Computational protocol: The actin-microtubule cross-linking activity of Drosophila Short stop is regulated by intramolecular inhibition

Similar protocols

Protocol publication

[…] The mean fraction of overlap between tubulin and EGFP-tagged Shot was calculated using Mander's coefficient (described in ) and the JACoP plug-in in ImageJ (National Institutes of Health [NIH], Bethesda, MD). Prior to analysis, images were thresholded. The Mander's coefficient varies from zero to one, with zero representing nonoverlapping images and one representing complete overlap/colocalization. All images used for analysis were acquired from live cells by TIRF microscopy using identical microscope settings.To exclude the possibility that overexpression artifacts affected the degree of colocalization between the EGFP-tagged Shot constructs and mCherry-tubulin, we compared the Mander's coefficient with the fluorescence intensity per cell area (Figure S4). The correlation between the Mander's coefficient and the fluorescence per cell area in cells expressing Shot A-EGFP, FKBP-Shot-FRB-EGFP (DMSO treated), and ShotGAS2-EGFP was statistically significant, indicating that the extent of colocalization increased with increasing expression levels of these constructs. In contrast, correlation between the Mander's coefficient and the fluorescence intensity per cell area was not statistically significant for ShotΔEFhand-EGFP, ShotΔGAS2-EGFP, and ShotΔRod-EGFP, indicating that microtubule localization of these constructs was independent of expression level and did not reflect an artifact of overexpression. […]

Pipeline specifications

Software tools JACoP, ImageJ
Application Microscopic phenotype analysis
Organisms Drosophila melanogaster