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 and (4) identify cellular factors that regulate spastin and katanin targeting and enzymatic activity. 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 recently 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. Due to the high sequence homology, we expect that this mechanism is shared with other microtubule-severing AAA enzymes. Our integrated study also provides insight into the many katanin mutants identified from classic genetic studies on meiosis where the katanin gene (also known as mei-1) was first identified as well as the many spastin disease mutations found in hereditary spastic paraplegia patients.
| 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 |
