Fig 1: PPP1R35 localizes to the proximal end of the centriolar lumen above the cartwheel.(a,b) 3D structured illumination microscopy (3DSIM) micrographs of U2OS cells expressing GFP-PPP1R35 (treated with digitonin to remove the cytoplasmic PPP1R35 population) and co-stained with CEP152 (a) and various other markers (b) labeling the distal (POC5, POC1B, CETN1) and proximal (SAS6, CEP135, CPAP, CEP250) regions of the centriole. The cartoon depiction of the centriole is alongside the images to assist in orienting the 3DSIM micrographs. Scale bars, 250 nm. (c) By measuring the distances between the fluorescence maxima of PPP1R35 and various centriolar markers on the same z-plane, the distance between GFP-PPP1R35 and the corresponding marker was determined. The fluorescence intensity along the line drawn in the micrograph (green, GFP-PPP1R35; red, POC5; white, CETN1) is plotted as a function of the distance along the line. The grey lines are centered at the fluorescence maxima and the distance between these lines corresponds to the distance between GFP-PPP1R35 and the corresponding marker (POC5 in this example). (d) By combining the analysis of the micrographs with these measurements, the position of PPP1R35 was mapped on to the centriole and the distances are shown in a Tukey box and whiskers plot, with the whiskers representing datum within an interquartile range of 1.5 and the band in the box as the mean. (e) A cartoon depiction of the localization of PPP1R35 in the centriole relative to the markers depicted in (b) and (d) with the average distances and standard deviations noted.10.7554/eLife.37846.009Figure 2—source data 1.Source data for Figure 2d and e (PPP1R35 mapping measurements).