Mechanism of microtubule severing enzymes

National Institute Of Neurological Disorders And Stroke

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Abstract

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. This year we have continued our studies into the structure of microtubule severing enzymes in complex with their regulators using cryo-EM and single molecule fluorescence imaging and have identified mechanisms of their regulation by accessory factors and posttranslational modifications. Specifcially, we discovered that glutamylation and glycylation are antagonistic rheostats with glycylation protecting microtubules from severing (Szczesna et al., 2022). Katanin exhibits graded and divergent responses to glutamylation on the alpha and beta-tubulin tails, and these act combinatorially. Glutamylation on the alpha-tail is purely stimulatory, while glutamylation on the beta-tail elicits a biphasic response dictated by the recognition of the glutamylated tail by the katanin hexamer central pore. Elements distal to the katanin AAA core sense -tubulin tyrosination, and detyrosination downregulates severing. This multivalent microtubule recognition that enables katanin to read multiple tubulin modification inputs explains in vivo observations and illustrates how effectors can integrate tubulin code signals to produce diverse functional outcomes in cells.