Microtubule glutamylation stabilizes BBs differentially from BB stability proteins. (A) SIM imaging reveals that glutamylation is enriched at the posterior and anterior triplet microtubules. (left) Averaged SIM image of BB glutamylation (GT335). n = 110 BBs. (middle) Heatmap representation. (right) Schematic localization of BB glutamylation. (B) Representative SIM images showing variable glutamylation in foci and horseshoe patterns at individual BBs. (C, left) Interpolated intensity heatmap of the mean SIM image reveals enrichment of glutamylation at the BB posterior and anterior faces. Quantification of a linear (middle, a.) and radial linescans (left, b.). (A–C) Bars, 0.25 µm. (D) The timing and rate of incorporation of BB glutamylation and Bld10 is similar to Fop1. Glutamylation levels depict the incorporation of glutamylation relative to BB separation. (right) Quantification of BB stability factor and microtubule glutamylation incorporation relative to the mother–daughter BB separation. Bar, 1 µm. (E) BB microtubule glutamylation is significantly reduced in ttll1,9Δ cells. BBs and cilia stained for glutamylated microtubules (red) and BBs for centrin (green). Bar, 0.5 µm. (F) Quantification of BB glutamylation levels in WT and ttll1,9Δ cells. n = 150 BBs. (G, top) Loss of BB microtubule glutamylation causes BB loss. Representative fluorescence images of BB rows in WT and ttll1,9Δ cells at 30°C and 37°C in cell cycle–arrested conditions. (bottom) BB frequency in WT and ttll1,9Δ cells. n = 300 rows. Bar, 1 µm. (H) Inhibition of ciliary beating prevents temperature-induced BB loss in ttll1,9Δ cells. Representative BB rows in WT and ttll1,9Δ cells in untreated or NiCl₂-treated media. n = 300 rows. (I) Loss of Poc1 or Fop1 increases BB glutamylation levels. Bar, 0.5 µm. n = 150 BBs. (J) Loss of both BB glutamylation and Poc1 increases BB disassembly. Quantification of BB frequency in WT and ttll1,9Δ, poc1Δ cells at 30°C and 37°C. n = 200 BBs. (A–J) Mean ± SEM; *, P < 0.01.