spindle orientation via caveolin-1

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Protocol publication

[…] For detection of Gαi1 and caveolin-1, cells were fixed with 4% paraformaldehyde followed by incubation with methanol at −20 °C for 10 min or 0.2% Triton X-100 at room temperature for 10 min. For detection of active β1-integrin, cells were incubated with medium including an anti-active β1-integrin antibody at 4 °C for 5 min. Subsequently, the cells were fixed with 4% paraformaldehyde followed by incubation with 0.2% Triton X-100 at room temperature for 10 min. The cells were blocked with 5% bovine serum albumin at 37 °C for 30 min, incubated with primary antibodies at 4 °C overnight, washed, and then incubated for 1 h with secondary antibodies (Alexa Fluor 488- or 594 goat anti-mouse, anti-rabbit or anti-rat IgG (Molecular Probes)). Fluorescent labelling was visualized using an inverted wide-field fluorescence microscope (IX81-ZDC, Olympus, Japan). Three-dimensional images (0.2 μm intervals) were obtained using an Olympus oil immersion objective lens (UPLSAPO 60XO/NA1.35) and computationally processed by three-dimensional deconvolution using Meta Morph software. For , and , images were obtained using an inverted confocal fluorescence microscope (Leica Microsystems, TCS SP8 with Hybrid Detector) and LAS-X software. Images were analysed using Image J software. [...] Images of mitotic cells were obtained using the inverted wide-field fluorescence microscope and analysed for the spindle angle against a respective micropattern using Image J software. Spindle angles were calculated as the orthogonal oriented angle of aligned chromosomes. To quantify the cortical intensity of caveolin-1 or Gαi1, we first extracted the cellular edge region using a manual thresholding, and then the signal intensity for each angle from the centre within the edge region was was measured by using ImageJ ‘line' command. We normalized the intensity by dividing the raw intensity with the sum of the intensity of all angles. The cortical intensity-based prediction was conducted as explained in the ‘Theoretical analysis of spindle orientation' except the forces pulling a pole of the spindle at a given direction are proportional to the signal intensity of caveolin-1 or Gαi1. […]

Pipeline specifications

Software tools LAS X, ImageJ
Application Microscopic phenotype analysis