Microtubule (MT) length and location is tightly controlled in cells. One novel family of MT-associated proteins that regulates MT dynamics is the MT-severing enzymes. In this work, we investigate how katanin (p60), believed to be the first discovered severing enzyme, binds and severs MTs via single molecule total internal reflection fluorescence microscopy. We find that severing activity depends on katanin concentration. We also find that katanin can remove tubulin dimers from the ends of MTs, appearing to depolymerize MTs. Strikingly, katanin localizes and severs at the interface of GMPCPP-tubulin and GDP-tubulin suggesting that it targets to protofilament-shift defects. Finally, we observe that binding duration, mobility, and oligomerization are ATP dependent.

Microtubule dynamics in cells are regulated by associated proteins that can be either stabilizers or destabilizers. A class of destabilizers that is important in a large number of cellular activities is the microtubule-severing enzymes, yet little is known about how they function. Katanin p60 was the first ATPase associated with microtubule severing. Here, we investigate the activity of katanin severing using a GFP-labeled human version. We quantify the effect of katanin concentration on katanin binding and severing activity. We find that free tubulin can inhibit severing activity by interfering with katanin binding to microtubules. The inhibition is mediated by the sequence of the tubulin and specifically depends on the carboxy-terminal tails. We directly investigate the inhibition effect of tubulin carboxy-terminal tails using peptide sequences of α-, β-, or detyrosinated α-tubulin tails that have been covalently linked to bovine serum albumin. Our results show that β-tubulin tails are the most effective at inhibiting severing, and that detyrosinated α-tubulin tails are the least effective. These results are distinct from those for other severing enzymes and suggest a scheme for regulation of katanin activity in cells dependent on free tubulin concentration and the modification state of the tubulin.

The evolutionarily divergent class of kinetoplastid organisms has a set of unconventional kinetochore proteins that drive chromosome segregation, but it is unclear which components interact with spindle microtubules. Llauró et al. now identify KKT4 as the first microtubule-binding kinetochore protein in Trypanosoma brucei, a major human pathogenic parasite.

Microtubule reorganization often results from the loss of polymer induced through breakage or active destruction by energy-using enzymes. Pre-existing defects in the microtubule lattice likely lower structural integrity and aid filament destruction. Using large-scale molecular simulations, we model diverse microtubule fragments under forces generated at specific positions to locally crush the filament. We show that lattices with 2% defects are crushed and severed by forces three times smaller than defect-free ones. We validate our results with direct comparisons of microtubule kinking angles during severing. We find a high statistical correlation between the angle distributions from experiments and simulations indicating that they sample the same population of structures. Our simulations also indicate that the mechanical environment of the filament affects breaking: local mechanical support inhibits healing after severing, especially in the case of filaments with defects. These results recall reports of microtubule healing after flow-induced bending and corroborate prior experimental studies that show severing is more likely at locations where microtubules crossover in networks. Our results shed new light on mechanisms underlying the ability of microtubules to be destroyed and healed in the cell, either by external forces or by severing enzymes wedging dimers apart. © 2016 Wiley Periodicals, Inc.

Disassembling microtubules can generate movement independently of motor enzymes, especially at kinetochores where they drive chromosome motility. A popular explanation is the ‘conformational wave’ model, in which protofilaments pull on the kinetochore as they curl outward from a disassembling tip. But whether protofilaments can work efficiently via this spring-like mechanism has been unclear. By modifying a previous assay to use recombinant tubulin and feedback-controlled laser trapping, we directly demonstrate the spring-like elasticity of curling protofilaments. Measuring their mechanical work output suggests they carry ~25% of the energy of GTP hydrolysis as bending strain, enabling them to drive movement with efficiency similar to conventional motors. Surprisingly, a β-tubulin mutant that dramatically slows disassembly has no effect on work output, indicating an uncoupling of disassembly speed from protofilament strain. These results show the wave mechanism can make a major contribution to kinetochore motility and establish a direct approach for measuring tubulin mechano-chemistry.DOI: http://dx.doi.org/10.7554/eLife.28433.001