Specificity of A1aY1 binder toward tyrosinated microtubules. (A) Confocal image stacks of U2OS cells stably expressing TagRFP-T A1aY1, indicated as “Binder” (magenta) along with GFP control, vasohibin2-X1-GFP-2A-SVBP, TTLL5-EYFP, catalytically dead TTLL5-EYFP, TTLL4-EYFP, and TTLL7-EYFP, indicated as “Enzyme” (gray) along with SiR-tubulin, indicated as “Microtubules” (green). Scale bars = 10 µm. Line scans of SiR-tubulin (green) and TagRFP-T A1aY1 (magenta) fluorescence intensity signal for each panel as indicated by the yellow line. Cartoon representation of α/β-tubulin in the right panel indicates the PTM state of microtubules for the respective experiment. (B) Quantification of Pearson’s coefficient (R-value) to calculate the correlation for a fraction of microtubules detected by TagRFP-T A1aY1 versus SiR-tubulin label, represented as “% overlap of intensity SiR-tubulin versus TagRFP-T.” A typical diffused cytoplasmic signal will have ∼60% or less overlap, whereas a complete colocalization will show near 100% overlap. n = 25 cells; each dot represents data from one cell. (C) NMR structure of A1aY1 (gray surface representation) and α-tubulin CTT peptide (blue cartoon representation); the C-terminal, tyrosine, and glutamic acid residues are indicated. (D and E) Closer view of the terminal tyrosine (Y451) and polyglutamylation site glutamic acid (E445) with key interacting residues from A1aY1 binder as indicated. A.U., arbitrary units; Fl., fluorescence; MT, microtubule.