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. 2020 Feb 24;30(4):610-623.e5.
doi: 10.1016/j.cub.2019.12.022. Epub 2020 Jan 9.

Non-enzymatic Activity of the α-Tubulin Acetyltransferase αTAT Limits Synaptic Bouton Growth in Neurons

Courtney E Coombes  1 , Harriet A J Saunders  2 , Anirudh G Mannava  1 , Dena M Johnson-Schlitz  3 , Taylor A Reid  1 , Sneha Parmar  1 , Mark McClellan  1 , Connie Yan  4 , Stephen L Rogers  5 , Jay Z Parrish  4 , Michael Wagenbach  6 , Linda Wordeman  6 , Jill Wildonger  7 , Melissa K Gardner  8
Affiliations

Non-enzymatic Activity of the α-Tubulin Acetyltransferase αTAT Limits Synaptic Bouton Growth in Neurons

Courtney E Coombes et al. Curr Biol. .

Abstract

Neuronal axons terminate as synaptic boutons that form stable yet plastic connections with their targets. Synaptic bouton development relies on an underlying network of both long-lived and dynamic microtubules that provide structural stability for the boutons while also allowing for their growth and remodeling. However, a molecular-scale mechanism that explains how neurons appropriately balance these two microtubule populations remains a mystery. We hypothesized that α-tubulin acetyltransferase (αTAT), which both stabilizes long-lived microtubules against mechanical stress via acetylation and has been implicated in promoting microtubule dynamics, could play a role in this process. Using the Drosophila neuromuscular junction as a model, we found that non-enzymatic dαTAT activity limits the growth of synaptic boutons by affecting dynamic, but not stable, microtubules. Loss of dαTAT results in the formation of ectopic boutons. These ectopic boutons can be similarly suppressed by resupplying enzyme-inactive dαTAT or by treatment with a low concentration of the microtubule-targeting agent vinblastine, which acts to suppress microtubule dynamics. Biophysical reconstitution experiments revealed that non-enzymatic αTAT1 activity destabilizes dynamic microtubules but does not substantially impact the stability of long-lived microtubules. Further, during microtubule growth, non-enzymatic αTAT1 activity results in increasingly extended tip structures, consistent with an increased rate of acceleration of catastrophe frequency with microtubule age, perhaps via tip structure remodeling. Through these mechanisms, αTAT enriches for stable microtubules at the expense of dynamic ones. We propose that the specific suppression of dynamic microtubules by non-enzymatic αTAT activity regulates the remodeling of microtubule networks during synaptic bouton development.

Keywords: Drosophila; acetylation; microtubule; microtubule aging; neuromuscular junction; neuron; synaptic bouton; αTAT1.

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Conflict of interest statement

Declaration of Interests The authors declare no competing interests.

Figures

Figure 1.
Figure 1.. dαTAT limits the growth of synaptic boutons at the NMJ.
Axon terminals on muscle 4 in 3rd instar larvae are outlined by HRP (magenta). Scale bars, 10 μm. (A) Neuronally over-expressed dαTAT-GFP (green) localizes to synaptic boutons (arrows highlight a subset of dαTAT-GFP-positive boutons). (B and C) Neuronal and muscle microtubules are acetylated in control (B) but not dαTATKO larvae (C). The 6–11B-1 antibody (green) recognizes acetylated microtubules. (D) Boutons at the NMJ contain a core of stable microtubules (solid purple lines) bound by Futsch (gold circles). Dynamic microtubules (blue dashed lines) grow and retract in the bouton periphery. (E and F) Representative images of synaptic terminals in control (E) and dαTATKO (F) larvae. Ectopic satellite boutons (arrowheads) sprout from axon terminals in dαTATKO larvae. (G-J) Quantification of total and satellite bouton numbers per NMJ 4 in control and experimental conditions as indicated (single-factor ANOVA with post-hoc Tukey; error bars: SEM). (G) Neuronal expression of dαTAT (O/E dαTAT) rescues the change in total and satellite bouton numbers in dαTATKO larvae. (H,I) Neuronal expression of wild-type (O/E dαTAT) or catalytically inactive dαTAT (O/E dαTATGG) suppresses the formation of ectopic boutons in response to high-potassium stimulation. (J) Ectopic boutons in dαTATKO larvae are suppressed by the expression of dαTATGG, indicating dαTAT limits bouton growth independently of its enzymatic activity (See also Figure S1). n.s. = not significant, *p = 0.01–0.05, **p = 0.01–0.001, ***p = 0.001–0.0001, ****p < 0.0001.
Figure 2.
Figure 2.. Increased microtubule growth stimulates the formation of satellite boutons in dαTAT knockout animals.
(A) In neurons, Futsch labels long-lived microtubules, some of which form loops (arrowheads). Scale bar, 10 μm. (B) The loss of dαTAT does not affect the number of Futsch-positive stable microtubule loops (“Futsch loops”) per NMJ 4 (top) (p=0.66, Mann-Whitney U test; error bars: SEM). The number of boutons with diffuse Futsch signal is also similar to controls in dαTATKO larvae (bottom) (p=0.83, Mann-Whitney U test; error bars: SEM). (C) Bundled microtubules at the NMJ core extend into ~25% of terminal boutons in control and dαTAT knockout animals. Top: Example of bundled microtubule core (arrowhead) extending into a terminal bouton (dotted circle). Bottom: Quantification of terminal boutons into which the bundled microtubule core has extended (n=12 control and 15 dαTATKO boutons). Scale bar, 10 μm. (D and E) Vinblastine suppresses the formation of ectopic boutons, including satellite boutons, in dαTATKO larvae and in response to high-potassium stimulation (single-factor ANOVA with post-hoc Tukey; error bars: SEM). n.s. = not significant, *p = 0.01–0.05, **p = 0.01–0.001, ***p = 0.001–0.0001, ****p < 0.0001.
Figure 3.
Figure 3.. Non-enzymatic αTAT1 activity increases the catastrophe frequency of dynamic microtubules.
(A) Top: In vitro reconstitution experiments in which GMPCPP-stabilized seed templates (red) were adhered to a coverslip (black), and then dynamic GTP-microtubules (green) were grown from the seeds. Bottom: Representative kymographs of dynamic microtubules in control experiments (left), and in experiments with 1.0 μM αTAT1 (center), and with 1.5 μM αTAT1 (right). The kymographs display slices from time-lapse videos of dynamic microtubules such that position is across the x-axis and time is down the y-axis. Catastrophe frequency was calculated as the inverse of the microtubule lifetime. See also Videos S1–S2. (B) Top: Catastrophe frequencies of control dynamic microtubules and dynamic microtubules incubated with 1.0 μM αTAT1 and 1.5 μM αTAT1 (p=9×10−11, single-factor ANOVA; error bars: SEM). Bottom: Growth rates for control dynamic microtubules and dynamic microtubules incubated with 1.0 μM αTAT1 and 1.5 μM αTAT1 (p=1.8×10−09, single-factor ANOVA, error bars: SEM). Microtubule growth rate was calculated from the slope of the growth event. (C) Western blots showing detection of RPE1 cell lysates (left), and purified αTAT1 (right) by anti-αTAT1 antibody (LifeSpan BioSciences #LS-C116215). Blot smearing likely reflects αTAT1 isoforms inside of cells. (D) Estimates of αTAT1 concentration inside of RPE1 cells (green), placed in context of the in vitro microtubule dynamics results. (E) Left: Representative TIRF images of αTAT1-GFP (green) binding to Taxol-stabilized GDP-microtubules (blue) grown from stabilized seed templates (red). Right: Quantitative average fluorescence line scans indicating the localization of αTAT1-GFP (green) on Taxol-stabilized GDP-microtubules (blue) (n=144 microtubules, error bars 95% confidence intervals). Line scans of microtubules oriented with minus-end to the left and plus-end to the right; microtubules used in the analysis were ~4 μm long, with additional lengths shown in Figure S2D.
Figure 4.
Figure 4.. αTAT1 does not efficiently depolymerize stable microtubules.
(A) GMPCPP extensions were grown from coverslip-attached seed templates to evaluate the effect of αTAT1 on GMPCPP stabilized microtubules. (B) Example kymographs of green GMPCPP microtubules extending from red GMPCPP seeds in the presence of imaging buffer alone (left) and with 1.5 μM αTAT1 (center). Positive control experiments with 20 nM MCAK demonstrates robust depolymerization (right). (Scale bars 2 μm (horiz.) and 1 min (vertical)). (C) Measured shortening rates of GMPCPP extensions with just buffer (yellow), and with 1.5 μM αTAT1 (magenta) (p=0.20, t-test; error bars: SEM) (D) Taxol-stabilized GDP-microtubule extensions from coverslip-attached seed templates were observed to evaluate the effect of αTAT1 on Taxol stabilized GDP-microtubules. (E) Example kymographs of green GDP-Taxol Extensions from red GMPCPP seeds in control (left), 1.6 μM αTAT1 (center), and positive control (20 nM MCAK, right) experiments. (Scale bars 2 μm (horiz.) and 1 min (vertical)). (F) Measured shortening rates of GDP-Taxol extensions with imaging buffer alone (yellow), and with αTAT1 (magenta) (p=0.30, t-test; error bars: SEM). (G) Top: GDP extensions (red) were grown from coverslip-attached seed templates (grey), capped with GMPCPP-tubulin (blue), and then new dynamic (GTP) extensions were grown (green), to simultaneously evaluate the effect of αTAT1 on dynamic and stable microtubules in the presence of free tubulin. Bottom: Example kymographs in control (left), 1.6 μM αTAT1 (center), and 40 nM MCAK (right) experiments. (Scale bars 5 μm (horiz.) and 5 min (vertical)). See also Videos S3–S5. (H) Quantification of microtubule length over time for each microtubule type (top: capped GDP; middle: GMPCPP cap; bottom: dynamic GTP). Sample sizes (average number microtubules per time step): Capped GDP (top): Control 27, αTAT1 21, MCAK 19; GMPCPP (middle): Control 81, αTAT1 29, MCAK 54; Dynamic (bottom): Control 49, αTAT1 55, MCAK 0 (no growth).
Figure 5.
Figure 5.. αTAT1 alters microtubule tip structures.
(A) Representative cryo-electron microscopy images of GMPCPP microtubules incubated with buffer alone (left) or 1.0 μM αTAT1 (right). Yellow dotted lines highlight microtubule tips. (B) Measured tip tapers (difference in length between longest visible protofilament and shortest visible protofilament) show that the tip tapers of GMPCPP microtubules treated with αTAT1 were on average 2.1-fold larger than untreated control microtubules (p=6.4×10−12, t-test; error bars: SEM). (C) Left: Line scans of Rhodamine-labeled GMPCPP microtubule tips (insets, scale bar 1 μm) were collected for microtubules incubated in buffer alone (top) and with αTAT1 (bottom). The average line scans were then fit to an error function to calculate a “tip standard deviation” (σtip), which reflects the rate that fluorescence at the microtubule tip drops off to background. A slower drop-off (larger σtip) is reflective of a more tapered end. All microtubules included in this measurement were ~ 3 μm long. Right: Fitted tip standard deviations (p=0.02, Z= 2.25; error bars: 95% confidence intervals). (D) Top: Tip structures of dynamic microtubules were analyzed using TIRF microscopy. Bottom: Representative images of short and long dynamic microtubule extensions (green) suggest that short microtubules have similar tip configurations in the presence and absence of αTAT1 (blue arrows). However, in the presence of αTAT1, longer microtubules frequently showed curled tip structures that were not present in the controls (yellow arrows). (E) Left: Line scans of dynamic microtubule tips were collected for short (top) and long (bottom) microtubules that were incubated in buffer alone (blue) and with αTAT1 (magenta). The average line scans were then fit to an error function to calculate a “tip standard deviation” (σtip) at the microtubule plus-end, which reflects the rate that fluorescence at the microtubule tip drops off to background. A slower drop-off (larger σtip) is reflective of a more tapered end. Right: There was little difference in the tip standard deviation between control and αTAT1 for the short microtubules (p=0.96, Z=0.048). However, the tip standard deviation was 42% larger for αTAT1-treated long microtubules as compared to long microtubule controls (p<10−5, Z= 9.76). (Error bars: 95% confidence intervals).
Figure 6.
Figure 6.. αTAT1 speeds the aging of dynamic microtubules.
(A) Microtubule lifetimes prior to catastrophe were plotted as a cumulative probability distribution, and then fitted to a Gamma probability distribution, which describes an aging process. (B) The fitting parameters for the Gamma distribution are (top) the number of steps required prior to a catastrophe event, which are similar between controls and αTAT1 treatments (p=0.46, Z=0.0967), and (bottom) the rate parameter for each step, which generally describes the speed of aging. The difference in rate parameters between control and αTAT1 treatment suggests that αTAT1 speeds microtubule aging (p=0.00035, Z=3.387). (Error bars: 95% confidence intervals). (C) Right: Schematic summarizing the proposed mechanism of dynamic microtubule aging by αTAT1: αTAT1 alters the tip structure of older microtubules, leading to increased tip tapering and associated protofilament curvature (curvature not shown in cartoon). This in turn leads to rapid aging and a higher catastrophe frequency in the presence of αTAT1. Left: A similar effect on stabilized microtubules has little effect on microtubule length.
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