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.     

The Katanin Microtubule Severing Protein in Plants

Katanin is a heterodimeric microtubule （MT） severing protein that uses energy from ATP hydrolysis to generate internal breaks along MTs. Katanin p60, one of the two subunits, possesses ATPase and MT-binding/severing activities, and the p 80 subunit is responsible for targeting of katanin to certain subcellular locations. In animals, katanin plays an important role in the release of MTs from their nucleation sites in the centrosome. It is also involved in severing MTs into smaller fragments which can serve as templates for further polymerization to increase MT number during meiotic and mitotic spindle assembly. Katanin homologs are present in a wide variety of plant species. The Arabidopsis katanin homolog has been shown to possess ATP-dependent MT severing activity in vitro and exhibit a punctate localization pattern at the cell cortex and the perinuclear region. Disruption of katanin functions by genetic mutations causes a delay in the disappearance of the perinuclear MT array and results in an aberrant organization of cortical MTs in elongating cells. Consequently, katanin mutations lead to defects in cell elongation, cellulose microfibril deposition, and hormonal responses. Studies of katanin in plants provide new insights into our understanding of its roles in cellular functions.     

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.     

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.     

The Non-Catalytic Domains of Drosophila Katanin Regulate Its Abundance and Microtubule-Disassembly Activity

Microtubule severing is a biochemical reaction that generates an internal break in a microtubule and regulation of microtubule severing is critical for cellular processes such as ciliogenesis, morphogenesis, and meiosis and mitosis. Katanin is a conserved heterodimeric ATPase that severs and disassembles microtubules, but the molecular determinants for regulation of microtubule severing by katanin remain poorly defined. Here we show that the non-catalytic domains of Drosophila katanin regulate its abundance and activity in living cells. Our data indicate that the microtubule-interacting and trafficking (MIT) domain and adjacent linker region of the Drosophila katanin catalytic subunit Kat60 cooperate to regulate microtubule severing in two distinct ways. First, the MIT domain and linker region of Kat60 decrease its abundance by enhancing its proteasome-dependent degradation. The Drosophila katanin regulatory subunit Kat80, which is required to stabilize Kat60 in cells, conversely reduces the proteasome-dependent degradation of Kat60. Second, the MIT domain and linker region of Kat60 augment its microtubule-disassembly activity by enhancing its association with microtubules. On the basis of our data, we propose that the non-catalytic domains of Drosophila katanin serve as the principal sites of integration of regulatory inputs, thereby controlling its ability to sever and disassemble microtubules.     

Katanin controls mitotic and meiotic spindle length

Accurate control of spindle length is a conserved feature of eukaryotic cell division. Lengthening of mitotic spindles contributes to chromosome segregation and cytokinesis during mitosis in animals and fungi. In contrast, spindle shortening may contribute to conservation of egg cytoplasm during female meiosis. Katanin is a microtubule-severing enzyme that is concentrated at mitotic and meiotic spindle poles in animals. We show that inhibition of katanin slows the rate of spindle shortening in nocodazole-treated mammalian fibroblasts and in untreated Caenorhabditis elegans meiotic embryos. Wild-type C. elegans meiotic spindle shortening proceeds through an early katanin-independent phase marked by increasing microtubule density and a second, katanin-dependent phase that occurs after microtubule density stops increasing. In addition, double-mutant analysis indicated that gamma-tubulin-dependent nucleation and microtubule severing may provide redundant mechanisms for increasing microtubule number during the early stages of meiotic spindle assembly.     

Microtubule severing by the katanin complex is activated by PPFR-1–dependent MEI-1 dephosphorylation

Katanin is an evolutionarily conserved microtubule (MT)-severing complex implicated in multiple aspects of MT dynamics. In Caenorhabditis elegans, the katanin homologue MEI-1 is required for meiosis, but must be inactivated before mitosis. Here we show that PPFR-1, a regulatory subunit of a trimeric protein phosphatase 4 complex, enhanced katanin MT-severing activity during C. elegans meiosis. Loss of ppfr-1, similarly to the inactivation of MT severing, caused a specific defect in meiosis II spindle disassembly. We show that a fraction of PPFR-1 was degraded after meiosis, contributing to katanin inactivation. PPFR-1 interacted with MEL-26, the substrate recognition subunit of the CUL-3 RING E3 ligase (CRL3^MEL-26), which also targeted MEI-1 for post-meiotic degradation. Reversible protein phosphorylation of MEI-1 may ensure temporal activation of the katanin complex during meiosis, whereas CRL3^MEL-26-mediated degradation of both MEI-1 and its activator PPFR-1 ensure efficient katanin inactivation in the transition to mitosis.     

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 contributes to interspecies spindle length scaling in Xenopus

Bipolar spindles must separate chromosomes by the appropriate distance during cell division, but mechanisms determining spindle length are poorly understood. Based on a 2D model of meiotic spindle assembly, we predicted that higher localized microtubule (MT) depolymerization rates could generate the shorter spindles observed in egg extracts of X. tropicalis compared to X. laevis. We found that katanin-dependent MT severing was increased in X. tropicalis, which, unlike X. laevis, lacks an inhibitory phosphorylation site in the katanin p60 catalytic subunit. Katanin inhibition lengthened spindles in both species. In X. tropicalis, k-fiber MT bundles that connect to chromosomes at their kinetochores extended through spindle poles, disrupting them. In both X. tropicalis extracts and the spindle simulation, a balance between k-fiber number and MT depolymerization is required to maintain spindle morphology. Thus, mechanisms have evolved in different species to scale spindle size and coordinate regulation of multiple MT populations in order to generate a robust steady-state structure.     

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.     

Katanin regulates dynamics of microtubules and biogenesis of motile cilia

The in vivo significance of microtubule severing and the mechanisms governing its spatial regulation are not well understood. In Tetrahymena, a cell type with elaborate microtubule arrays, we engineered null mutations in subunits of the microtubule-severing complex, katanin. We show that katanin activity is essential. The net effect of katanin on the polymer mass depends on the microtubule type and location. Although katanin reduces the polymer mass and destabilizes the internal network of microtubules, its activity increases the mass of ciliary microtubules. We also show that katanin reduces the levels of several types of post-translational modifications on tubulin of internal and cortical microtubules. Furthermore, katanin deficiencies phenocopy a mutation of beta-tubulin that prevents deposition of polymodifications (glutamylation and glycylation) on microtubules. We propose that katanin preferentially severs older, post-translationally modified segments of microtubules.     

Arabidopsis katanin binds microtubules using a multimeric microtubule-binding domain.

Katanin is a heterodimeric protein that mediates ATP-dependent destabilization of microtubules in animal cells. In plants, the catalytic subunit of Arabidopsis thaliana katanin (AtKSS, Arabidopsis thaliana Katanin Small Subunit) has been identified and its microtubule-severing activity has been demonstrated in vitro. In vivo, plant katanin plays a role in the organization of cortical microtubules, but the way it achieves this function is unknown. To go further in our understanding of the mechanisms by which katanin severs microtubules, we analyzed the functional domains of Arabidopsis katanin. We characterized the microtubule-binding domain of katanin both in vitro and in vivo. It corresponds to a poorly conserved sequence between plant and animal katanins that is located in the N-terminus of the protein. This domain interacts with cortical microtubules in vivo and has a low affinity for microtubules in vitro. We also observed that katanin microtubule-binding domain oligomerizes into trimers. These results show that, besides being involved in the interaction of katanin with microtubules, the microtubule-binding domain may also participate in the oligomerization of katanin. At the structural level, we observed that AtKSS forms ring-shaped oligomers.     

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.     

N-Terminal Phosphorylation of p60 Katanin Directly Regulates Microtubule Severing

Proteins of the AAA (ATPases associated with various cellular activities) family often have complex modes of regulation due to their central position in important cellular processes. p60 katanin, an AAA protein that severs and depolymerizes microtubules, is subject to multiple modes of regulation including a phosphorylation in the N-terminal domain involved in mitotic control of severing. Phosphorylation decreases severing activity in Xenopus egg extracts and is involved in controlling spindle length. Here, we show that the evolutionarily divergent N-terminal domains of p60 have maintained hotspots of mitotic kinase regulation. By reconstituting in vitro severing reactions, we show that phosphomimetic modification at amino acid position 131 in Xenopus laevis p60 decreases severing and microtubule-stimulated ATPase activity without affecting the binding affinity of p60 for microtubules. At high concentrations of the phosphomimetic mutant p60, wild-type levels of activity could be observed, indicating a more switch-like threshold of activity that is controlled by regulating oligomerization on the microtubule. This provides a cellular mechanism for high local concentrations of p60, like those found on spindle poles, to maintain severing activity while most of the protein is inhibited. Overall, we have shown that the modular domain architecture of AAA proteins allows for precise control of cellular activities with simple modifications.     

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.     

KL1 is a novel microtubule severing enzyme that regulates mitotic spindle architecture

Katanin is a microtubule severing enzyme with demonstrated roles in a variety of cellular activities including mitosis. Here we identify the closely related, but relatively uncharacterized human protein, Katanin-like 1 (KL1), as a novel mitotic regulator. Over expression of KL1 in tissue culture cells results in the complete disassembly of cellular microtubules strongly suggesting that it is an active microtubule severing protein. During mitosis, the localization of KL1 is restricted to spindle poles and is notably absent from centrosomes. This is in clear contrast to conventional Katanin whose localization extends from centrosomes onto poles. Consistent with its localization, siRNA depletion of KL1 from U2OS cells results in a specific and significant reduction in the density of microtubules at spindle poles and significantly increases spindle length. Depletion of KL1 also alters the distribution of gamma-tubulin at centrosomes/spindle poles. Despite its impact on spindle morphology, we could find no evidence that KL1 influences anaphase chromosome motility. Based on our findings, we propose that KL1-mediated microtubule severing is utilized to generate microtubule seeds within the poles and that loss of this activity alters the normal balance of motor-generated forces that determine spindle length.     

PF15p is the chlamydomonas homologue of the Katanin p80 subunit and is required for assembly of flagellar central microtubules

Numerous studies have indicated that the central apparatus plays a significant role in regulating flagellar motility, yet little is known about how the central pair of microtubules or their associated projections assemble. Several Chlamydomonas mutants are defective in central apparatus assembly. For example, mutant pf15 cells have paralyzed flagella that completely lack the central pair of microtubules. We have cloned the wild-type PF15 gene and confirmed its identity by rescuing the motility and ultrastructural defects in two pf15 alleles, the original pf15a mutant and a mutant generated by insertional mutagenesis. Database searches using the 798-amino-acid polypeptide predicted from the complete coding sequence indicate that the PF15 gene encodes the Chlamydomonas homologue of the katanin p80 subunit. Katanin was originally identified as a heterodimeric protein with a microtubule-severing activity. These results reveal a novel role for the katanin p80 subunit in the assembly and/or stability of the central pair of flagellar microtubules.     

KTN80 confers precision to microtubule severing by specific targeting of katanin complexes in plant cells

The microtubule (MT)‐severing enzyme katanin triggers dynamic reorientation of cortical MT arrays that play crucial functions during plant cell morphogenesis, such as cell elongation, cell wall biosynthesis, and hormonal signaling. MT severing specifically occurs at crossover or branching nucleation sites in living Arabidopsis cells. This differs from the random severing observed along the entire length of single MTs in vitro and strongly suggests that a precise control mechanism must exist in vivo. However, how katanin senses and cleaves at MT crossover and branching nucleation sites in vivo has remained unknown. Here, we show that the katanin p80 subunit KTN80 confers precision to MT severing by specific targeting of the katanin p60 subunit KTN1 to MT cleavage sites and that KTN1 is required for oligomerization of functional KTN80–KTN1 complexes that catalyze severing. Moreover, our findings suggest that the katanin complex in Arabidopsis is composed of a hexamer of KTN1–KTN80 heterodimers that sense MT geometry to confer precise MT severing. Our findings shed light on the precise control mechanism of MT severing in plant cells, which may be relevant for other eukaryotes.     

A Common Substrate Recognition Mode Conserved between Katanin p60 and VPS4 Governs Microtubule Severing and Membrane Skeleton Reorganization

Katanin p60 (kp60), a microtubule-severing enzyme, plays a key role in cytoskeletal reorganization during various cellular events in an ATP-dependent manner. We show that a single domain isolated from the N terminus of mouse katanin p60 (kp60-NTD) binds to tubulin. The solution structure of kp60-NTD was determined by NMR. Although their sequence similarities were as low as 20%, the structure of kp60-NTD revealed a striking similarity to those of the microtubule interacting and trafficking (MIT) domains, which adopt anti-parallel three-stranded helix bundle. In particular, the arrangement of helices 2 and 3 is well conserved between kp60-NTD and the MIT domain from Vps4, which is a homologous protein that promotes disassembly of the endosomal sorting complexes required for transport III membrane skeleton complex. Mutation studies revealed that the positively charged surface formed by helices 2 and 3 binds tubulin. This binding mode resembles the interaction between the MIT domain of Vps4 and Vps2/CHMP1a, a component of endosomal sorting complexes required for transport III. Our results show that both the molecular architecture and the binding modes are conserved between two AAA-ATPases, kp60 and Vps4. A common mechanism is evolutionarily conserved between two distinct cellular events, one that drives microtubule severing and the other involving membrane skeletal reorganization.