Colocalization analysis

For colocalization analysis, we first defined a mask of the signal in either channel using the ImageJ tool Find Maxima and generated a mask using the output Maxima Within Tolerance that best described the signal. Colocalization within the regions specified by the independent masks of each channel was quantified using the Coloc 2 plug-in for Fiji for Figure 1. This program automates thresholding and reports statistically significant thresholded colocalization coefficients tM₁ and tM₂ as defined in Costes et al. (2004 ). We used the confined displacement algorithm (Ramirez et al., 2010) implemented by the Genome Damage and Stability Centre (GDSC) plug-in for ImageJ by Alex Herbert (University of Sussex, Brighton, United Kingdom) for Figure 2, which calculates colocalization coefficients tM₁ and tM₂ for both channels following a pixel randomization assessment. These operations were applied to deconvolved images of confocal z-stacks obtained using Nikon A1 confocal microscope running Nikon Elements software.