Megan E. Bailey

Drosophila Katanin-60 Depolymerizes and Severs at Microtubule Defects

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.

Katanin Severing and Binding Microtubules Are Inhibited by Tubulin Carboxy Tails

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.

Invited review: Microtubule severing enzymes couple atpase activity with tubulin GTPase spring loading.

Microtubules are amazing filaments made of GTPase enzymes that store energy used for their own self‐destruction to cause a stochastically driven dynamics called dynamic instability. Dynamic instability can be reproduced in vitro with purified tubulin, but the dynamics do not mimic that observed in cells. This is because stabilizers and destabilizers act to alter microtubule dynamics. One interesting and understudied class of destabilizers consists of the microtubule‐severing enzymes from the ATPases Associated with various cellular Activities (AAA+) family of ATP‐enzymes. Here we review current knowledge about GTP‐driven microtubule dynamics and how that couples to ATP‐driven destabilization by severing enzymes. We present a list of challenges regarding the mechanism of severing, which require development of experimental and modeling approaches to shed light as to how severing enzymes can act to regulate microtubule dynamics in cells.

Modeling the effects of lattice defects on microtubule breaking and healing

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.

Purification and Biophysical Analysis of Microtubule-Severing Enzymes In Vitro

Microtubule-severing enzymes are a novel class of microtubule regulators. They are enzymes from the ATPases associated with various cellular activities family (AAA+) that utilize ATP to cut microtubules into smaller filaments. Discovered over 20 years ago, there are still many open questions about severing enzymes. Both cellular and biochemical studies need to be pursued to fully understand how these enzymes function mechanistically in the cell. Here, we present methods to express, purify, and test the biophysical nature of these proteins in vitro to begin to address the biochemical and biophysical mechanisms of this important and novel group of microtubule destabilizers.

The kinetoplastid kinetochore protein KKT4 is an unconventional microtubule tip–coupling protein

Kinetochores are multiprotein machines that drive chromosome segregation by maintaining persistent, load-bearing linkages between chromosomes and dynamic microtubule tips. Kinetochores in commonly studied eukaryotes bind microtubules through widely conserved components like the Ndc80 complex. However, in evolutionarily divergent kinetoplastid species such as Trypanosoma brucei, which causes sleeping sickness, the kinetochores assemble from a unique set of proteins lacking homology to any known microtubule-binding domains. Here, we show that the T. brucei kinetochore protein KKT4 binds directly to microtubules and maintains load-bearing attachments to both growing and shortening microtubule tips. The protein localizes both to kinetochores and to spindle microtubules in vivo, and its depletion causes defects in chromosome segregation. We define a microtubule-binding domain within KKT4 and identify several charged residues important for its microtubule-binding activity. Thus, despite its lack of significant similarity to other known microtubule-binding proteins, KKT4 has key functions required for driving chromosome segregation. We propose that it represents a primary element of the kinetochore–microtubule interface in kinetoplastids.