Fop1 localizes asymmetrically at BBs. (A) Fop1 is enriched at the posterior side of BBs. Inset of Sas6a-labeled (green; Sas6a:GFP) and Fop1-labeled (red; Fop1:mCherry) BBs. Bar, 1 µm. (B) Schematic showing a representative, polarized BB. KF, kinetodesmal fiber; PC, postciliary microtubules. (C) Fop1 is enriched at the posterior side of BBs. (left) Averaged two-color, colocalization of Fop1 (red) relative to centroid localizing, Sas6a (green). (middle) Linescan from a Gaussian fit curve to the mean localization. (right) Schematic representation of Fop1’s BB localization. n = 45 BBs. Bar, 0.5 µm. (D, left) SIM image of Fop1’s enrichment at the posterior (triplet microtubules 8 and 9) and anterior (triplet microtubules 4 and 5) triplet microtubules with an enrichment for posterior microtubules. Averaged SIM image of Fop1-labeled (red; Fop1:mCherry) BBs. n = 328 BBs. Bar, 0.25 µm. (middle) Heatmap illustrating intensity differences. (right) Schematic representation of Fop1 localization. (E) Representative SIM images showing dynamic Fop1 localization at individual BBs. Bar, 0.25 µm. (F, left) Interpolated intensity map of averaged Fop1 SIM images. Linear (middle) and radial (left) linescans. Arrow a in left panel corresponds to the linear linescan. Arrow b corresponds to the radial linescan. Asterisks denote levels distinct from posterior facing peak (*, P < 0.01). (G) Fop1:GFP immuno-EM localization in BB cross-sectional view. (right) Schematic of red dots denote Fop1’s localization based on the relative distribution of 25 gold particles to represent the total quantified gold label. Numbers denote the region where Fop1 was measured. n = 125 gold particles in 21 BBs. Bar, 100 nm.