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, 164 (5), 911-21

Graded Control of Microtubule Severing by Tubulin Glutamylation

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Graded Control of Microtubule Severing by Tubulin Glutamylation

Max L Valenstein et al. Cell.

Abstract

Microtubule-severing enzymes are critical for the biogenesis and maintenance of complex microtubule arrays in axons, spindles, and cilia where tubulin detyrosination, acetylation, and glutamylation are abundant. These modifications exhibit stereotyped patterns suggesting spatial and temporal control of microtubule functions. Using human-engineered and differentially modified microtubules we find that glutamylation is the main regulator of the hereditary spastic paraplegia microtubule severing enzyme spastin. Glutamylation acts as a rheostat and tunes microtubule severing as a function of glutamate number added per tubulin. Unexpectedly, glutamylation is a non-linear biphasic tuner and becomes inhibitory beyond a threshold. Furthermore, the inhibitory effect of localized glutamylation propagates across neighboring microtubules, modulating severing in trans. Our work provides the first quantitative evidence for a graded response to a tubulin posttranslational modification and a biochemical link between tubulin glutamylation and complex architectures of microtubule arrays such as those in neurons where spastin deficiency causes disease.

Conflict of interest statement

COMPETING FINANCIAL INTERESTS

The authors declare no competing financial interests.

Figures

Figure 1.
Figure 1.. Graded regulation of spastin-catalyzed microtubule severing by tubulin glutamylation.
(A–E). Left, spastin-catalyzed microtubule severing of unmodified microtubules (A) and microtubules with increasing glutamylation levels (B–E). Scale bar, 2 μm. Right, reversed-phase LC-MS of microtubules used in severing assays shown on the left (Experimental Procedures). There are one detectable α (α1Β) and two β (βI and βIVb) isoforms in these preparations. Throughout, numbers of glutamates added to α and β-tubulin isoforms are indicated in grey and green, respectively. The weighted mean of the number of glutamates (E>) added to α- and β-tubulin (overall and separately for the two β isoforms) are denoted α+<nE> and, β+<nE>, respectively (Experimental Procedures). See also Figure S1.
Figure 2.
Figure 2.. Spastin-catalyzed microtubule severing displays a biphasic response to tubulin glutamylation levels.
(A) Spastin-catalyzed microtubule severing varies with the number of glutamates on tubulin tails. E> on α and β-tubulin, indicated in grey and green, respectively. Error bars, S.E.M. (n > 23 microtubules from multiple chambers). Severing rates normalized to that of unmodified microtubules (Experimental Procedures). (B-C) Modulation of β-tubulin glutamylation levels is sufficient to increase (B) or decrease (C) microtubule severing activity while α-tubulin glutamylation levels are kept constant (for mass spectra, see Figures S2A and S2B). Error bars, S.E.M. (n = 23 microtubules from multiple chambers for both B and C). **** p < 0.0001; *** p < 0.001. Top panels, progress curves of severing reactions; bottom panels, severing rates as in (A). (D) Microtubule severing activity is highly sensitive to β-tubulin glutamylation levels (for mass spectra, see Figure S2C). Error bars, S.E.M. (n = 23 microtubules from multiple chambers). † p > 0.01 Top panels, progress curves of severing reactions; bottom panels, severing rates as in (A). (E) An increase in β-tubulin glutamylation is sufficient to induce a biphasic response in microtubule severing when α-tubulin glutamylation is constant (for mass spectra, see Figure S2D). Error bars, S.E.M. (n = 24 microtubules from multiple chambers). **** p < 0.0001; ** p < 0.01. Top panels, progress curves of severing reactions; bottom panels, severing rates as in (A).
Figure 3.
Figure 3.. Spastin-mediated microtubule severing requires the β-, but not the α-tubulin C-terminal tail.
(A) Left, normalized microtubule severing rates (left axis) and binding affinity (right axis) for human recombinant microtubules missing either the α- or β-tail (Figure S3A). Error bars, S.E.M. (n > 34 or 70 microtubules from multiple chambers for severing and binding assays, respectively). Right, engineered human microtubules severed by spastin. Arrows indicate severing sites. Scale bar, 2 μm. See also Figure S3. (B) Left, normalized microtubule severing rates (left axis) and binding affinity (right axis) for human recombinant tyrosinated and detyrosinated microtubules. Error bars, S.E.M. (n > 15 microtubules for severing assays; n > 96 microtubules for binding assays, from multiple chambers). Right, overlaid reversed-phase LC-MS of tyrosinated (gray) and detyrosinated (yellow) microtubules corresponding to the α-tubulin peak.
Figure 4.
Figure 4.. α-tubulin acetylation does not affect spastin-mediated microtubule severing.
(A) Left, normalized spastin severing activity with unmodified and acetylated human microtubules. Error bars, S.E.M. (n = 50 and 27 for unmodified and acetylated microtubules, respectively, from multiple chambers). Right, overlaid reversed-phase LC-MS of unmodified (gray) and acetylated (magenta) α-tubulin showing the 42 Da mass shift corresponding to the acetylated species. No mass change is detected in β-tubulin (not shown).
Figure 5.
Figure 5.. Competition between affinity and specific activity gives rise to the biphasic regulation of spastin-catalyzed microtubule severing by tubulin glutamylation.
(A) Spastin affinity for microtubules increases with the number of glutamates on tubulin tails. Spastin binding on modified microtubules normalized to that for unmodified microtubules). <nE> on α and β-tubulin are indicated as in Figure 1. Error bars, S.E.M. (n = 50 microtubules from multiple chambers for each <nE>). (B) Spastin microtubule affinity increases linearly with glutamate numbers on tubulin (R2 = 0.99). Left, microtubule severing rates, kobs, right, microtubule association constants, Ka for various <nE> normalized to those for unmodified microtubules. Error bars represent S.E.M. (C) Microtubule severing rates as a function of spastin concentration for microtubules with various glutamylation levels. Increased glutamylation induces earlier and more abrupt onset of anti-cooperativity of microtubule severing. Error bars, S.E.M. (n > 21 microtubules from multiple chambers for each E>). Grey shading, region of transition from cooperative to anti-cooperative behavior. (D) Microtubule severing activities for different glutamylation levels at constant number of bound spastin molecules per tubulin. The specific activity of bound spastin decreases with increased glutamylation. Error bars, S.E.M. (n > 20 microtubules from multiple chambers for each <nE>). (E) Competition between glutamylation-induced increase in microtubule affinity and decrease in enzyme specific activity yields a biphasic response of spastin severing to microtubule glutamylation levels. See also Figure S4.
Figure 6.
Figure 6.. Free poly-glutamic acid inhibits spastin microtubule severing
(A) Microtubule severing rates in the presence of 0.75 – 3 kDa poly-glutamic acid (6 to 23 glutamates). Error bars, S.E.M. (n > 24 microtubules from multiple chambers) (B) Microtubule severing rates in the presence of 3 – 15 kDa poly-glutamic acid (23 to 115 glutamates). Error bars, S.E.M. (n > 24 microtubules from multiple chambers). See also Figure S5.
Figure 7.
Figure 7.. Spastin-catalyzed severing activity is modulated by the glutamylation levels of neighboring microtubules.
(A) Spastin association with unmodified and glutamylated microtubules (top, panels E> ~ 6.8 E, bottom panels, <nE> ~ 22.3 E). Magenta, unmodified microtubules; yellow, glutamylated microtubules (<nE> of 6.8 or 22.3); green, DyLight 488 labeled spastin. Scale bar, 2 μm. (B) Spastin severing activity in chambers with mixed microtubule populations. Severing activity for unmodified microtubules in isolation or in the presence of equimolar concentrations of microtubules with defined glutamylation levels (E> of 6.8, 10.9, 18.5 or 22.3). Error bars, S.E.M. (n > 17 microtubules from multiple chambers).

Comment in

  • Getting a Grip on Microtubules
    J Schaletzky et al. Cell 164 (5), 836-7. PMID 26919420.
    Posttranslational modifications control microtubule behavior, yet assigning roles to particular signals was hampered by lack of defined in vitro systems. In this issue of …

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