The structured core of human β tubulin confers isotype-specific polymerization properties

Abstract

Diversity in cytoskeleton organization and function may be achieved through variations in primary sequence of tubulin isotypes. Recently, isotype functional diversity has been linked to a "tubulin code" in which the C-terminal tail, a region of substantial sequence divergence between isotypes, specifies interactions with microtubule-associated proteins. However, it is not known whether residue changes in this region alter microtubule dynamic instability. Here, we examine recombinant tubulin with human β isotype IIB and characterize polymerization dynamics. Microtubules with βIIB have catastrophe frequencies approximately threefold lower than those with isotype βIII, a suppression similar to that achieved by regulatory proteins. Further, we generate chimeric β tubulins with native tail sequences swapped between isotypes. These chimeras have catastrophe frequencies similar to that of the corresponding full-length construct with the same core sequence. Together, our data indicate that residue changes within the conserved β tubulin core are largely responsible for the observed isotype-specific changes in dynamic instability parameters and tune tubulin's polymerization properties across a wide range.

Figure 1.

Purification of recombinant α/βIIB tubulin heterodimers. (A) Purification scheme. (B) SDS-PAGE analysis (1, lysate; 2, supernatant; 3 and 4, nickel affinity: flow-through (3); elution (4); 5, TEV-digested protein elution from nickel affinity column (a band corresponding to the added TEV protein is at ∼25 kD); 6–8, TOG affinity: flow-through (6); elution (7), 20× amount in lane 7 (8); Coomassie stain). (C) Western blot (WB) analyses. Full blots are provided in Fig. S1 A. (D) Elution profiles from size-exclusion chromatography. Peak volume: 14.4 ml (α/βIIB); 14.3 ml (bovine tubulin, used as reference). Void volume (V₀) is 7 ml. A.U., arbitrary units. (E and F) TIRF images of taxol-stabilized (E) or GMPCPP (F) microtubules. Bars, 3 µm. (G) SDS-PAGE analysis of tubulin sedimentation in the presence of allocolchicine (+Allo) or 3% DMSO control (−Allo). Pellet (P) and supernatant (S) fractions are indicated. (H) Representative equilibrium binding curve for α/βIIB with allocolchicine from one experiment is shown. K_d = 1.8 ± 0.42 µM (n = 3, mean ± SD). Fig. S1 C shows data averaged from all three experiments and fitted to a single curve.

Figure 2.

Single-filament TIRF analysis of α/βIIB tubulin polymerization properties. (A) TIRF assay schematic. Microtubule extensions (red) and GMPCPP seeds (green) are shown with plus ends (+) and minus ends (−) indicated. TIRF image overlays (B and D) and kymographs (C and E) of microtubule extensions (red) growing from seeds (green; total tubulin: 6 µM [B and C] and 13 µM [D and E]). (F and G) Plus-end polymerization rates (F) and catastrophe frequencies (G) for microtubules composed of α/βIIB at different total tubulin concentrations. The data were pooled from at least three independent experiments. Overlay images (H–J) and kymographs (K–M) of mixed α/βIIB and α/βIII microtubules (α/βIIB:α/βIII ratio, 3:1 [H and K], 1:1 [I and L], or 1:3 [J and M]; total tubulin, 10.5 µM). (N and O) Plus-end polymerization rate (N) and catastrophe frequency (O) for mixed microtubules (α/βIIB:α/βIII ratio, 1:1, total tubulin, 10.5 µM) with α/βIIB shown for reference. The data were pooled from at least two independent experiments. Bars: (horizontal) 3 µm; (vertical) 2 min. Error bars are SD. For catastrophe frequency (f_cat), SD were calculated as f_cat/$\sqrt{n}$ (assuming a Poisson distribution), where n is the number of catastrophe events. Table 1 summarizes these measurements.

Figure 3.

Design and characterization of chimeric β tubulin constructs. (A) Design of tail-swapped β tubulin constructs, with amino acid sequence derived from α/βIIB (black) and α/βIII (gray). (B) Schematic of tubulin heterodimer indicating β tubulin C-terminal tail (black). Amino acid sequences from the C terminus of βIIB and βIII are shown. (C) SDS-PAGE analysis (1, nickel affinity elution; 2, TOG affinity elution; Coomassie stain). (D) Western blot (WB) analysis. Full blots are provided in Fig. S2 A. (E) Protein elution profiles from size-exclusion chromatography. Peak volume: 14.2 ml (α/βIIB-tail-III); 14.1 ml (α/βIII-tail-IIB). Void volume (V₀) is 7 ml. A.U., arbitrary units. (F and G) TIRF images of taxol-stabilized (F) or GMPCPP (G) microtubules. Bars, 3 µm.

Figure 4.

Single-filament TIRF analysis of chimeric β tubulins. TIRF image overlays (A, C, E, and G) and kymographs (B, D, F, and H) showing microtubule extensions (red) growing from GMPCPP seeds (green) assembled with α/βIIB-tail-III (A–D) or α/βIII-tail-IIB (E–H). (I and J) Plus-end polymerization rates (I) and catastrophe frequencies (J) for chimeric α/βIIB-tail-III or α/βIII-tail-IIB microtubules at different free tubulin concentrations. Catastrophe frequency measurements for full-length α/βIIB (blue dashed line) and α/βIII (red dashed line) are shown for reference. The data were pooled from at least three independent experiments. Error bars are SD. For catastrophe frequency (f_cat), SD were calculated as f_cat/$\sqrt{n}$ (assuming a Poisson distribution), where n is the number of catastrophe events. Bars: (horizontal) 3 µm; (vertical) 2 min. Table 1 summarizes these measurements.