Article Figures & Data
Figures
- Figure 1. Gene-edited NCI-H460 cells expressing βIII-tubulin modified at the C-terminal tail region.
(A) Schematic outlining the gene editing approach to replace the endogenous βIII-tubulin protein with either the full-length βIII-tubulin protein with GFP tag (ZB3), the βIII-tubulin protein truncated at the C-terminal tail with GFP tag (ZB3Δ), or the βIII-tubulin body with a βI-tubulin C-terminal tail sequence with GFP tag (ZB3/CB1); ZFN1 and ZFN2: plasmids encoding zinc-finger nucleases; pDNR: donor cassette plasmid encoding the insertion sequence. (B) Representative Western blot of the gene-edited clones, which have knocked out expression of the endogenous βIII-tubulin protein (55 kD) and expression of the higher molecular weight GFP-tagged modified βIII-tubulin proteins (∼82 kD). NCI-H460 parental cells, which endogenously express βIII-tubulin, are presented as a control. These Western blots are replicated in Fig S1C to measure the isotype composition of the gene-edited clones.
- Figure S1. Modification of the β-tubulin C-terminal tail does not affect the tubulin isotype composition.
(A) Expression of the modified β-tubulin proteins in gene-edited cells expressing the full-length βIII-tubulin protein (ZB3), truncated βIII-tubulin protein (ZB3Δ), and βIII-tubulin body with βI-tubulin tail (ZB3/CB1) compared with the parental cell line using antibodies against GFP (upper blot) and S55 of βIII-tubulin (middle blot) with GAPDH as a loading control. Representative Western blot of two independent experiments. (B) Quantitation of the Western blot in (A) for two independent experiments. Graph gives the mean ± SEM. (C) Western blot of the β-tubulin isotype expression in gene-edited NCI-H460 cells. The protein expression of the βI-, βII-, βIII-, and βIV-tubulin isotypes together with the expression of the modified tubulin proteins (GFP) and total tubulin levels for each clone are shown. The GFP, βIII-tubulin and GAPDH Western blots are replicated in Fig 1B.
- Figure 2. Modification of the βIII-tubulin C-terminal tail does not affect the incorporation of the protein into the microtubule network, the microtubule architecture, or the cellular proliferation rate.
(A) Representative immunofluorescence and live-cell images of the microtubule architecture in gene-edited NCI-H460 cell clones expressing the full-length βIII-tubulin protein (ZB3), truncated βIII-tubulin protein (ZB3Δ), or βIII-tubulin body with the βI-tubulin tail (ZB3/CB1) compared with the NCI-H460 parental cell line (H460). For interphase microtubule architecture, immunofluorescence staining was performed for α-tubulin detection. Individual channels are presented as grayscale images and the merged image with each channel colored accordingly. Left panel: modified βIII-tubulin proteins; center panel: α-tubulin; right panel: merged image of modified βIII-tubulin proteins (green) and α-tubulin (red). Scale bar 25 μm; and far right panel: higher magnification images of the mitotic spindle in live cells (GFP imaging). Scale bar 10 μm. Representative of three independent experiments. (B) Proliferation rates of gene-edited clones as measured by the BrdU assay and normalized to cell number. Mean ± SEM of four independent experiments, no significant difference.
- Figure S2. Modification of the βIII-tubulin C-terminal tail does not affect tubulin polymerization or the cellular proliferation rate.
Representative Western blot (A) and densitometric quantitation (B and C) of the level of polymerized total tubulin (α-tubulin) (B) and polymerized modified βIII-tubulin proteins (GFP) (C) in gene-edited NCI-H460 cells expressing either the full-length βIII-tubulin protein (ZB3) or truncated βIII-tubulin protein (ZB3Δ) compared with the parental cell line (H460), which endogenously expresses βIII-tubulin. Mean ± SEM of four independent experiments, no significant difference. S, soluble fraction; P, polymerized fraction. (D) Cell number for NCI-H460 cells expressing the full-length βIII-tubulin proteins (ZB31 and ZB34), truncated βIII-tubulin protein (ZB3Δ1 and ZB3Δ2), or the βIII-tubulin body with βI-tubulin C-terminal tail (ZB3/CB12 and ZB3/CB13) measured periodically over 96 h by trypan blue dye exclusion and cell counting.
- Figure S3. Modification of the β-tubulin C-terminal tail does not alter tubulin partitioning and the C-terminal tag does not affect microtubule dynamics.
(A) Representative Western blot (i) and densitometric quantitation (ii) of the level of polymerized tubulin in normal growth conditions in gene-edited NCI-H460 cells expressing either the full-length βIII-tubulin protein (ZB3), truncated βIII-tubulin (ZB3Δ), or βIII-tubulin body with the βI-tubulin C-terminal tail (ZB3/CB1). Mean ± SEM of four independent experiments, no significant difference. (B) Microtubule assembly parameters in gene-edited NCI-H460 cells expressing the full-length βIII-tubulin protein (ZB3) compared with the parental cell line (H460) as measured by EB3-mCherry motion and particle tracking. The microtubule assembly rate (i), microtubule growth length (ii), microtubule growth duration (iii), and the number of microtubule growth events (iv) are presented as the mean ± SEM of at least 50 cells in each of three independent experiments for each tubulin modification. No statistically significant difference.
- Figure 3. The β-tubulin C-terminal tail modulates microtubule assembly in an isotype-dependent manner.
(A) Representative images of EB3-transfected gene-edited cells (i). The image processing involved in measuring the assembly parameters: image appearance following the processing of raw images (ii), identification of microtubule plus ends by the particle tracking analysis (iii), and microtubule assembly events colored according to assembly rate (iv). Scale bar 5 μm. (B) Microtubule assembly parameters in gene-edited NCI-H460 cells expressing the full-length βIII-tubulin protein (ZB3), truncated βIII-tubulin protein (ZB3Δ), and βIII-tubulin body with βI-tubulin tail (ZB3/CB1) as measured by EB3-mCherry motion and particle tracking. The microtubule assembly rate (i), microtubule growth length (ii), microtubule growth duration (iii), and the number of microtubule growth events (iv) were calculated as the average value per cell and are presented as the per-cell mean ± SEM of at least 50 cells in each of three independent experiments for each tubulin modification. *P < 0.05, ***P < 0.001, and ****P < 0.0001 relative to cells expressing the full-length protein (ZB3).
- Figure S4. The β-tubulin C-terminal tail affects microtubule assembly measured by spatiotemporal image correlation spectroscopy analysis.
(A) Schematic outline of spatiotemporal image correlation analysis showing time-lapse images are acquired of cells transiently transfected with EB3 (i); time-lapse images are subdivided into overlapping voxels (ii); the autocorrelation function of GFP, autocorrelation function of EB3, or the cross-correlation function of EB3 with GFP is calculated and the velocities extracted per voxel (iii); and voxels are shifted in space and time to produce a vector map indicating directional GFP and EB3 movement (iv). (B) Measures of microtubule motion by spatiotemporal image correlation spectroscopy. (i) Speed of directional microtubule motion as measured by the autocorrelation function for tubulin-GFP time-lapse images of gene-edited cells expressing modified βIII-tubulin proteins. (ii) Speed of microtubule assembly as measured by the autocorrelation function for EB3-mCherry time-lapse images of gene-edited cells expressing modified βIII-tubulin proteins. (iii) Number of microtubule growth events as measured by the autocorrelation function for EB3-mCherry time-lapse images of gene-edited cells expressing modified βIII-tubulin proteins. Graphs give the median, box gives the 25th to the 75th percentile, and whiskers give the minimum and maximum values of at least 15 cells from 2 independent experiments. **P < 0.05 and **P < 0.01 relative to cells expressing the full-length βIII-tubulin protein.
- Figure 4. The β-tubulin C-terminal tail modulates the coordination of microtubule assembly in an isotype-dependent manner.
(A) Representative images and STICCS analysis of gene-edited NCI-H460 cells expressing the full-length βIII-tubulin protein (ZB3), truncated βIII-tubulin protein (ZB3Δ), and βIII-tubulin body with βI-tubulin tail (ZB3/CB1). (i) Raw images of tubulin-GFP (left) and EB3-RFP (center) and merged image (right) at a single time point. (ii) STICCS vector maps showing the velocity of the movement in a single time block. The direction of the arrows indicates the direction of movement of the tubulin-GFP (left), EB3-RFP (center), and cross-correlation of GFP and EB3 (right); and the color indicates the speed of movement. Scale bar 5 μm. (B) Representative close-up images of the regions of the ZB3 cells indicated by the white boxes for microtubule and EB3 movement between the time t0 and Δt for the GFP (left panel), EB3 (center panel), and merged (right panel) channel showing microtubule assembly that do (i) and do not (ii) correlate with tubulin movement, together with the STICCS vector maps corresponding to these ROI and TOI regions (lower panel) for the GFP autocorrelation (left panel), EB3 autocorrelation (center panel), and cross-correlation between GFP and EB3 (right panel). Blue arrows indicate microtubule assembly events that are (i) and are not (ii) correlated. Microtubule assembly along existing microtubule fibers (ii) is shown as noncorrelating microtubule assembly. Conversely, explorative microtubule growth where the GFP and EB3 signals proceed at the similar rate into new territory (ii) is measured as a correlated STICCS event (i). Scale bar 2 μm. (C) Microtubule assembly dynamics as measured by STICCS, showing the speed of cross-correlated movement between the microtubule and EB3 channels (i) and the proportion of microtubule assembly events that are cross-correlated with the movement of microtubules (ii). Graphs give the median, box gives the 25th to the 75th percentile, and whiskers give the minimum and maximum values of at least 10 cells from two independent experiments. *P < 0.05, **P < 0.01, ***P < 0.001, and ****P < 0.0001 relative to cells expressing the full-length βIII-tubulin protein. Corresponding values are presented in Table S1.
- Figure S5. Modification of the β-tubulin C-terminal tail does not affect the partitioning of tubulin between the soluble and polymerized fractions in response to paclitaxel treatment.
Representative Western blot (A) and densitometric quantitation (B) of the level of polymerized total tubulin (measured by α-tubulin levels) and polymerized modified βIII-tubulin (measured by GFP levels) in gene-edited NCI-H460 cells expressing either the full-length βIII-tubulin protein (ZB3), truncated βIII-tubulin (ZB3Δ), or βIII-tubulin body with the βI-tubulin C-terminal tail (ZB3/CB1) and treated with paclitaxel (6 or 20 nM for 4 h). Mean ± SEM of four independent experiments, no significant difference.
- Figure 5. The β-tubulin C-terminal tail modulates the sensitivity of microtubules to paclitaxel.
(A) Microtubule assembly parameters in gene-edited NCI-H460 cells expressing either the full-length βIII-tubulin protein (ZB3), truncated βIII-tubulin protein (ZB3Δ), or βIII-tubulin body with βI-tubulin C-terminal tail (ZB3/CB1) cultured in normal growth media as measured by EB3-mCherry motion and particle tracking in response to paclitaxel treatment (TXL, 6 or 20 nM for 2 h). The microtubule assembly rate (i), microtubule growth length (ii), microtubule growth duration (iii), and number of microtubule growth events (iv) are presented as the mean ± SEM of at least 50 cells in each of three independent experiments for each tubulin modification. *P < 0.05, ***P < 0.001, and ****P < 0.0001 relative to cells expressing the full-length protein (ZB3); #P < 0.05 relative to untreated cells expressing the same type of tubulin modification. (B) Microtubule assembly dynamics as measured by spatiotemporal image correlation spectroscopy for cells treated with paclitaxel (20 nM for 1 or 2 h), showing the speed of cross-correlated movement between the microtubule and EB3 channels (i) and the proportion of microtubule assembly events that are cross-correlated with the microtubule movement (ii). Graphs give the median, box gives the 25th to the 75th percentile, and whiskers give the minimum and maximum values of at least 15 cells from two independent experiments. *P < 0.05,**P < 0.01, ***P < 0.001, and ****P < 0.0001 relative to cells expressing the full-length βIII-tubulin protein; #P < 0.05 relative to untreated cells expressing the same type of tubulin modification. Measurements of untreated cells are reproduced from Fig 3. Corresponding values are presented in Table S1.
- Figure S6. Modification of the β-tubulin C-terminal tail alters MCAK activity.
(A) MCAK activity in control shRNA (CtrlSH2) and βIII-tubulin-targeted shRNA-expressing (βIIISH4) NCI-H460 cells as measured by normalized α-tubulin fluorescence. Mean ± SEM of at least 100 cells of each type in each of three independent experiments. ****P < 0.0001 relative to control cells (CtrlSH2). (B) MCAK activity in CHO cells overexpressing the GFP protein (GFP), βI-tubulin-GFP protein (B1), βIII-tubulin-GFP protein (B3), βIII-tubulin lacking the C-terminal tail with GFP tag (B3Δ), or the βIII-tubulin body with βI-tubulin C-terminal tail with GFP tag (B3/CB1). Mean ± SEM of normalized α-tubulin fluorescence of at least 100 cells expressing each type of modified tubulin. *P < 0.05 relative to cells expressing the full-length βIII-tubulin protein (B3). #P < 0.05 relative to the full-length βI-tubulin protein (B1).
- Figure 6. The βI-tubulin C-terminal tail confers resistance to MCAK-mediated microtubule depolymerization.
(A) MCAK activity in two sets of expression-matched gene-edited NCI-H460 cells as measured by normalized α-tubulin fluorescence. Mean ± SEM of at least 100 cells of each type in each of three independent experiments. Solid and striped bars indicate different sets of expression-matched gene-edited clones. *P < 0.05, ***P < 0.001, and ****P < 0.0001 relative to cells expressing the full-length βIII-tubulin protein.
- Figure S7. Modification of the β-tubulin C-terminal tail does not affect the partitioning of tubulin between the soluble and polymerized fractions in response to nocodazole treatment or tubulin posttranslational modifications.
(A) Representative Western blot (i) and densitometric quantitation (ii) of the level of polymerized total tubulin (measured by α-tubulin levels) and polymerized modified βIII-tubulin (measured by GFP levels) in gene-edited NCI-H460 cells expressing modified βIII-tubulin proteins and treated with nocodazole for 4 h. Mean ± SEM of four independent experiments, no significant difference. (B) Western blot of acetylated (AcTub) and tyrosinated (TyrTub) tubulin levels in gene-edited NCI-H460 cells expressing modified β-tubulin proteins. Representative of three independent experiments.
- Figure 7. The β-tubulin C-terminal tail region spatiotemporally regulates microtubule dynamics in an isotype-dependent manner.
Schematic of a model summarizing findings that the β-tubulin C-terminal tails regulate microtubule growth and assembly. Compared with the ubiquitous βI-tubulin C-terminal tail, the βIII-tubulin C-terminal tail increases the propensity of microtubules to grow and depolymerize in normal growth conditions and under the influence of MCAK or microtubule-stabilizing agents. The C-terminal tail region also modulates the collective dynamics of microtubules such that the β-tubulin isotype C-terminal tails confer unique dynamic properties to microtubules. E, Energy; TXL, paclitaxel; ↑ and ↓, increased or decreased levels, respectively, for the indicated parameters.
Supplementary Materials
