Katanin Is Responsible for the M-Phase Microtubule-severing Activity in Xenopus Eggs

Microtubules are dynamic structures whose proper rearrangement during the cell cycle is essential for the positioning of membranes during interphase and for chromosome segregation during mitosis. The previous discovery of a cyclin B/cdc2-activated microtubule-severing activity in M-phase Xenopus egg extracts suggested that a microtubule-severing protein might play an important role in cell cycle-dependent changes in microtubule dynamics and organization. However, the isolation of three different microtubule-severing proteins, p56, EF1α, and katanin, has only confused the issue because none of these proteins is directly activated by cyclin B/cdc2. Here we use immunodepletion with antibodies specific for a vertebrate katanin homologue to demonstrate that katanin is responsible for the majority of M-phase severing activity in Xenopus eggs. This result suggests that katanin is responsible for changes in microtubules occurring at mitosis. Immunofluorescence analysis demonstrated that katanin is concentrated at a microtubule-dependent structure at mitotic spindle poles in Xenopus A6 cells and in human fibroblasts, suggesting a specific role in microtubule disassembly at spindle poles. Surprisingly, katanin was also found in adult mouse brain, indicating that katanin may have other functions distinct from its mitotic role.     

Cytokinesis in bloodstream stage Trypanosoma brucei requires a family of katanins and spastin

Microtubule severing enzymes regulate microtubule dynamics in a wide range of organisms and are implicated in important cell cycle processes such as mitotic spindle assembly and disassembly, chromosome movement and cytokinesis. Here we explore the function of several microtubule severing enzyme homologues, the katanins (KAT80, KAT60a, KAT60b and KAT60c), spastin (SPA) and fidgetin (FID) in the bloodstream stage of the African trypanosome parasite, Trypanosoma brucei. The trypanosome cytoskeleton is microtubule based and remains assembled throughout the cell cycle, necessitating its remodelling during cytokinesis. Using RNA interference to deplete individual proteins, we show that the trypanosome katanin and spastin homologues are non-redundant and essential for bloodstream form proliferation. Further, cell cycle analysis revealed that these proteins play essential but discrete roles in cytokinesis. The KAT60 proteins each appear to be important during the early stages of cytokinesis, while downregulation of KAT80 specifically inhibited furrow ingression and SPA depletion prevented completion of abscission. In contrast, RNA interference of FID did not result in any discernible effects. We propose that the stable microtubule cytoskeleton of T. brucei necessitates the coordinated action of a family of katanins and spastin to bring about the cytoskeletal remodelling necessary to complete cell division.     

The Drosophila homologue of the hereditary spastic paraplegia protein, spastin, severs and disassembles microtubules

Hereditary spastic paraplegias (HSPs), a group of neurodegenerative disorders characterized by lower-extremity spasticity and weakness, are most commonly caused by mutations in the spastin gene, which encodes a AAA+ ATPase related to the microtubule-severing protein katanin. A Drosophila homolog of spastin (D-spastin) was identified recently, and D-spastin RNAi-treated or genetic null flies show neurological defects, and protein overexpression decreases the density of cellular microtubules. Elucidating spastin's function and disease mechanism will require a more detailed understanding of its structure and biochemical mechanism. Here, we have investigated the effects of D-spastin, individual D-spastin domains, and D-spastin proteins bearing disease mutations on microtubules in cellular and in vitro assays. We show that D-spastin, like katanin, displays ATPase activity and uses energy from ATP hydrolysis to sever and disassemble microtubules; disease mutations abolish or partially interfere with these activities.     

The spindle assembly function of Caenorhabditis elegans katanin does not require microtubule-severing activity

Katanin is a heterodimeric microtubule-severing protein that is conserved among eukaryotes. Loss-of-function mutations in the Caenorhabditis elegans katanin catalytic subunit, MEI-1, cause specific defects in female meiotic spindles. To determine the relationship between katanin's microtubule-severing activity and its role in meiotic spindle formation, we analyzed the MEI-1(A338S) mutant. Unlike wild-type MEI-1, which mediated disassembly of microtubule arrays in Xenopus fibroblasts, MEI-1(A338S) had no effect on fibroblast microtubules, indicating a lack of microtubule-severing activity. In C. elegans, MEI-1(A338S) mediated assembly of extremely long bipolar meiotic spindles. In contrast, a nonsense mutation in MEI-1 caused assembly of meiotic spindles without any poles as assayed by localization of the spindle-pole protein, ASPM-1. These results indicated that katanin protein, but not katanin's microtubule-severing activity, is required for assembly of acentriolar meiotic spindle poles. To understand the nonsevering activities of katanin, we characterized the N-terminal domain of the katanin catalytic subunit. The N-terminal domain was necessary and sufficient for binding to the katanin regulatory subunit. The katanin regulatory subunit in turn caused a dramatic change in the microtubule-binding properties of the N-terminal domain of the catalytic subunit. This unique bipartite microtubule-binding structure may mediate the spindle-pole assembly activity of katanin during female meiosis.     

cDNA isolation, characterization, and protein intracellular localization of a katanin-like p60 subunit from Arabidopsis thaliana

Summary Katanin, a heterodimeric protein with ATP-dependent microtubule-severing activity, localizes to the centrosome in animal cells. Widespread occurrence is suspected as several species contain homologs to the katanin p60 subunit. Recently we isolated anArabidopsis thaliana cDNA with significant identity to the p60 subunit of sea urchin katanin. Like p60, the encoded protein is a member of the AAA superfamily of ATPases, containing the Walker ATP binding consensus and the signature AAA minimal consensus sequences within a single larger AAA/CAD amino acid motif. Phylogenetic analysis placed the encoded protein in the AAA subfamily of cytoskeleton-interactive proteins, where it formed a strongly supported clade with 4 other members identified as katanin p60 subunits. The clone was named AtKSSArabidopsis thaliana kataninlike protein small subunit). Western blots, performed using a polyclonal antibody raised against recombinant AtKSS, revealed AtKSS is present in protein extracts of all Arabidopsis organs examined. To evaluate potential interactions between AtKSS and the cytoskeleton, the intracellular localization of AtKSS was correlated with that of tubulin. AtKSS was found in perinuclear regions during interphase, surrounding the spindle poles during mitosis, but was absent from the preprophase band and phragmoplast microtubule arrays. These data support the thesis that AtKSS is an Arabidopsis homolog of the p60 subunit of katanin. Its cell cycle-dependent distribution is consistent with microtubule-severing activity, but additional studies will better define its role.     

Making more microtubules by severing: a common theme of noncentrosomal microtubule arrays?

Two related enzymes, katanin and spastin, use the energy from ATP hydrolysis to sever microtubules. Two new studies (one in this issue; see McNally et al., p. 881) show that microtubule severing by katanin provides a means for increasing microtubule density in meiotic spindles. Interestingly, loss of spastin leads to a sparser microtubule array in axons and synaptic boutons. Together, these studies hint at a wider role for microtubule-severing enzymes in the formation and organization of noncentrosomal microtubule arrays by generating new seeds for microtubule growth.     

Katanin-mediated microtubule severing can be regulated by multiple mechanisms

Microtubules are essential for a wide range of cellular processes that vary between cell types. Katanin is a microtubule-severing protein that carries out an essential role in meiotic spindles in Caenorhabditis elegans and a non-essential role in mitotic spindles of vertebrates. In contrast to these M-phase associated roles, katanin is also essential for post-mitotic differentiation events in vertebrate neurons and in Arabidopsis. This diversity of function suggests that katanin's activity might be regulated by multiple mechanisms. Because katanin is active in M-phase Xenopus extracts but not in interphase extracts, we assayed for regulators of katanin's activity in these extracts. The microtubule-severing activity of purified katanin was inhibited by interphase Xenopus extracts. Fractionation revealed that this inhibition was due to at least 4 separable components, one of which contains the MAP4 homolog, XMAP230. Inhibition of katanin-mediated microtubule-disassembly activity by the XMAP230-containing fraction was reversible by cyclinB/cdk1, suggesting one possible mechanism for the increased severing activity observed in M-phase Xenopus extracts. In a previous study, spindle pole association by katanin was essential for its activity during mitosis suggesting that katanin's activity might also be regulated by co-localization with an activator. The polo-like kinase, Plx1, co-localized with katanin at spindle poles in vivo and purified Plx1 increased the microtubule-severing activity of katanin in vitro. These in vitro experiments illustrate the potential complexity of the regulation of katanin's activity in vivo and may explain how katanin can carry out widely different functions in different cell types.     

The microtubule-severing proteins spastin and katanin participate differently in the formation of axonal branches

Neurons express two different microtubule-severing proteins, namely P60-katanin and spastin. Here, we performed studies on cultured neurons to ascertain whether these two proteins participate differently in axonal branch formation. P60-katanin is more highly expressed in the neuron, but spastin is more concentrated at sites of branch formation. Overexpression of spastin dramatically enhances the formation of branches, whereas overexpression of P60-katanin does not. The excess spastin results in large numbers of short microtubules, whereas the excess P60-katanin results in short microtubules intermingled with longer microtubules. We hypothesized that these different microtubule-severing patterns may be due to the presence of molecules such as tau on the microtubules that more strongly shield them from being severed by P60-katanin than by spastin. Consistent with this hypothesis, we found that axons depleted of tau show a greater propensity to branch, and that this is true whether or not the axons are also depleted of spastin. We propose that there are two modes by which microtubule severing is orchestrated during axonal branch formation, one based on the local concentration of spastin at branch sites and the other based on local detachment from microtubules of molecules such as tau that regulate the severing properties of P60-katanin.     

Severing of stable microtubules by a mitotically activated protein in Xenopus egg extracts.

Eukaryotic cells disassemble and reorganize their cytoskeleton during the cell cycle and in response to environmental cues. Disassembly of the actin cytoskeleton is aided by proteins that sever filamentous actin, but microtubule-severing proteins thus far have not been identified. Here, we describe an activity in extracts from Xenopus eggs that rapidly severs stable microtubules along their length. Severing is elicited by a protein(s) whose activity is greatly stimulated during mitosis through a posttranslational mechanism. The microtubule-severing factor may be involved in disassembling the interphase microtubule network prior to constructing the mitotic spindle.     

Samurai sword sets spindle size

Although the parts list is nearly complete for many cellular structures, mechanisms that control their size remain poorly understood. Loughlin and colleagues now show that phosphorylation of a single residue of katanin, a microtubule-severing protein, largely accounts for the difference in spindle length between two closely related frogs.     

Katanin disrupts the microtubule lattice and increases polymer number in C. elegans meiosis

Katanin is a heterodimer that exhibits ATP-dependent microtubule-severing activity in vitro. In Xenopus egg extracts, katanin activity correlates with the addition of cyclin B/cdc2, suggesting a role for microtubule severing in the disassembly of long interphase microtubules as the cell prepares for mitosis. However, studies from plant cells, cultured neurons, and nematode embryos suggest that katanin could be required for the organization or postnucleation processing of microtubules, rather than the dissolution of microtubule structures. Here we reexamine katanin's role by studying acentrosomal female meiotic spindles in C. elegans embryos. In mutant embryos lacking katanin, microtubules form around meiotic chromatin but do not organize into bipolar spindles. By using electron tomography, we found that katanin converts long microtubule polymers into shorter microtubule fragments near meiotic chromatin. We further show that turning on katanin during mitosis also creates a large pool of short microtubules near the centrosome. Furthermore, the identification of katanin-dependent microtubule lattice defects supports a mechanism involving an initial perforation of the protofilament wall. Taken together, our data suggest that katanin is used during meiotic spindle assembly to increase polymer number from a relatively inefficient chromatin-based microtubule nucleation pathway.     

Mutations of a redundant alpha-tubulin gene affect Caenorhabditis elegans early embryonic cleavage via MEI-1/katanin-dependent and -independent pathways

The C. elegans zygote supports both meiosis and mitosis within a common cytoplasm. The meiotic spindle is small and is located anteriorly, whereas the first mitotic spindle fills the zygote. The C. elegans microtubule-severing complex, katanin, is encoded by the mei-1 and mei-2 genes and is solely required for oocyte meiotic spindle formation; ectopic mitotic katanin activity disrupts mitotic spindles. Here we characterize two mutations that rescue the lethality caused by ectopic MEI-1/MEI-2. Both mutations are gain-of-function alleles of tba-2 alpha-tubulin. These tba-2 alleles do not prevent MEI-1/MEI-2 microtubule localization but do interfere with its activity. TBA-1 and TBA-2 are redundant for viability, but when katanin activity is limiting, TBA-2 is preferred over TBA-1 by katanin. This is similar to what we previously reported for the beta-tubulins. Removing both preferred alpha- and beta-isoforms results in normal development, suggesting that the katanin isoform preferences are not absolute. We conclude that while the C. elegans embryo expresses redundant alpha- and beta-tubulin isoforms, they nevertheless have subtle functional specializations. Finally, we identified a dominant tba-2 allele that disrupts both meiotic and mitotic spindle formation independently of MEI-1/MEI-2 activity. Genetic studies suggest that this tba-2 mutation has a "poisonous" effect on microtubule function.     

Creative destruction of the microtubule array

Comment on: Mukherjee S, et al. Cell Cycle 2012; 11:2359-66.Typical cells contain a dense array of microtubules that serves as a structural backbone and also provides a substrate against which molecular motor proteins generate force. Cells transitioning through the cell cycle or undergoing significant morphological changes must be able to tear apart the microtubule array and reconstruct it into new configurations, either partially or completely. The microtubule field was revolutionized in the 1980s with the introduction of the dynamic instability model,¹ now broadly recognized as a fundamental mechanism by which microtubule populations are reconfigured.² Dynamic instability involves the catastrophic disassembly of microtubules, generally from their plus ends, as well as the rapid reassembly of microtubules and selective stabilization of particular ones. Microtubules can be stabilized along their length by binding to various proteins and can be attached at their minus ends to structures such as the centrosome and “captured” at their plus ends by proteins in the cell’s cortex.² Given the contribution of these stabilizing and anchoring factors, additional mechanisms beyond dynamic instability are required to tear down previous microtubule structures so that new ones can be constructed. Borrowing from the field of economics, we refer to this as creative destruction.Various proteins such as stathmin³ and kinesin-13⁴ contribute to creative destruction by promoting loss of tubulin subunits from the ends of the microtubules. We find especially interesting a category of AAA enzymes called microtubule-severing proteins that use the energy of ATP hydrolysis to yank at tubulin subunits within the microtubule, thereby causing the lattice to break.⁵ If this occurs along the length of the microtubule, the microtubule will be severed into pieces. If this occurs at either of the two ends of the microtubule, the microtubule will lose subunits from that end. The first discovered and best-studied microtubule-severing proteins are katanin and spastin.Thanks to David Sharp and his colleagues at Albert Einstein College of Medicine, as well as other workers in the field, we now know that cells express at least five other AAA proteins with potential microtubule-severing properties, on the basis of sequence similarity to katanin and spastin in the AAA region.⁵ Two of these, called katanin-like-1 and katanin-like-2, are very similar to katanin. The three others are similar to one another, collectively termed fidgetins (fidgetin, fidgetin-like-1 and fidgetin-like-2). One possibility is that all seven of the microtubule-severing proteins are regulated similarly and are functionally redundant with one another. A more compelling possibility is that, while there is some functional redundancy, there is also a division of labor, with each severing protein displaying distinct properties and carrying out its own duties. Thus far, Sharp’s studies on mitosis support the latter scenario, with katanin, fidgetin and spastin having characteristic distributions within the spindle, resulting in unique phenotypes when depleted.⁶In a new article, Sharp’s group has confirmed that fidgetin has microtubule-severing properties. Interestingly, fidgetin depolymerizes microtubules preferentially from the minus end.⁷ In addition, the new work shows that in human U2OS cells, fidgetin targets to the centrosome, where most minus ends of microtubules are clustered, suggesting a scenario by which fidgetin suppresses microtubule growth from the centrosome as well as attachment to it. Consistent with this scenario, the authors show that experimental depletion of fidgetin reduces that speed of poleward tubulin flux as well as the speed of anaphase A chromatid-to-pole motion and also results in an increase in both the number and length of astral microtubules. Notably, this contrasts with katanin, which favors the plus ends of microtubules, for example, at the chromosome during cell division⁶ and at the leading edge of motile cells.⁸The authors close their article by pointing out that microtubule-severing is important beyond mitosis, for example, in the restructuring of the microtubule array in neurons and migrating cells, and we would point to plants as well.⁹ We previously described a mechanism called “cut and run,” wherein the severing of microtubules is important for motility within the microtubule array, as short microtubules are more mobile than long ones.⁹ Now, inspired by the work of Sharp and colleagues, we envision “creative destruction” as another way of understanding the crucial roles played by a diversity of microtubule-severing proteins in cells.     

Basic Fibroblast Growth Factor Elicits Formation of Interstitial Axonal Branches via Enhanced Severing of Microtubules

The formation of interstitial axonal branches involves the severing of microtubules at sites where new branches form. Here we wished to ascertain whether basic fibroblast growth factor (bFGF) enhances axonal branching through alterations in proteins involved in the severing of microtubules. We found that treatment of cultured hippocampal neurons with bFGF heightens expression of both katanin and spastin, which are proteins that sever microtubules in the axon. In addition, treatment with bFGF enhances phosphorylation of tau at sites expected to cause it to dissociate from microtubules. This is important because tau regulates the access of katanin to the microtubule. In live-cell imaging experiments, axons of neurons treated with bFGF displayed greater numbers of dynamic free ends of microtubules, as well as greater numbers of short mobile microtubules. Entirely similar enhancement of axonal branching, short microtubule transport, and frequency of microtubule ends was observed when spastin was overexpressed in the neurons. Depletion of either katanin or spastin with siRNA diminished but did not eliminate the enhancement in branching elicited by bFGF. Collectively, these results indicate that bFGF enhances axonal branch formation by augmenting the severing of microtubules through both a spastin-based mode and a katanin-based mode.     

An essential role for katanin in severing microtubules in the neuron

Several lines of evidence suggest that microtubules are nucleated at the neuronal centrosome, and then released for transport into axons and dendrites. Here we sought to determine whether the microtubule-severing protein known as katanin mediates microtubule release from the neuronal centrosome. Immunomicroscopic analyses on cultured sympathetic neurons show that katanin is present at the centrosome, but is also widely distributed throughout the neuron. Microinjection of an antibody that inactivates katanin results in a dramatic accumulation of microtubules at the centrosome, indicating that katanin is indeed required for microtubule release from the centrosome. However, the antibody also causes an inhibition of axon outgrowth that is more immediate than expected on this basis alone. It may be that katanin severs microtubules throughout the cell body to keep them sufficiently short to be efficiently transported into developing processes. Consistent with this idea, there were significantly fewer free ends of microtubules in the cell bodies of neurons that had been injected with the katanin antibody compared with controls. These results indicate that microtubule-severing by katanin is essential for releasing microtubules from the neuronal centrosome, and also for regulating the length of the microtubules after their release.     

WDR62 localizes katanin at spindle poles to ensure synchronous chromosome segregation

Mutations in the WDR62 gene cause primary microcephaly, a pathological condition often associated with defective cell division that results in severe brain developmental defects. The precise function and localization of WDR62 within the mitotic spindle is, however, still under debate, as it has been proposed to act either at centrosomes or on the mitotic spindle. Here we explored the cellular functions of WDR62 in human epithelial cell lines using both short-term siRNA protein depletions and long-term CRISPR/Cas9 gene knockouts. We demonstrate that WDR62 localizes at spindle poles, promoting the recruitment of the microtubule-severing enzyme katanin. Depletion or loss of WDR62 stabilizes spindle microtubules due to insufficient microtubule minus-end depolymerization but does not affect plus-end microtubule dynamics. During chromosome segregation, WDR62 and katanin promote efficient poleward microtubule flux and favor the synchronicity of poleward movements in anaphase to prevent lagging chromosomes. We speculate that these lagging chromosomes might be linked to developmental defects in primary microcephaly.     

Katanin maintains meiotic metaphase chromosome alignment and spindle structure in vivo and has multiple effects on microtubules in vitro

Assembly of Caenorhabditis elegans female meiotic spindles requires both MEI-1 and MEI-2 subunits of the microtubule-severing ATPase katanin. Strong loss-of-function mutants assemble apolar intersecting microtubule arrays, whereas weaker mutants assemble bipolar meiotic spindles that are longer than wild type. To determine whether katanin is also required for spindle maintenance, we monitored metaphase I spindles after a fast-acting mei-1(ts) mutant was shifted to a nonpermissive temperature. Within 4 min of temperature shift, bivalents moved off the metaphase plate, and microtubule bundles within the spindle lengthened and developed a high degree of curvature. Spindles eventually lost bipolar structure. Immunofluorescence of embryos fixed at increasing temperature indicated that MEI-1 was lost from spindle microtubules before loss of ASPM-1, indicating that MEI-1 and ASPM-1 act independently at spindle poles. We quantified the microtubule-severing activity of purified MEI-1/MEI-2 complexes corresponding to six different point mutations and found a linear relationship between microtubule disassembly rate and meiotic spindle length. Previous work showed that katanin is required for severing at points where two microtubules intersect in vivo. We show that purified MEI-1/MEI-2 complexes preferentially sever at intersections between two microtubules and directly bundle microtubules in vitro. These activities could promote parallel/antiparallel microtubule organization in meiotic spindles.     

Spastin's Microtubule-Binding Properties and Comparison to Katanin

Spastin and katanin are ring-shaped hexameric AAA ATPases that sever microtubules, and thus crucially depend on a physical interaction with microtubules. For the first time, we report here the microtubule binding properties of spastin at the single-molecule level, and compare them to katanin. Microscopic fluorescence assays showed that human spastin bound to microtubules by ionic interactions, and diffused along microtubules with a diffusion coefficient comparable to katanin. The microscopic measurement of landing and dissociation rates demonstrated the ionic character of the interaction, which could be mapped to a patch of three lysine residues outside of the catalytic domain of human spastin. This motif is not conserved in Drosophila spastin or katanin, which also bound by non-catalytic parts of the protein. The binding affinities of spastin and katanin were nucleotide-sensitive, with the lowest affinities under ADP,, the highest under ATP-γS conditions. These changes correlated with the formation of higher oligomeric states, as shown in biochemical experiments and electron microscopic images. Vice versa, the artificial dimerization of human spastin by addition of a coiled coil led to a constitutively active enzyme. These observations suggest that dimer formation is a crucial step in the formation of the active complex, and thus the severing process by spastin.     

Katanin p60 Contributes to Microtubule Instability around the Midbody and Facilitates Cytokinesis in Rat Cells

The completion of cytokinesis is crucial for mitotic cell division. Cleavage furrow ingression is followed by the breaking and resealing of the intercellular bridge, but the detailed mechanism underlying this phenomenon remains unknown. Katanin is a microtubule-severing protein comprised of an AAA ATPase subunit and an accessory subunit designated as p60 and p80, respectively. Localization of katanin p60 was observed at the midzone to midbody from anaphase to cytokinesis in rat cells, and showed a ring-shaped distribution in the gap between the inside of the contractile ring and the central spindle bundle in telophase. Katanin p60 did not bind with p80 at the midzone or midbody, and localization was shown to be dependent on microtubules. At the central spindle and the midbody, no microtubule growth plus termini were seen with katanin p60, and microtubule density was inversely correlated with katanin p60 density in the region of katanin p60 localization that seemed to lead to microtubule destabilization at the midbody. Inhibition of katanin p60 resulted in incomplete cytokinesis by regression and thus caused the appearance of binucleate cells. These results suggest that katanin p60 contributes to microtubule instability at the midzone and midbody and facilitates cytokinesis in rat cells.