Cells constantly assemble and disassemble their microtubule cytoskeleton through the concerted action of microtubule polymerases, depolymerases, crosslinkers and severing enzymes. Microtubule severing enzymes spastin and katanin generate internal breaks in microtubules. They are are critical in a wide range of cell biological processes including biogenesis of neuronal and non-centrosomal microtubule arrays, phototropism, spindle scaling, chromosome segregation, and control of centriole and cilia numbers. Mutations in microtubule severing enzymes cause severe neurodegenerative and neurodevelopmental disorders. The mechanism used by these enzymes to destabilize the microtubule and their effect on microtubule dynamics and the morphology of microtubule networks is still poorly understood.
We aim (1) to understand the structural transitions that spastin and katanin undergo during microtubule disassembly; (2) characterize the mechanism of ATP hydrolysis in the katanin and spastin hexamers during the microtubule severing reaction and how they are coupled to the mechanical work of tubulin dimer removal from the microtubule lattice; (3) establish the effects of tubulin modifications on microtubule severing; (4) characterize the effects of microtubule severing enzymes on microtubule dynamics and architecture; (5) develop a comprehensive understanding of how spastin and katanin disease mutations associated with hereditary spastic paraplegia and microcephaly, respectively, affect protein structure and function and (6) identify cellular factors that regulate spastin and katanin. Despite it being a basic mechanism to destabilize microtubules, we know very little about severing, not in small part due to the lack of any structural information. The mechanism of destabilizing microtubules from their ends is far better understood, in large part due to the wealth of structural information on the molecular machines involved, obtained by X-ray crystallography and electron microscopy. A mechanistic approach to the study of microtubule severing enzymes will provide a new framework for analyses and design of cellular studies. Moreover, insights into the mechanism of action of severing enzymes will likely hold implications for AAA ATPase in general, a large class of proteins still poorly understood, despite the fact that every major pathway in the human body contains an AAA ATPase. We reported the first X-ray structure of the monomeric AAA katanin module and cryo-EM reconstructions of the hexamer in two conformations (Zehr et al., Nature Struct. & Molec. Biol. 2017). These revealed an unexpected asymmetric arrangement of the AAA domains mediated by structural elements unique to microtubule severing enzymes that are critical for their function. Our cryo-EM reconstructions at 4.4 and 6 resolution of the katanin hexamer revealed an open spiral and a closed ring conformations of the AAA core, depending on the nucleotide occupancy of a gating protomer that closes a 40 wide gate in the katanin hexamer. Together with solution small-angle X-ray scattering (SAXS) reconstructions, our integrated structural study allowed us to advance a model whereby katanin makes multivalent interactions with the microtubule through its AAA core, flexible MIT domains and a newly defined linker element that crowns the AAA ring, and engages the C-terminal tails of tubulin through conserved pore loops that gradually pull tubulin dimers out of the microtubule lattice by cycling between open spiral and closed AAA ring conformations. More recently, we also reported the cryo-EM structure of the hereditary spastic paraplegia (HSP) protein spastin in complex with its substrate (Sandate et al., Nature Struct. & Molec. Biol. 2019). This structure revealed for the first time how a severing enzyme engages the tubulin substrate and shed light on how concurrent nucleotide and substrate binding organizes the conserved spastin pore loops into an ordered allosteric network that supports tubulin tail translocation to pull the tubulin dimer out of the microtubule and sever it. The majority of the residues in this allosteric network are mutated in HSP patients, underscoring their importance to spastin function. Our comprehensive structural analysis of all reported HSP-associated spastin missense mutations in its AAA core provides a framework for understanding spastin molecular dysfunction. My laboratory also discovered that severing enzymes spastin and katanin regulate microtubule dynamics by promoting the exchange of tubulin dimers along the microtubule shaft (Vemu et al. Science 2018). Combining single-molecule TIRF and electron microscopy we showed that spastin and katanin introduce nanoscale damage throughout the microtubule by active extraction of tubulin heterodimers that is repaired spontaneously by GTP-tubulin incorporation. As a result, the microtubule shaft is rejuvenated with GTP-tubulin islands that stabilize it against depolymerization and newly severed ends emerge with a high-density of GTP-tubulin that protects against depolymerization. The stabilization of the newly severed plus-ends and the higher rescue rates synergize to amplify microtubule number and mass. Thus, our work identified microtubule-severing enzymes spastin and katanin as biological agents responsible for the generation of GTP- tubulin islands within microtubules and demonstrated that microtubule-severing enzymes alone can amplify microtubule number and mass by promoting GTP-tubulin incorporation in 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 can explain why loss of spastin and katanin results in loss of microtubule mass in systems that are dependent on non-centrosomal microtubule generation.
| Vemu, Annapurna; Szczesna, Ewa; Zehr, Elena A et al. (2018) Severing enzymes amplify microtubule arrays through lattice GTP-tubulin incorporation. Science 361: |
| Zehr, Elena; Szyk, Agnieszka; Piszczek, Grzegorz et al. (2017) Katanin spiral and ring structures shed light on power stroke for microtubule severing. Nat Struct Mol Biol : |
| Valenstein, Max L; Roll-Mecak, Antonina (2016) Graded Control of Microtubule Severing by Tubulin Glutamylation. Cell 164:911-21 |
