Effect of tyrosination sensor on microtubule function. (A) Observation of microtubule dynamics using tyrosination sensor (Tag-RFP-T_A1aY1). The green arrow indicates microtubule pause and the orange arrow shows dynamic instability behavior. Full movie of the frames can be found in Video 1. Scale bar = 5 µm and time in minutes:seconds as indicated. (B) Histogram of EB3 comet velocities, representing the microtubule plus-end growth rates (microns/second). The average growth velocities for EB3 comets are 0.36 ± 0.1 µm/s (n = 468), 0.35 ± 0.09 µm/s (n = 450), and 0.19 ± 0.06 µm/s (n = 414) for cells with the tyrosination sensor (red), without binder (blue), and with 0.5 µM SiR-tubulin (gray), respectively. (C) CLIP170 staining (green) of U2OS cells in the presence of A1aY1 binder (red) or VASH2-SVBP (gray) with DAPI (blue) as indicated. Scale bars = 10 µm; zoom panel scale bar = 5 µm. (D and E) Distribution of K560-SNAP and KIF1A-1-393-LZ-SNAP motor velocities and run lengths with (red) and without (blue) A1aY1 binder. Representative kymograph of K560-SNAP and KIF1a-LZ-SNAP motors with and without A1aY1 binder as indicated. Scale bar x axis = 10 µm and y axis = 10 s. The average velocity of K560-SNAP is 0.75± 0.16 µm, n = 220 (with A1aY1-GFP) and 0.77 ± 0.19 µm, n = 234 (without A1aY1) and KIF1A-1-393-LZ-SNAP are 2.25 ± 0.50 µm, n = 338 (with A1aY1-GFP) and 2.37 ± 0.62 µm, n = 330 (without A1aY1). The average run length of K560-SNAP is 1.8 µm (with A1aY1-GFP) or 1.5 µm (without A1aY1), and the average run length of KIF1A-1-393-LZ-SNAP is 10.1 µm (with A1aY1-GFP) or 9.8 µm (without A1aY1). n represents the number of motor particles analyzed.