Katanin Activity Research Articles

Katanin activity in tau envelopes negatively correlates with ATP levels.

Katanin is an ATP-dependent microtubule severing enzyme involved in neuronal development and function. Neurodegeneration-related protein tau cooperatively forms dense envelopes on microtubules. These envelopes can protect the microtubule lattice from katanin-induced microtubule disassembly. Neurodegenerative processes and aging are accompanied by changes in the level of intracellular ATP, however, how is katanin-tau interplay regulated by ATP level is unclear. Here we show that in presence of katanin, ATP decrease causes enhanced disassembly of tau envelope-coated microtubules. Performing katanin-driven severing experiments at 3 and 10 mM ATP, we found that katanin ATP-ase activity on bare microtubules was identical. However, counterintuitively, in presence of tau envelopes, katanin severed microtubules significantly faster in the presence of 3 mM ATP compared to the increased ATP level. These results suggest that the protective function of tau envelopes is ATP-dependent, and links decreased ATP levels with microtubule disassembly.

STRIPAK regulation of katanin microtubule severing in the Caenorhabditis elegans embryo.

Microtubule severing plays important role in cell structure and cell division. The microtubule severing protein katanin, composed of the MEI-1/MEI-2 subunits in Caenorhabditis elegans, is required for oocyte meiotic spindle formation; however, it must be inactivated for mitosis to proceed as continued katanin expression is lethal. Katanin activity is regulated by 2 ubiquitin-based protein degradation pathways. Another ubiquitin ligase, HECD-1, the homolog of human HECTD1/HECT domain E3 ubiquitin protein ligase 1, regulates katanin activity without affecting katanin levels. In other organisms, HECD-1 is a component of the striatin-interacting kinase phosphatase complex, which affects cell proliferation and a variety of signaling pathways. Here we conducted a systematic screen of how mutations in striatin-interacting kinase phosphatase components affect katanin function in C. elegans. Striatin-interacting kinase phosphatase core components (FARL-11, CASH-1, LET-92, and GCK-1) were katanin inhibitors in mitosis and activators in meiosis, much like HECD-1. By contrast, variable components (SLMP-1, OTUB-2) functioned as activators of katanin activity in mitosis, indicating they may function to alter striatin-interacting kinase phosphatase core function. The core component CCM-3 acted as an inhibitor at both divisions, while other components (MOB-4, C49H3.6) showed weak interactions with katanin mutants. Additional experiments indicate that katanin may be involved with the centralspindlin complex and a tubulin chaperone. HECD-1 shows ubiquitous expression in the cytoplasm throughout meiosis and early development. The differing functions of the different subunits could contribute to the diverse functions of the striatin-interacting kinase phosphatase complex in C. elegans and other organisms.

Wdr47, Camsaps, and Katanin cooperate to generate ciliary central microtubules

The axonemal central pair (CP) are non-centrosomal microtubules critical for planar ciliary beat. How they form, however, is poorly understood. Here, we show that mammalian CP formation requires Wdr47, Camsaps, and microtubule-severing activity of Katanin. Katanin severs peripheral microtubules to produce central microtubule seeds in nascent cilia. Camsaps stabilize minus ends of the seeds to facilitate microtubule outgrowth, whereas Wdr47 concentrates Camsaps into the axonemal central lumen to properly position central microtubules. Wdr47 deficiency in mouse multicilia results in complete loss of CP, rotatory beat, and primary ciliary dyskinesia. Overexpression of Camsaps or their microtubule-binding regions induces central microtubules in Wdr47−/− ependymal cells but at the expense of low efficiency, abnormal numbers, and wrong location. Katanin levels and activity also impact the central microtubule number. We propose that Wdr47, Camsaps, and Katanin function together for the generation of non-centrosomal microtubule arrays in polarized subcellular compartments.

The Mammalian Family of Katanin Microtubule-Severing Enzymes.

The katanin family of microtubule-severing enzymes is critical for cytoskeletal rearrangements that affect key cellular processes like division, migration, signaling, and homeostasis. In humans, aberrant expression, or dysfunction of the katanins, is linked to developmental, proliferative, and neurodegenerative disorders. Here, we review current knowledge on the mammalian family of katanins, including an overview of evolutionary conservation, functional domain organization, and the mechanisms that regulate katanin activity. We assess the function of katanins in dividing and non-dividing cells and how their dysregulation promotes impaired ciliary signaling and defects in developmental programs (corticogenesis, gametogenesis, and neurodevelopment) and contributes to neurodegeneration and cancer. We conclude with perspectives on future katanin research that will advance our understanding of this exciting and dynamic class of disease-associated enzymes.

WDR47 protects neuronal microtubule minus ends from katanin-mediated severing.

Axons and dendrites are long extensions of neurons that contain arrays of noncentrosomal microtubules. Calmodulin-regulated spectrin-associated proteins (CAMSAPs) bind to and stabilize free microtubule minus ends and are critical for proper neuronal development and function. Previous studies have shown that the microtubule-severing ATPase katanin interacts with CAMSAPs and limits the length of CAMSAP-decorated microtubule stretches. However, how CAMSAP and microtubule minus end dynamics are regulated in neurons is poorly understood. Here, we show that the neuron-enriched protein WDR47 interacts with CAMSAPs and is critical for axon and dendrite development. We find that WDR47 accumulates at CAMSAP2-decorated microtubules, is essential for maintaining CAMSAP2 stretches, and protects minus ends from katanin-mediated severing. We propose a model where WDR47 protects CAMSAP2 at microtubule minus ends from katanin activity to ensure proper stabilization of the neuronal microtubule network.

An anchoring complex recruits katanin for microtubule severing at the plant cortical nucleation sites

Microtubules are severed by katanin at distinct cellular locations to facilitate reorientation or amplification of dynamic microtubule arrays, but katanin targeting mechanisms are poorly understood. Here we show that a centrosomal microtubule-anchoring complex is used to recruit katanin in acentrosomal plant cells. The conserved protein complex of Msd1 (also known as SSX2IP) and Wdr8 is localized at microtubule nucleation sites along the microtubule lattice in interphase Arabidopsis cells. Katanin is recruited to these sites for efficient release of newly formed daughter microtubules. Our cell biological and genetic studies demonstrate that Msd1-Wdr8 acts as a specific katanin recruitment factor to cortical nucleation sites (but not to microtubule crossover sites) and stabilizes the association of daughter microtubule minus ends to their nucleation sites until they become severed by katanin. Molecular coupling of sequential anchoring and severing events by the evolutionarily conserved complex renders microtubule release under tight control of katanin activity.

External Mechanical Cues Reveal a Katanin-Independent Mechanism behind Auxin-Mediated Tissue Bending in Plants.

Tissue folding is a central building block of plant and animal morphogenesis. In dicotyledonous plants, hypocotyl folds to form hooks after seedling germination that protects their aerial stem cell niche during emergence from soil. Auxin response factors and auxin transport are reported to play a key role in this process. Here, we show that the microtubule-severing enzyme katanin contributes to hook formation. However, by exposing hypocotyls to external mechanical cues mimicking the natural soil environment, we reveal that auxin response factors ARF7/ARF19, auxin influx carriers, and katanin are dispensable for apical hook formation, indicating that these factors primarily play the role of catalyzers of tissue bending in the absence of external mechanical cues. Instead, our results reveal the key roles of the non-canonical TMK-mediated auxin pathway, PIN efflux carriers, and cellulose microfibrils as components of the core pathway behind hook formation in the presence or absence of external mechanical cues.

Design, synthesis, antitumor activities and biological studies of novel diaryl substituted fused heterocycles as dual ligands targeting tubulin and katanin

Microtubule is one of the important targets for cancer treatment. A novel class of diaryl substituted imidazo[4,5-c]pyridin-2-ones and imidazo[4,5-c]pyridines were designed based on combination principles by merging the structures of β-lactams and purine-type compounds known as tubulin polymerization inhibitor and katanin activity up-regulator, respectively. Their antitumor activities were evaluated in vitro and the mechanism was elucidated, leading to the identification of 1,6-diaryl-1H-imidazo[4,5-c]pyridin-2(3H)-one 20b as the first bifunctional agent that can target both tubulin and katanin simultaneously. The in vivo assays verified that compound 20b significantly inhibited xenograft tumor growth with good pharmacokinetic characteristics, demonstrating a promising potential for further development into anti-tumor drug candidates with a unique mechanism of dual-targeting microtubule.

Structural basis for disassembly of katanin heterododecamers

The reorganization of microtubules in mitosis, meiosis, and development requires the microtubule-severing activity of katanin. Katanin is a heterodimer composed of an ATPase associated with diverse cellular activities (AAA) subunit and a regulatory subunit. Microtubule severing requires ATP hydrolysis by katanin's conserved AAA ATPase domains. Whereas other AAA ATPases form stable hexamers, we show that katanin forms only a monomer or dimers of heterodimers in solution. Katanin oligomers consistent with hexamers of heterodimers or heterododecamers were only observed for an ATP hydrolysis-deficient mutant in the presence of ATP. X-ray structures of katanin's AAA ATPase in monomeric nucleotide-free and pseudo-oligomeric ADP-bound states revealed conformational changes in the AAA subdomains that explained the structural basis for the instability of the katanin heterododecamer. We propose that the rapid dissociation of katanin AAA oligomers may lead to an autoinhibited state that prevents inappropriate microtubule severing or that cyclical disassembly into heterodimers may critically contribute to the microtubule-severing mechanism.

Katanin Effects on Dynamics of Cortical Microtubules and Mitotic Arrays in Arabidopsis thaliana Revealed by Advanced Live-Cell Imaging.

Katanin is the only microtubule severing protein identified in plants so far. Previous studies have documented its role in regulating cortical microtubule organization during cell growth and morphogenesis. Although, some cell division defects are reported in KATANIN mutants, it is not clear whether or how katanin activity may affect microtubule dynamics in interphase cells, as well as the progression of mitosis and cytokinesis and the orientation of cell division plane (CDP). For this reason, we characterized microtubule organization and dynamics in growing and dividing cotyledon cells of Arabidopsis ktn1-2 mutant devoid of KATANIN 1 activity. In interphase epidermal cells of ktn1-2 cortical microtubules exhibited aberrant and largely isotropic organization, reduced bundling and showed excessive branched microtubule formation. End-wise microtubule dynamics were not much affected, although a significantly slower rate of microtubule growth was measured in the ktn1-2 mutant where microtubule severing was completely abolished. KATANIN 1 depletion also brought about significant changes in preprophase microtubule band (PPB) organization and dynamics. In this case, many PPBs exhibited unisided organization and splayed appearance while in most cases they were broader than those of wild type cells. By recording PPB maturation, it was observed that PPBs in the mutant narrowed at a much slower pace compared to those in Col-0. The form of the mitotic spindle and the phragmoplast was not much affected in ktn1-2, however, the dynamics of both processes showed significant differences compared to wild type. In general, both mitosis and cytokinesis were considerably delayed in the mutant. Additionally, the mitotic spindle and the phragmoplast exhibited extensive rotational motions with the equatorial plane of the spindle being essentially uncoupled from the division plane set by the PPB. However, at the onset of its formation the phragmoplast undergoes rotational motion rectifying the expansion of the cell plate to match the original cell division plane. Conclusively, KATANIN 1 contributes to microtubule dynamics during interphase, regulates PPB formation and maturation and is involved in the positioning of the mitotic spindle and the phragmoplast.

Modifications of Alpha and Beta Carboxy-Terminal Tails Regulate Microtubule Severing by Katanin

Microtubules are part of a dynamic cytoskeletal network that is constantly being reorganized to control cell processes such as neuronal development and maintenance, cell division, and cargo transport. Many stabilizing and destabilizing enzymes function to reorganize these networks for the specific needs of the cell in a spatiotemporal manner. Katanin p60 is a microtubule destabilizing enzyme from the ATPases Associated with various Activities (AAA+) family. It recognizes the tubulin carboxy-terminal tails (CTTs) to sever microtubules. Our lab has previously shown free tubulin dimers and CTTs alone can inhibit katanin severing. We seek to determine the manner that tubulin CTTs sequence can regulate katanin activity using polypeptide sequences of CTTs of different tubulin isoforms. We find that the sequence's ionic, hydrophobic, and steric features play a role in determining katanin's activity.

Regulation of katanin activity in the ciliate Tetrahymena thermophila.

Katanin is a microtubule severing protein that functions as a heterodimer composed of an AAA domain catalytic subunit, p60, and a regulatory subunit, a WD40 repeat protein, p80. Katanin-dependent severing of microtubules is important for proper execution of key cellular activities including cell division, migration, and differentiation. Published data obtained in Caenorhabditis elegans, Xenopus and mammals indicate that katanin is regulated at multiple levels including transcription, posttranslational modifications (of both katanin and microtubules) and degradation. Little is known about how katanin is regulated in unicellular organisms. Here we show that in the ciliated protist Tetrahymena thermophila, as in Metazoa, the localization and activity of katanin requires specific domains of both p60 and p80, and that the localization of p60, but not p80, is sensitive to the levels of microtubule glutamylation. A prolonged overexpression of either a full length, or a fragment of p80 containing WD40 repeats, partly phenocopies a knockout of p60, indicating that in addition to its activating role, p80 could also contribute to the inhibition of p60. We also show that the level of p80 depends on the 26S proteasome activity.

Purine-Type Compounds Induce Microtubule Fragmentation and Lung Cancer Cell Death through Interaction with Katanin.

Microtubule targeting agents (MTAs) constitute a class of drugs for cancer treatment. Despite many MTAs have been proven to significantly improve the treatment outcomes of various malignancies, resistance has usually occurred. By selection from a two million entry chemical library based on the efficacy and safety, we identified purine-type compounds that were active against lung small cell lung cancer (NSCLC). The purine compound 5a (GRC0321) was an MTA with good effects against NSCLC. Lung cancer cells H1975 treated with 5a could induce microtubule fragmentation, leading to G2/M cell cycle arrest and intrinsic apoptosis. Compound 5a directly targeted katanin and regulated the severing activity of katanin, which cut the cellular microtubules into short pieces and activated c-Jun N-terminal kinases (JNK). The microtubule fragmenting effect of 5a is a unique mechanism in MTAs. It might overcome the resistance problems that most of the MTAs have faced.

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.

Katanin Activity is Regulated by Common Cellular Components

Proper organization of the microtubule cytoskeletal network is required to perform necessary cellular functions. Network organization is achieved through remodeling by microtubule associated proteins (MAPs) that control microtubule dynamics by selectively stabilizing and destabilizing microtubules. Stabilizing MAPs are relatively well understood, while less is known about destabilizing MAPs, such as severing enzymes. Katanin, the first-discovered microtubule severing enzyme, is a AAA+ enzyme that oligomerizes into hexamers and uses ATP hydrolysis to sever microtubules. Using single molecule biophysics techniques in vitro we investigate how katanin and its severing activity can be regulated by the type of microtubule, free tubulin, ATP concentration, and the neuronal MAP tau isoform 4RL. This work provides evidence that katanin activity is regulated by common cellular contents that will ultimately affect microtubule organization in cells.

SPIRAL2 Determines Plant Microtubule Organization by Modulating Microtubule Severing

SummaryOne of the defining characteristics of plant growth and morphology is the pivotal role of cell expansion. While the mechanical properties of the cell wall determine both the extent and direction of cell expansion, the cortical microtubule array plays a critical role in cell wall organization and, consequently, determining directional (anisotropic) cell expansion [1–6]. The microtubule-severing enzyme katanin is essential for plants to form aligned microtubule arrays [7–10]; however, increasing severing activity alone is not sufficient to drive microtubule alignment [11]. Here, we demonstrate that katanin activity depends upon the behavior of the microtubule-associated protein (MAP) SPIRAL2 (SPR2). Petiole cells in the cotyledon epidermis exhibit well-aligned microtubule arrays, whereas adjacent pavement cells exhibit unaligned arrays, even though SPR2 is found at similar levels in both cell types. In pavement cells, however, SPR2 accumulates at microtubule crossover sites, where it stabilizes these crossovers and prevents severing. In contrast, in the adjacent petiole cells, SPR2 is constantly moving along the microtubules, exposing crossover sites that become substrates for severing. Consequently, our study reveals a novel mechanism whereby microtubule organization is determined by dynamics and localization of a MAP that regulates where and when microtubule severing occurs.

USP47 and C Terminus of Hsp70-Interacting Protein (CHIP) Antagonistically Regulate Katanin-p60-Mediated Axonal Growth

Katanin is a heterodimeric enzyme that severs and disassembles microtubules. While the p60 subunit has the enzyme activity, the p80 subunit regulates the p60 activity. The microtubule-severing activity of katanin plays an essential role in axonal growth. However, the mechanisms by which neuronal cells regulate the expression of katanin-p60 remains unknown. Here we showed that USP47 and C terminus of Hsp70-interacting protein (CHIP) antagonistically regulate the stability of katanin-p60 and thereby axonal growth. USP47 was identified as a katanin-p60-specific deubiquitinating enzyme for its stabilization. We also identified CHIP as a ubiquitin E3 ligase that promotes proteasome-mediated degradation of katanin-p60. Moreover, USP47 promoted axonal growth of cultured rat hippocampal neurons, whereas CHIP inhibited it. Significantly, treatment with basic fibroblast growth factor (bFGF), an inducer of axonal growth, increased the levels of USP47 and katanin-p60, but not CHIP. Consistently, bFGF treatment resulted in a marked decrease in the level of ubiquitinated katanin-p60 and thereby in the promotion of axonal growth. On the other hand, the level of USP47, but not CHIP, decreased concurrently with that of katanin-p60 as axons reached their target cells. These results indicate that USP47 plays a crucial role in the control of axonal growth during neuronal development by antagonizing CHIP-mediated katanin-p60 degradation.

Katanin P60 Targets Microtubules with Defects

Microtubules form and reorganize a variety of networks in cells, including the mitotic spindle, cilia, and neuronal processes. The organization and remodeling of microtubule architecture is regulated by microtubule-associated proteins (MAPs). Microtubule severing enzymes are a novel class of MAPs that regulate filaments by cutting them anywhere along their length. Microtubule severing enzymes, such as Katanin, oligomerize into hexamers and hydrolyze ATP to sever microtubules. Previous work has shown that Katanin specifically targets defects in the microtubules. Using in vitro single molecule assays with GFP labeled Katanin p60, the catalytic subunit of Katanin, we show that Katanin severs microtubules made in high salt more frequently. High salt microtubules have been shown to have fewer than the typical 13 protofilaments and a high incidence of protofilament shifts. We also measured the activity of Katanin on subtilisin treated microtubules (normal and high-salt) which have the c-terminal tails of alpha and beta tubulin cleaved off. Using these types of microtubules has given us insight into what types of defects Katanin might target and a better understanding of its mechanism of severing.

The spindle assembly function ofCaenorhabditis eleganskatanin 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.

IGF-1 participates differently in regulation of severing activity of katanin and spastin.

Spastin and p60-katanin are AAA family proteins that participate in microtubule severing, while lipotransin, another AAA family protein is a hormone sensitive lipase interacting protein. Sequence alignment analysis suggests that lipotransin and human p60-katanin are the orthologs of each other. Studies identified that insulin may negatively regulate ATPase activity of lipotransin. To reveal the effects of insulin on regulation of severing activity of p60-katanin and spastin, hippocampal neurons over-expressing spastin and p60-katanin were treated with IGF-1. Changes in neuronal branching by considering the total process lengths and average process numbers were quantitatively analyzed. According to the results of this study, total process lengths of hippocampal neurons and average process numbers remained similar in control and p60-katanin over-expressing neurons upon IGF-1 treatment, while significant decrease was observed in spastin over-expressing neurons. This study indicated that IGF-1 participates differently in the regulation of spastin and p60-katanin in terms of neuronal branching.