Figure 1: Automated analysis for quantification of epithelia-specific parameters.
From: Defining functional interactions during biogenesis of epithelial junctions
(a) Cell morphology changes during cell−cell adhesion. Diagrams are shown as lateral view (left), top view (middle) or representation of homophilic binding of E-cadherin (right). Addition of calcium ions induces assembly of E-cadherin puncta at the interface between neighbouring cells. F-actin re-organization is shown as appearance of junctional actin (co-localizes with E-cadherin) and circumferential thin bundles that compact towards junctions. (b) Immunofluorescence image showing E-cadherin localization at newly formed junctions (30 min; green), junctional actin and adjacent thin bundles (red). (c–g) Validation of automated image analysis (Supplementary Fig. 1). Positive and negative controls are cells induced to form junctions with standard calcium medium (30 min std.Ca2+) or maintained without cell−cell contacts (0 min std.Ca2+), respectively. (c) Keratinocytes were treated to generate images with E-cadherin staining as punctate (5 min std.Ca2+), reduced (PMA treatment) or increased cytoplasmic staining (active Arf6 expression). (d) Quantification of E-cad parameter as % thresholded area. (e) To validate image segmentation of F-actin pools, keratinocytes were pre-treated with blebbistatin (Bleb.) or Y27632 and junctions induced for 30 min in the presence of the drugs. (f,g) Images were quantified to generate the parameter Jun-A (F-actin pool co-localizing with E-cadherin at junctions, (f) or Cyt-A (that is, F-actin pool outside junctions and nucleus, (g). Data from three independent replicates; error bars represent standard error of the means. Scale bars, 25 μm. Statistical analysis t-test paired two samples, assuming equal variance; *P<0.02; **P<0.003; ***P<0.0004.
