Spastin is a dual-function enzyme that severs microtubules and promotes their regrowth to increase the number and mass of microtubules

Abstract

The remodeling of the microtubule cytoskeleton underlies dynamic cellular processes, such as mitosis, ciliogenesis, and neuronal morphogenesis. An important class of microtubule remodelers comprises the severases-spastin, katanin, and fidgetin-which cut microtubules into shorter fragments. While severing activity might be expected to break down the microtubule cytoskeleton, inhibiting these enzymes in vivo actually decreases, rather increases, the number of microtubules, suggesting that severases have a nucleation-like activity. To resolve this paradox, we reconstituted Drosophila spastin in a dynamic microtubule assay and discovered that it is a dual-function enzyme. In addition to its ATP-dependent severing activity, spastin is an ATP-independent regulator of microtubule dynamics that slows shrinkage and increases rescue. We observed that spastin accumulates at shrinking ends; this increase in spastin concentration may underlie the increase in rescue frequency and the slowdown in shortening. The changes in microtubule dynamics promote microtubule regrowth so that severed microtubule fragments grow, leading to an increase in the number and mass of microtubules. A mathematical model shows that spastin's effect on microtubule dynamics is essential for this nucleation-like activity: spastin switches microtubules into a state where the net flux of tubulin onto each polymer is positive, leading to the observed exponential increase in microtubule mass. This increase in the microtubule mass accounts for spastin's in vivo phenotypes.

Keywords: microtubule dynamics; microtubule nucleation; severase; severing enzyme; spastin.

Fig. 1.

Drosophila spastin modulates microtubule dynamics. (A) Experimental setup of the in vitro microtubule dynamic assay using IRM. The tubulin concentration was 8 μM. (B and C) Example kymographs of the dynamic microtubule assay. (B) Tubulin alone control with 1 mM AMP-PNP. (C) In the presence of 50 nM spastin + 1 mM AMP-PNP. Yellow dashed lines marked the position of GMP-CPP seeds on the kymographs. The shrinkage rate in the presence of spastin is slower than the tubulin alone control (arrowheads). A rescue event is marked with an arrow. (D and E) Comparison of growth rate and shrinkage rate in the presence of 50 nM spastin with tubulin alone control under no adenosine nucleotide and 1 mM AMP-PNP conditions. In each box, the midline shows the median value, the top and bottom lines show the 75th and 25th percentiles, and the whiskers are 1.5 interquartile ranges from the 75th and 25th percentiles. Outliers (outside the whisker range) are represented as empty circles. Sample size N indicates the number of microtubules analyzed in each condition. (F and G) Catastrophe and rescue frequency in the presence or absence of 50 nM spastin under no adenosine nucleotide or 1 mM AMP-PNP conditions. Error bars represent the SD. Sample size n = 4 independent experiments. MT, microtubule.

Fig. 2.

Spastin severs dynamic microtubules and increases the total mass and number of microtubules. (A) Time series of dynamic microtubules severed and amplified by spastin using IRM imaging. Breakage and regrowth of microtubules occurred when treated with 50 nM spastin and 1 mM ATP. (B) Example of total microtubule mass increasing over time in the dynamic microtubule-severing assay. The black line shows the relative total microtubule mass normalized by time = 0 s. The increase of total microtubule mass was fitted by a single-exponential growth curve (red dashed line).

Fig. 3.

Asymmetric behaviors of the two microtubule ends generated after severing. (A) Representative time series of dynamic severed microtubule. The corresponding kymograph is in B. The microtubule plus end is to the right. After the breakage occurred, the newly generated plus end (yellow arrowheads) shrank and rescued, while the new minus end directly regrew without detectable shrinkage taking place. (C) Quantification of the percentages of nonshrinking and shrinking ends generated by severing. The majority of new plus ends shrank after cut (mean ± SD; 69 ± 5%), and newly generated minus ends are mostly nonshrinking (80 ± 3%). Measurements were quantified from four independent experiments. The probability of a shrinking plus end rescuing within 0.5 μm (the shrinking length that can be confidently measured) is ∼15%, which was estimated from the rescue frequency and shrinkage rate measured in Table 2, assuming that rescue is a Poisson process. The error-corrected percentage of shrinking plus end is, therefore, 84%.

Fig. 4.

Mathematical model and microtubule length distribution. (A) Graphic representation of the dynamic instability with severing model. Rows 1 and 2 correspond to the canonical dynamic instability model. Rows 3 and 4 show that, when a microtubule is severed, the newly generated plus end starts shrinking. Here, we considered all of the minus ends as “neutral” ends where no growth or shrinkage takes place but that will disappear after the microtubules depolymerize completely. (B) Bulk measurement of microtubule length in the presence of severing with microtubule spin-down assay. Upper shows the example epifluorescence images before and 10 min after initiating severing reaction. Red indicates TAMRA-labeled GMP-CPP microtubule seeds. Green indicates tubulin (Alexa-488–labeled antitubulin antibody). Microtubule density increased notably 10 min after introducing 50 nM spastin. Lower shows the length distribution of 0 min (Left) and 10 min (Right) after the severing reaction. Severing shortens the mean length and changes the distribution from an exponential-like to a more compact peak-like function. The black line in Left indicates the exponential fit. Mean lengths ± SD: 5.5 ± 4.4 μm (0 min) and 4.0 ± 2.1 μm (10 min). Numbers of microtubules measured: 319 (0 min) and 377 (10 min). MT, microtubule.

Fig. 5.

Drosophila spastin tracks and concentrates on shrinking microtubule tips. (A) Representative time series showing that DyLight488-labeled spastin (shown in green) accumulates and remains associated with a microtubule plus end during shrinkage in the absence of ATP. Microtubules were visualized by IRM, and fluorescent spastin molecules were imaged by TIRF microscopy. Distinct foci were visible in both IRM and TIRF channels. Corresponding kymographs are shown in B. (C) Example time series of spastin molecules accumulating at a shrinking microtubule tip in the presence of 1 mM ATP. High-intensity foci localize at the shrinking plus end generated by severing (red arrows). Shrinkage stops in the last three frames, presumably due to a rescue event. The middle region, where the spastin signal is weak, corresponds to the GMP-CPP microtubule seed. (D) Line-scan fluorescence intensity profile of dynamic microtubule segments from C showing a high signal peak (red arrowheads) that corresponds to the high density of spastin that accumulates at the shrinking microtubule tip. Plus ends of microtubules are all pointing to the right.

Fig. 6.

Proposed model for increasing microtubule polymer mass by spastin. Tubulin subunits are removed from the microtubule lattice by spastin and lead to severing. Spastin molecules accumulate on the shrinking microtubule plus ends, potentially stabilizing the shrinking tip, and thus, they reduce the shrinkage rate and increase the rescue frequency. The promotion of rescue and slowdown of shrinkage lead to an increase in net flux onto the microtubules. When the average net flux becomes positive (unbounded growth regime), microtubule number and mass are increased by severing.