Severing enzymes amplify microtubule arrays through lattice GTP-tubulin incorporation

Severing to build microtubules Microtubules are essential intracellular polymers, built from tubulin subunits, that establish cell shape, move organelles, and segregate chromosomes during cell division. Vemu et al. show that microtubule-severing enzymes extract tubulin subunits along the microtubule shaft. This nanoscale damage is repaired by the incorporation of free tubulin, which stabilizes the microtubule against depolymerization. When extraction outpaces repair, microtubules are severed, emerging with stabilized ends composed of fresh tubulin. The severed microtubules act as templates for new microtubule growth, leading to amplification of microtubule number and mass. Thus, seemingly paradoxically, severing enzymes can increase microtubule mass in processes such as neurogenesis and mitotic spindle assembly. Science, this issue p. eaau1504 Spastin and katanin extract tubulin dimers from microtubules and amplify microtubule arrays via lattice repair. INTRODUCTION The microtubule cytoskeleton is continuously sculpted by polymerization, depolymerization, cross-linking, and microtubule severing. Microtubule severing generates internal breaks in microtubules through a poorly understood mechanism. It is mediated by the AAA [adenosine triphosphatases (ATPases) associated with various cellular activities] ATPases katanin, spastin, and fidgetin. Paradoxically, despite the destructive activity of severing enzymes, loss of severing enzyme activity leads to a decrease rather than an increase in microtubule mass. It was hypothesized that this severing enzyme–dependent increase in microtubule mass results from templated nucleation from the severed ends. This is an attractive hypothesis for a mechanism to generate microtubule mass, especially in the absence of centrosome-based nucleation as in neurons or meiotic spindles. However, for this amplification to operate, the guanosine diphosphate (GDP)–tubulin lattice exposed through severing would have to be stabilized. The GDP-microtubule lattice is the product of guanosine triphosphate (GTP)–tubulin polymerization and depolymerizes spontaneously when exposed in the absence of a stabilizing GTP cap. We examined this paradox by examining the effects of the severing enzymes spastin and katanin on microtubule structure and dynamics in vitro. RATIONALE Because light microscopy–based severing assays fail to capture ultrastructural features of severing intermediates due to resolution limitations, we used negative-stain transmission electron microscopy (TEM) to capture and image spastin- and katanin-mediated microtubule severing in vitro. We combined these experiments with quantitative analyses of tubulin and microtubule polymer dynamics by using total internal reflection fluorescence (TIRF) microscopy to understand the effects of severing on microtubule networks. RESULTS Our electron microscopy analyses coupled with TIRF microscopy revealed that spastin and katanin actively extract tubulin dimers out of the microtubule, introducing nanoscale damage along the microtubule, and that this action is counteracted by spontaneous, de novo incorporation of GTP-tubulin dimers from the soluble pool. Depending on the local balance between the rates of active tubulin extraction and passive repair, there are two non–mutually exclusive consequences: The microtubule is rejuvenated with GTP-tubulin islands that stabilize it against depolymerization, or severing proceeds to completion and the newly severed microtubule ends emerge with a high density of stabilizing GTP-tubulin. Consistent with this, we found that spastin and katanin activities increase rates of microtubule rescue and that rescues occur preferentially at sites of enzyme-dependent GTP-tubulin incorporation. Lastly and unexpectedly, we found that the incorporation of GTP-tubulin at severing sites ensures that the newly severed plus ends are stable because they emerge with a high density of GTP-tubulin that protects them against spontaneous depolymerization and promotes elongation. The synergy between the increased rescue rates and the stabilization of the newly severed ends leads to microtubule amplification. CONCLUSION Our study identifies the microtubule-severing enzymes spastin and katanin as biological agents that introduce GTP-tubulin islands within microtubules and demonstrates that microtubule-severing enzymes alone can amplify microtubule number and mass by promoting GTP-tubulin incorporation into the microtubule shaft, away from the dynamic ends long thought to be the sole locus of tubulin exchange. This microtubule-based amplification mechanism in the absence of a nucleating factor helps explain why the loss of spastin and katanin results in the loss of microtubule mass in systems that are dependent on noncentrosomal microtubule generation. Severing enzymes spastin and katanin amplify microtubule arrays by catalyzing tubulin exchange along the microtubule. Spastin and katanin sever and destabilize microtubules. Paradoxically, despite their destructive activity they increase microtubule mass in vivo. We combined single-molecule total internal reflection fluorescence microscopy and electron microscopy to show that the elemental step in microtubule severing is the generation of nanoscale damage throughout the microtubule by active extraction of tubulin heterodimers. These damage sites are repaired spontaneously by guanosine triphosphate (GTP)–tubulin incorporation, which rejuvenates and stabilizes the microtubule shaft. Consequently, spastin and katanin increase microtubule rescue rates. Furthermore, newly severed ends emerge with a high density of GTP-tubulin that protects them against depolymerization. The stabilization of the newly severed plus ends and the higher rescue frequency synergize to amplify microtubule number and mass. Thus, severing enzymes regulate microtubule architecture and dynamics by promoting GTP-tubulin incorporation within the microtubule shaft.