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. 2017 Dec 1;28(25):3564-3572.
doi: 10.1091/mbc.E17-02-0124. Epub 2017 Oct 11.

Tubulin Isoform Composition Tunes Microtubule Dynamics

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Free PMC article

Tubulin Isoform Composition Tunes Microtubule Dynamics

Annapurna Vemu et al. Mol Biol Cell. .
Free PMC article

Abstract

Microtubules polymerize and depolymerize stochastically, a behavior essential for cell division, motility, and differentiation. While many studies advanced our understanding of how microtubule-associated proteins tune microtubule dynamics in trans, we have yet to understand how tubulin genetic diversity regulates microtubule functions. The majority of in vitro dynamics studies are performed with tubulin purified from brain tissue. This preparation is not representative of tubulin found in many cell types. Here we report the 4.2-Å cryo-electron microscopy (cryo-EM) structure and in vitro dynamics parameters of α1B/βI+βIVb microtubules assembled from tubulin purified from a human embryonic kidney cell line with isoform composition characteristic of fibroblasts and many immortalized cell lines. We find that these microtubules grow faster and transition to depolymerization less frequently compared with brain microtubules. Cryo-EM reveals that the dynamic ends of α1B/βI+βIVb microtubules are less tapered and that these tubulin heterodimers display lower curvatures. Interestingly, analysis of EB1 distributions at dynamic ends suggests no differences in GTP cap sizes. Last, we show that the addition of recombinant α1A/βIII tubulin, a neuronal isotype overexpressed in many tumors, proportionally tunes the dynamics of α1B/βI+βIVb microtubules. Our study is an important step toward understanding how tubulin isoform composition tunes microtubule dynamics.

Figures

FIGURE 1:
FIGURE 1:
Isoform composition and purity of tubulin isolated from brain tissue and tsA201 cells using the TOG affinity method. (A) Mass spectra and SDS–polyacrylamide gel (inset) of tubulin isolated from mouse brain. Number of posttranslationally added glutamates is indicated for the glutamylated species. (B) Mass spectra and SDS–polyacrylamide gel (inset) tubulin isolated from tsA201 cells (see Materials and Methods).
FIGURE 2:
FIGURE 2:
Dynamics of brain and α1B/βI+βIVb microtubules. (A) Kymographs showing typical microtubule growth for brain and α1B/βI+βIVb tubulin at 6 µM. Horizontal and vertical scale bars, 5 µm and 5 min, respectively. (B) Left: kymographs showing a typical depolymerization event for brain and α1B/βI+βIVb microtubules. Horizontal and vertical scale bars, 5 µm and 2 s, respectively. Right: Tukey plot showing plus-end depolymerization rates at 6 µM tubulin; n = 12 and 27 events for brain and α1B/βI+βIVb microtubules, respectively. (C) Plus-end growth rates as a function of varying tubulin concentration for brain (gray) and α1B/βI+βIVb microtubules (blue). (D) Plus-end dynamics of brain (gray) and α1B/βI+βIVb (blue) microtubules at 6 µM tubulin. From left to right: box-whisker plot (whiskers indicate minimum and maximum) showing plus-end growth rates for brain and α1B/βI+βIVb microtubules; n = 38 and 191 events for brain and α1B/βI+βIVb tubulin, respectively. Plus-end catastrophe frequencies; n = 20 and 69 microtubules for brain and α1B/βI+βIVb tubulin, respectively. Plus-end microtubule mean lengths; n = 49 and 102 events for brain and α1B/βI+βIVb tubulin, respectively. Plus-end microtubule mean lifetimes; n = 49 and 102 events for brain and α1B/βI+βIVb tubulin, respectively. (E) Minus-end dynamics of brain and α1B/βI+βIVb tubulin at 6 µM tubulin. From left to right: box-whisker plot (whiskers indicate minimum and maximum) showing minus-end growth rates for brain and α1B/βI+βIVb tubulin; n = 12 and 84 events for brain and α1B/βI+βIVb tubulin, respectively. Minus-end catastrophe frequencies; n = 10 and 33 microtubules for brain and α1B/βI+βIVb tubulin, respectively. Minus-end microtubule mean lengths; n = 9 and 10 events for brain and α1B/βI+βIVb, respectively. Minus-end microtubule mean lifetimes; n = 9 and 10 events for brain and α1B/βI+βIVb, respectively. ** and ****, p values < 0.01 and < 0.0001, respectively, determined by unpaired t test.
FIGURE 3:
FIGURE 3:
Cryoelectron microscopy of α1B/βI+βIVb microtubules. (A) Cross-section of the cryo-EM map (gray density) and model of GMPCPP human α1B/βI+βIVb microtubules (three protofilaments shown). A central protofilament (Pf2) makes lateral contacts with adjacent protofilaments (Pf1 and Pf3); α-tubulin, orange, β-tubulin, red (Pf1, Pf3); α-tubulin, cyan; β-tubulin, purple (Pf2). (B) β-Tubulin helix H7 and GMPCPP (left, purple) and α-tubulin helix H7 and GTP (right, cyan) and their corresponding experimental densities (gray density). (C) Gallery of polymerizing brain (bovine [Cytoskeleton] and mouse, TOG-affinity purified) and α1B/βI+βIVb microtubule ends. Similar architectures are observed, including short and long taper/curved region lengths. Scale bar: 20 nm. (D) Quantification of the length of the curved/tapered region for brain (bovine [Cytoskeleton] and mouse, TOG-affinity purified) and α1B/βI+βIVb microtubule ends; n = 79, 130, and 95 for mouse brain, bovine brain, and α1B/βI+βIVb microtubule ends, respectively. ** and *p value < 0.01 or < 0.05, respectively, as determined by the Mann-Whitney test. (E) Histogram showing curved/tapered region length frequency of α1B/βI+βIVb microtubule ends. (F) Gallery of tubulin rings in dynamic preparations of commercial bovine brain (Cytoskeleton) microtubules (top), TOG-affinity purified mouse brain, microtubules (middle), and α1B/βI+βIVb microtubules (bottom) showing rings of different diameters and orientations. Scale bar: 20 nm. (G) Quantification of maximum ring diameter from brain (bovine [Cytoskeleton] and mouse, TOG-affinity purified) and α1B/βI+βIVb dynamic microtubule preparations; n = 48, 151, and 240 for bovine brain, mouse brain, and α1B/βI+βIVb. **** p value < 0.0001 determined by the Mann-Whitney test.
FIGURE 4:
FIGURE 4:
EB1-GFP comet analysis on brain and α1B/βI+βIVb microtubules. (A) TIRF microscopy images of EB1-GFP comets at the ends of growing brain microtubules (left) and α1B/βI+βIVb (right) microtubules at different growth speeds. From top to bottom: brain microtubule growth speeds are 0.8, 2.1, and 4.2 µm/min (n = 26, 13, and 15 microtubules, respectively). From top to bottom: α1B/βI+βIVb microtubule growth speeds are 0.7, 2.3, and 4.3 µm/min (n = 24, 12, and 10 microtubules, respectively). Scale bar: 2 µm. (B) Averaged fluorescence intensity profiles of EB1-GFP comets at different growth speeds of brain (left) and α1B/βI+βIVb (right) microtubules. n = 420, 333, and 421 comet profiles from 12, 13, and 15 brain microtubules at growth speeds of 0.8, 2.1, and 4.2 µm/min, respectively and n = 364, 351, and 234 comet profiles for 15, 12, and 10 α1B/βI+βIVb microtubules at growth speeds of 0.7, 2.3, and 4.3 µm/min, respectively. (C) EB1-GFP comet tail lengths as a function of microtubule growth speeds for brain (gray) and α1B/βI+βIVb (blue) microtubules. Comet tail lengths were obtained by single exponential fits to the averaged intensity profiles (see Materials and Methods).
FIGURE 5:
FIGURE 5:
Modulation of α1B/βI+βIVb tubulin dynamics by addition of neuronal α1A/βIII tubulin. (A) Left panel: box-whisker plot (whiskers indicate minimum and maximum) showing plus-end growth rates at 6 µM tubulin; n = 191, 258, 432, 377, and 203 events for α1B/βI+βIVb, 75% α1B/βI+βIVb 25% α1A/βIII, 50% α1B/βI+βIVb 50% α1A/βIII, 25% α1B/βI+βIVb 75% α1A/βIII, and α1A/βIII tubulin, respectively. Right panel: plus-end catastrophe frequencies; n = 69, 77, 113, 94, and 85 microtubules for α1B/βI+βIVb, 75% α1B/βI+βIVb 25% α1A/βIII, 50% α1B/βI+βIVb 50% α1A/βIII, 25% α1B/βI+βIVb 75% α1A/βIII, and α1A/βIII tubulin, respectively. ** and ****, p values < 0.01 and < 0.0001, respectively, determined by unpaired t test. (B) Left panel: box-whisker plot (whiskers indicate minimum and maximum) showing minus-end growth rates at 6 µM tubulin; n = 84, 66, 206, 91, and 93 events for α1B/βI+βIVb, 75% α1B/βI+βIVb 25% α1A/βIII, 50% α1B/βI+βIVb 50% α1A/βIII, 25% α1B/βI+βIVb 75% α1A/βIII, and α1A/βIII tubulin, respectively. Right panel: minus-end catastrophe frequencies; n = 33, 34, 59, 30, and 40 microtubules for α1B/βI+βIVb, 75% α1B/βI+βIVb 25% α1A/βIII, 50% α1B/βI+βIVb 50% α1A/βIII, 25% α1B/βI+βIVb 75% α1A/βIII, and α1A/βIII tubulin, respectively.

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