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 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 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.     

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.     

Microtubule Severing by Katanin p60 AAA+ ATPase Requires the C-terminal Acidic Tails of Both α- and β-Tubulins and Basic Amino Acid Residues in the AAA+ Ring Pore

The microtubule (MT) network is highly dynamic and undergoes dramatic reorganizations during the cell cycle. Dimers of α- and β-tubulins rapidly polymerize to and depolymerize from the end of MT fibrils in an intrinsic GTP-dependent manner. MT severing by ATP-driven enzymes such as katanin and spastin contributes significantly to microtubule dynamics, and it has been shown that katanin p60, a AAA+ family protein, has ATPase and MT-severing activities. The mechanism of MT severing by katanin p60 is poorly understood, and the residues in katanin p60 and tubulins important for severing activity were therefore explored in this study. MT-severing activity, but not ATPase activity, was inhibited by mutations of the conserved aromatic residue and the flanking basic residues in the pore region of the katanin p60 hexameric ring. When the acidic residue-rich C-terminal unstructured segment of either α- or β-tubulin was removed, polymerized MTs were resistant to katanin p60 treatment. Interactions between katanin p60 and the mutant MTs, on the other hand, were unaffected. Taken together, these findings led us to propose that the interactions between the positively charged residues of katanin p60 and the acidic tails of both tubulins are essential for efficient severing of MTs.     

An essential role for katanin p80 and microtubule severing in male gamete production

Katanin is an evolutionarily conserved microtubule-severing complex implicated in multiple aspects of microtubule dynamics. Katanin consists of a p60 severing enzyme and a p80 regulatory subunit. The p80 subunit is thought to regulate complex targeting and severing activity, but its precise role remains elusive. In lower-order species, the katanin complex has been shown to modulate mitotic and female meiotic spindle dynamics and flagella development. The in vivo function of katanin p80 in mammals is unknown. Here we show that katanin p80 is essential for male fertility. Specifically, through an analysis of a mouse loss-of-function allele (the Taily line), we demonstrate that katanin p80, most likely in association with p60, has an essential role in male meiotic spindle assembly and dissolution and the removal of midbody microtubules and, thus, cytokinesis. Katanin p80 also controls the formation, function, and dissolution of a microtubule structure intimately involved in defining sperm head shaping and sperm tail formation, the manchette, and plays a role in the formation of axoneme microtubules. Perturbed katanin p80 function, as evidenced in the Taily mouse, results in male sterility characterized by decreased sperm production, sperm with abnormal head shape, and a virtual absence of progressive motility. Collectively these data demonstrate that katanin p80 serves an essential and evolutionarily conserved role in several aspects of male germ cell development.     

The C-terminal α-helix of SPAS-1, a Caenorhabditis elegans spastin homologue, is crucial for microtubule severing

Spastin belongs to the meiotic subfamily, together with Vps4/SKD1, fidgetin and katanin, of AAA (ATPases associated with diverse cellular activities) proteins, and functions in microtubule severing. Interestingly, all members of this subgroup specifically contain an additional α-helix at the very C-terminal end. To understand the function of the C-terminal α-helix, we characterised its deletion mutants of SPAS-1, a Caenorhabditis elegans spastin homologue, in vitro and in vivo. We found that the C-terminal α-helix plays essential roles in ATP binding, ATP hydrolysing and microtubule severing activities. It is likely that the C-terminal α-helix is required for cellular functions of members of meiotic subgroup of AAA proteins, since the C-terminal α-helix of Vps4 is also important for assembly, ATPase activity and in vivo function mediated by ESCRT-III complexes.     

WDR62 regulates spindle dynamics as an adaptor protein between TPX2/Aurora A and katanin

WDR62 is a microcephaly-related, microtubule (MT)-associated protein (MAP) that localizes to the spindle pole and regulates spindle organization, but the underlying mechanisms remain elusive. Here, we show that WDR62 regulates spindle dynamics by recruiting katanin to the spindle pole and further reveal a TPX2–Aurora A–WDR62–katanin axis in cells. By combining cellular and in vitro experiments, we demonstrate that WDR62 shows preference for curved segments of dynamic GDP-MTs, as well as GMPCPP- and paclitaxel-stabilized MTs, suggesting that it recognizes extended MT lattice. Consistent with this property, WDR62 alone is inefficient in recruiting katanin to GDP-MTs, while WDR62 complexed with TPX2/Aurora A can potently promote katanin-mediated severing of GDP-MTs in vitro. In addition, the MT-binding affinity of WDR62 is autoinhibited through JNK phosphorylation-induced intramolecular interaction. We propose that WDR62 is an atypical MAP and functions as an adaptor protein between its recruiting factor TPX2/Aurora A and the effector katanin to orchestrate the regulation of spindle dynamics.     

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.     

Microtubules cut and run

There is broad agreement that cells reconfigure their microtubules through rapid bouts of assembly and disassembly, as described by the mechanism known as dynamic instability. However, many cell types have complex patterns of microtubule organization that are not entirely explicable by dynamic instability. There is growing evidence that microtubules can be moved into new patterns of organization by forces generated by molecular motor proteins. Studies on several cell types support a model called 'cut and run' in which long microtubules are stationary, but relatively short microtubules are mobile. In this model, cells mobilize their microtubules by severing them into short pieces, using enzymes such as katanin and spastin that break the lattice of the microtubule polymer. After being reorganized, the short microtubules can once again elongate and lose their mobility. Microtubule severing is also crucial for a variation of 'cut and run' in which the severed microtubules are reorganized by means of treadmilling.     

A Role for Katanin-mediated Axonemal Severing during Chlamydomonas Deflagellation

Deflagellation of Chlamydomonas reinhardtii, and other flagellated and ciliated cells, is a highly specific process that involves signal-induced severing of the outer doublet microtubules at a precise site in the transition region between the axoneme and basal body. Although the machinery of deflagellation is activated by Ca²⁺, the mechanism of microtubule severing is unknown. Severing of singlet microtubules has been observed in vitro to be catalyzed by katanin, a heterodimeric adenosine triphosphatase that can remove tubulin subunits from the walls of stable microtubules. We found that purified katanin induced an ATP-dependent severing of the Chlamydomonas axoneme. Using Western blot analysis and indirect immunofluorescence, we demonstrate that Chlamydomonas expresses a protein that is recognized by an anti-human katanin antibody and that this protein is localized, at least in part, to the basal body complex. Using an in vitro severing assay, we show that the protein(s) responsible for Ca²⁺-activated outer doublet severing purify with the flagellar-basal body complex. Furthermore, deflagellation of purified flagellar-basal body complexes is significantly blocked by the anti-katanin antibody. Taken together, these data suggest that a katanin-like mechanism may mediate the severing of the outer doublet microtubules during Chlamydomonas deflagellation.     

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.     

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.     

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.     

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.     

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.     

The Caenorhabditis elegans microtubule-severing complex MEI-1/MEI-2 katanin interacts differently with two superficially redundant beta-tubulin isotypes

The microtubule-severing protein complex katanin is required for a variety of important microtubule-base morphological changes in both animals and plants. Caenorhabditis elegans katanin is encoded by the mei-1 and mei-2 genes and is required for oocyte meiotic spindle formation and must be inactivated before the first mitotic cleavage. We identified a mutation, sb26, in the tbb-2 beta-tubulin gene that partially inhibits MEI-1/MEI-2 activity: sb26 rescues lethality caused by ectopic MEI-1/MEI-2 expression during mitosis, and sb26 increases meiotic defects in a genetic background where MEI-1/MEI-2 activity is lower than normal. sb26 does not interfere with MEI-1/MEI-2 microtubule localization, suggesting that this mutation likely interferes with severing. Tubulin deletion alleles and RNA-mediated interference revealed that TBB-2 and the other germline enriched beta-tubulin isotype, TBB-1, are redundant for embryonic viability. However, limiting MEI-1/MEI-2 activity in these experiments revealed that MEI-1/MEI-2 preferentially interacts with TBB-2-containing microtubules. Our results demonstrate that these two superficially redundant beta-tubulin isotypes have functionally distinct roles in vivo.     

PAR‐1 promotes microtubule breakdown during dendrite pruning in Drosophila

Pruning of unspecific neurites is an important mechanism during neuronal morphogenesis. Drosophila sensory neurons prune their dendrites during metamorphosis. Pruning dendrites are severed in their proximal regions. Prior to severing, dendritic microtubules undergo local disassembly, and dendrites thin extensively through local endocytosis. Microtubule disassembly requires a katanin homologue, but the signals initiating microtubule breakdown are not known. Here, we show that the kinase PAR‐1 is required for pruning and dendritic microtubule breakdown. Our data show that neurons lacking PAR‐1 fail to break down dendritic microtubules, and PAR‐1 is required for an increase in neuronal microtubule dynamics at the onset of metamorphosis. Mammalian PAR‐1 is a known Tau kinase, and genetic interactions suggest that PAR‐1 promotes microtubule breakdown largely via inhibition of Tau also in Drosophila. Finally, PAR‐1 is also required for dendritic thinning, suggesting that microtubule breakdown might precede ensuing plasma membrane alterations. Our results shed light on the signaling cascades and epistatic relationships involved in neurite destabilization during dendrite pruning.