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. 2016 May 23;213(4):425-33.
doi: 10.1083/jcb.201603050. Epub 2016 May 16.

The Structured Core of Human β Tubulin Confers Isotype-Specific Polymerization Properties

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Free PMC article

The Structured Core of Human β Tubulin Confers Isotype-Specific Polymerization Properties

Melissa C Pamula et al. J Cell Biol. .
Free PMC article

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.

Figures

Figure 1.
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. Kd = 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.
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 (fcat), SD were calculated as fcat/n (assuming a Poisson distribution), where n is the number of catastrophe events. Table 1 summarizes these measurements.
Figure 3.
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.
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 (fcat), SD were calculated as fcat/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.

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