Figure S5.
Cell viability assays and microtubule dynamics measurements. (A) Trypan blue staining of the wild-type U2OS cells and stable U2OS cells expressing the blue sensor or red sensor. In all the cases, the cells were viable around 95%. (B) Flow cytometry–based viability test for red sensor–expressing U2OS cells stained with DAPI and blue sensor–expressing U2OS cells stained with PI. In both the cases the percentage of dead cells (DAPI-positive or PI-positive cells) doesn’t exceed >2%, which is comparable to the wild-type U2OS cells. (C) U2OS cells stably expressing red sensor undergoes mitosis with cells imaged for different stages of the cell cycle (metaphase, anaphase and telophase). Binder-bound microtubules are shown in magenta, and DNA marked with DAPI is shown in cyan. Scale bars = 5 µm. (D) Histogram representing the microtubule growth rates (in blue) and depolymerization rates (in gray) obtained from the manual tracking of individual filaments in stable U2OS cells expressing the red tyrosination sensor. A total of 115 filaments analyzed from 15 cells. The growth rate (mean ± SD) and the depolymerization rate (mean ± SD) of microtubules are 0.32 ± 0.09 µm/s and 0.92 ± 0.18 µm/s respectively, in the cells. (E) Representative kymograph for a microtubule undergoing growth (polymerization), dynamic instability and catastrophe (depolymerization) events. The red star marks the rescue events followed by microtubule growth. The scale bar on the x axis = 2 µm and y axis = 50 s. (F) Microtubule dynamics from three different filaments (MT-1, 2, and 3 shown in green, blue, and red) from the stable U2OS cells. Respective paused, catastrophe, rescue, and growth phases of MT-1 (shown in green) are marked. (G) Catastrophe event frequency distribution per micrometer length of microtubule undergoing continuous catastrophe per minute, for a total of 130 filaments analyzed from 15 cells. (H) Quantification of total number of EB3-GFP comets per micrometer area per minute with and without A1aY1 binder. (I) Representative snapshots of movie frames from stable U2OS cells with the red tyrosination sensor (in grayscale), transiently expressing EB3-GFP (in green). The magenta and blue arrows for reference of typical EB3 comets at two selected microtubule plus ends. Scale bars = 5 µm, and time format (minutes:seconds) as indicated in Fig. 6 A. (J) Comparison of time-stack projections from TIRF movies of EB3 movement for control (without A1aY1 binder), with A1aY1 binder, and 0.5 μM SiR-tubulin + 10 μM Verapamil. Scale bar = 5 μm.

Cell viability assays and microtubule dynamics measurements. (A) Trypan blue staining of the wild-type U2OS cells and stable U2OS cells expressing the blue sensor or red sensor. In all the cases, the cells were viable around 95%. (B) Flow cytometry–based viability test for red sensor–expressing U2OS cells stained with DAPI and blue sensor–expressing U2OS cells stained with PI. In both the cases the percentage of dead cells (DAPI-positive or PI-positive cells) doesn’t exceed >2%, which is comparable to the wild-type U2OS cells. (C) U2OS cells stably expressing red sensor undergoes mitosis with cells imaged for different stages of the cell cycle (metaphase, anaphase and telophase). Binder-bound microtubules are shown in magenta, and DNA marked with DAPI is shown in cyan. Scale bars = 5 µm. (D) Histogram representing the microtubule growth rates (in blue) and depolymerization rates (in gray) obtained from the manual tracking of individual filaments in stable U2OS cells expressing the red tyrosination sensor. A total of 115 filaments analyzed from 15 cells. The growth rate (mean ± SD) and the depolymerization rate (mean ± SD) of microtubules are 0.32 ± 0.09 µm/s and 0.92 ± 0.18 µm/s respectively, in the cells. (E) Representative kymograph for a microtubule undergoing growth (polymerization), dynamic instability and catastrophe (depolymerization) events. The red star marks the rescue events followed by microtubule growth. The scale bar on the x axis = 2 µm and y axis = 50 s. (F) Microtubule dynamics from three different filaments (MT-1, 2, and 3 shown in green, blue, and red) from the stable U2OS cells. Respective paused, catastrophe, rescue, and growth phases of MT-1 (shown in green) are marked. (G) Catastrophe event frequency distribution per micrometer length of microtubule undergoing continuous catastrophe per minute, for a total of 130 filaments analyzed from 15 cells. (H) Quantification of total number of EB3-GFP comets per micrometer area per minute with and without A1aY1 binder. (I) Representative snapshots of movie frames from stable U2OS cells with the red tyrosination sensor (in grayscale), transiently expressing EB3-GFP (in green). The magenta and blue arrows for reference of typical EB3 comets at two selected microtubule plus ends. Scale bars = 5 µm, and time format (minutes:seconds) as indicated in Fig. 6 A. (J) Comparison of time-stack projections from TIRF movies of EB3 movement for control (without A1aY1 binder), with A1aY1 binder, and 0.5 μM SiR-tubulin + 10 μM Verapamil. Scale bar = 5 μm.

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