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.     

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.     

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.     

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 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.     

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.     

Identification of XMAP215 as a microtubule-destabilizing factor in Xenopus egg extract by biochemical purification

Microtubules (MTs) polymerized with GMPCPP, a slowly hydrolyzable GTP analogue, are stable in buffer but are rapidly depolymerized in Xenopus egg extracts. This depolymerization is independent of three previously identified MT destabilizers (Op18, katanin, and XKCM1/KinI). We purified the factor responsible for this novel depolymerizing activity using biochemical fractionation and a visual activity assay and identified it as XMAP215, previously identified as a prominent MT growth-promoting protein in Xenopus extracts. Consistent with the purification results, we find that XMAP215 is necessary for GMPCPP-MT destabilization in extracts and that recombinant full-length XMAP215 as well as an NH2-terminal fragment have depolymerizing activity in vitro. Stimulation of depolymerization is specific for the MT plus end. These results provide evidence for a robust MT-destabilizing activity intrinsic to this microtubule-associated protein and suggest that destabilization may be part of its essential biochemical functions. We propose that the substrate in our assay, GMPCPP-stabilized MTs, serves as a model for the pause state of MT ends and that the multiple activities of XMAP215 are unified by a mechanism of antagonizing MT pauses.     

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.     

Protein phosphatase 4 catalytic subunit regulates Cdk1 activity and microtubule organization via NDEL1 dephosphorylation

Protein phosphatase 4 catalytic subunit (PP4c) is a PP2A-related protein serine/threonine phosphatase with important functions in a variety of cellular processes, including microtubule (MT) growth/organization, apoptosis, and tumor necrosis factor signaling. In this study, we report that NDEL1 is a substrate of PP4c, and PP4c selectively dephosphorylates NDEL1 at Cdk1 sites. We also demonstrate that PP4c negatively regulates Cdk1 activity at the centrosome. Targeted disruption of PP4c reveals disorganization of MTs and disorganized MT array. Loss of PP4c leads to an unscheduled activation of Cdk1 in interphase, which results in the abnormal phosphorylation of NDEL1. In addition, abnormal NDEL1 phosphorylation facilitates excessive recruitment of katanin p60 to the centrosome, suggesting that MT defects may be attributed to katanin p60 in excess. Inhibition of Cdk1, NDEL1, or katanin p60 rescues the defective MT organization caused by PP4 inhibition. Our work uncovers a unique regulatory mechanism of MT organization by PP4c through its targets Cdk1 and NDEL1 via regulation of katanin p60 distribution.     

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.     

Severing at sites of microtubule crossover contributes to microtubule alignment in cortical arrays

The cortical microtubule (MT) array and its organization is important in defining the growth axes of plant cells. In roots, the MT array exhibits a net-like configuration in the division zone, and a densely-packed transverse alignment in the elongation zone. This transition is essential for anisotropic cell expansion and consequently has been the subject of intense study. Cotyledons exhibit a net-like array in pavement cells and a predominantly aligned array in the petioles, and provide an excellent system for determining the basis of plant MT organization. We show that in both kinds of MT array, growing MTs frequently encounter existing MTs. Although some steep-angled encounters result in catastrophes, the most frequent outcome of these encounters is successful negotiation of the existing MT by the growing MT to form an MT crossover. Surprisingly, the outcome of such encounters is similar in both aligned and net-like arrays. In contrast, aligned arrays exhibit a much higher frequency of MT severing events compared with net-like arrays. Severing events occur almost exclusively at sites where MTs cross over one another. This process of severing at sites of MT crossover results in the removal of unaligned MTs, and is likely to form the basis for the difference between a net-like and an aligned MT array.     

Alteration of oriented deposition of cellulose microfibrils by mutation of a katanin-like microtubule-severing protein

It has long been hypothesized that cortical microtubules (MTs) control the orientation of cellulose microfibril deposition, but no mutants with alterations of MT orientation have been shown to affect this process. We have shown previously that in Arabidopsis, the fra2 mutation causes aberrant cortical MT orientation and reduced cell elongation, and the gene responsible for the fra2 mutation encodes a katanin-like protein. In this study, using field emission scanning electron microscopy, we found that the fra2 mutation altered the normal orientation of cellulose microfibrils in walls of expanding cells. Although cellulose microfibrils in walls of wild-type cells were oriented transversely along the elongation axis, cellulose microfibrils in walls of fra2 cells often formed bands and ran in different directions. The fra2 mutation also caused aberrant deposition of cellulose microfibrils in secondary walls of fiber cells. The aberrant orientation of cellulose microfibrils was shown to be correlated with disorganized cortical MTs in several cell types examined. In addition, the thickness of both primary and secondary cell walls was reduced significantly in the fra2 mutant. These results indicate that the katanin-like protein is essential for oriented cellulose microfibril deposition and normal cell wall biosynthesis. We further demonstrated that the Arabidopsis katanin-like protein possessed MT-severing activity in vitro; thus, it is an ortholog of animal katanin. We propose that the aberrant MT orientation caused by the mutation of katanin results in the distorted deposition of cellulose microfibrils, which in turn leads to a defect in cell elongation. These findings strongly support the hypothesis that cortical MTs regulate the oriented deposition of cellulose microfibrils that determines the direction of cell elongation.     

Modeling anterograde and retrograde transport of short mobile microtubules from the site of axonal branch formation

This theoretical research is motivated by a recent model of microtubule (MT) transport put forward by Baas and Mozgova (Cytoskeleton 69:416–425, 2012). According to their model, in an axon all plus-end-distal mobile MTs move anterogradely while all minus-end-distal mobile MTs move retrogradely. Retrograde MT transport thus represents a mechanism by which minus-end-distal MTs are removed from the axon. We suggested equations that implement Baas and Mozgova’s model. We employed these equations to simulate transport of short mobile MTs from a region (such as the site of axonal branch formation) where MT severing activity results in generation of a large number of short MTs of both orientations. We obtained the exact and approximate transient solutions of these equations utilizing the Laplace transform technique. We applied the obtained solutions to calculate the average rates of anterograde and retrograde transport of short MTs.     

Microtubules, MAPs and plant directional cell expansion

Plant microtubules (MTs) polymerize and depolymerize in a process termed dynamic instability. This allows the assembly, reorganization, and disassembly of at least four MT arrays throughout the cell cycle. The cortical MT array lines the plasma membrane during interphase and plays a central role in directional cell expansion. Microtubule-associated proteins (MAPs) decorate cortical MTs with distinct patterns, regulating MT dynamic instability, MT severing, and other array-ordering processes. The Arabidopsis root has emerged as a highly useful system for identifying and studying cell-expansion-related MAPs. Here, we review how cortical MTs are thought to behave and become ordered in expanding root cells, and we discuss the emerging picture of how MAPs fundamentally govern MT ordering and directional growth processes.     

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.     

Centrosomal and non-centrosomal microtubules.

While microtubule (MT) arrays in cells are often focused at the centrosome, a variety of cell types contain a substantial number of non-centrosomal MTs. Epithelial cells, neurons, and muscle cells all contain arrays of non-centrosomal MTs that are critical for these cells' specialized functions. There are several routes by which non-centrosomal MTs can arise, including release from the centrosome, cytoplasmic assembly, breakage or severing, and stabilization from non-centrosomal sites. Once formed, MTs that are not tethered to the centrosome must be organized, which can be accomplished by means of self-organization or by capture and nucleation of MTs where they are needed. The presence of free MTs requires stabilization of minus ends, either by MT-associated proteins or by an end-capping complex. Although some of the basic elements of free MT formation and organization are beginning to be understood, a great deal of work is still necessary before we have a complete picture of how non-centrosomal MT arrays are assembled in specific cell types.     

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 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.     

Microtubule-associated proteins in higher plants

A variety of microtubule-associated proteins (MAPs) have been reported in higher plants. Microtubule (MT) polymerization starts from the γ-tubulin complex (γTuC), a component of the MT nucleation site. MAP200/MOR1 and katanin regulate the length of the MT by promoting the dynamic instability of MTs and cutting MTs, respectively. In construction of different MT structures, MTs are bundled or are associated with other components—actin filaments, the plasma membrane, and organelles. The MAP65 family and some of kinesin family are important in bundling MTs. MT plus-end-tracking proteins (+TIPs) including end-binding protein 1 (EB1), Arabidopsis thaliana kinesin 5 (ATK5), and SPIRAL 1 (SPR1) localize to the plus end of MTs. It has been suggested that +TIPs are involved in binding of MT to other structures. Phospholipase D (PLD) is a possible candidate responsible for binding of MTs to the plasma membrane. Many candidates have been reported as actin-binding MAPs, for example calponin-homology domain (KCH) family kinesin, kinesin-like calmodulin-binding protein (KCBP), and MAP190. RNA distribution and translation depends on MT structures, and several RNA-related MAPs have been reported. This article gives an overview of predicted roles of these MAPs in higher plants.     

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.