Computational protocol: Mitotic Internalization of Planar Cell Polarity Proteins Preserves Tissue Polarity

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Protocol publication

[…] Images were acquired with a Zeiss LSM510 laser-scanning microscope (Carl Zeiss MicroImaging) and a 100× oil objective (N.A. 1.4). For whole mount imaging Z-stacks of 5–15 planes (0.5µm) were captured. Representative single Z-planes are presented. Images were recorded at 1024×1024 square pixels representing a 92µm × 92µm area. E17.5 whole mount hair follicle images were collected through a 10× (N.A. 0.8) objective and Z-stacks of 10–15 planes (2.5µm) were captured. Z-stacks were projected using ImageJ software. Images of cultured keratinocytes were obtained with an Axioplan microscope (Carl Zeiss MicroImaging) equipped with an Orca-ER camera (Hamamatsu) using Metamorph software. RGB images were assembled in Adobe Photoshop CS2 v. 9.0.2 and panels were labelled in Adobe Illustrator CS2 v. 12.0.1.Quantification of co-localization between Celsr1 and E-Cadherin or trafficking markers in mitotic cells was performed in ImageJ using JACoP. M1 coefficients representing the percentage of Celsr1 co-localization per cell were calculated and plotted using Prism5 Software (GraphPad).Quantifications of Celsr1 distribution during cytokinesis were performed as follows. Dividing cells were selected from sum-of-slices projection images of wild-type or Vangl2Lp/Lp epidermis, manually segmented in ImageJ on the basis of E-cadherin fluorescence, and imported into MATLAB. For each cell, Celsr1 fluorescence levels were normalized, and coordinates of the centroid and perimeter were computed. The angles of all pixels relative to the cell centre within 20 pixels (1.8um) of the periphery and above a 20% intensity threshold, determined empirically to include the majority of Celsr1-containing vesicles, were plotted in a rose diagram.To quantify Celsr1 polarity in interphase cells, the mean intensity of Celsr1 along individual basal cells borders (manually identified by E-Cadherin labelling) was calculated using ImageJ software. The intensities were plotted against the angle of the cell borders relative to the A-P axis. […]

Pipeline specifications

Software tools ImageJ, MetaMorph, Adobe Illustrator, JACoP
Applications Miscellaneous, Laser scanning microscopy, Microscopic phenotype analysis
Organisms Mus musculus, Drosophila melanogaster, Homo sapiens, Caenorhabditis elegans