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Francis J. Mcnally - One of the best experts on this subject based on the ideXlab platform.
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Structural basis for disassembly of Katanin heterododecamers.
The Journal of biological chemistry, 2018Co-Authors: Stanley Nithianantham, Francis J. Mcnally, Jawdat Al-bassamAbstract: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.
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An Essential Role for Katanin p80 and Microtubule Severing in Male Gamete Production
2016Co-Authors: Danielle Rhodes, Jo D Merriner, Stephanie J. Smith, Brett J. Clark, Claire Borg, Belinda Whittle, Anne E. O’connor, Lee B. Smith, Francis J. Mcnally, David M. De KretserAbstract: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 progressiv
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an essential role for Katanin p80 and microtubule severing in male gamete production
PLOS Genetics, 2012Co-Authors: Liza Odonnell, Jo D Merriner, Danielle Rhodes, Stephanie J. Smith, Claire Borg, Belinda Whittle, Lee B. Smith, Brett Clark, Anne E Oconnor, Francis J. McnallyAbstract: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.
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The spindle assembly function of Caenorhabditis elegans Katanin does not require microtubule-severing activity.
Molecular biology of the cell, 2011Co-Authors: Karen Mcnally, Francis J. McnallyAbstract: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.
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Katanin controls mitotic and meiotic spindle length
Journal of Cell Biology, 2006Co-Authors: Karen Mcnally, Anjon Audhya, Karen Oegema, Francis J. McnallyAbstract: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.
Jennifer L. Ross - One of the best experts on this subject based on the ideXlab platform.
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Katanin catalyzes microtubule depolymerization independently of tubulin C-terminal tails.
Cytoskeleton (Hoboken N.J.), 2019Co-Authors: Liudmila Belonogov, Megan E. Bailey, Madison Tyler, Arianna Kazemi, Jennifer L. RossAbstract:Microtubule network remodeling is an essential process for cell development, maintenance, cell division, and motility. Microtubule-severing enzymes are key players in the remodeling of the microtubule network; however, there are still open questions about their fundamental biochemical and biophysical mechanisms. Here, we explored the ability of the microtubule-severing enzyme Katanin to depolymerize stabilized microtubules. Interestingly, we found that the tubulin C-terminal tail (CTT), which is required for severing, is not required for Katanin-catalyzed depolymerization. We also found that the depolymerization of microtubules lacking the CTT does not require ATP or Katanin's ATPase activity, although the ATP turnover enhanced depolymerization. We also observed that the depolymerization rate depended on the Katanin concentration and was best described by a hyperbolic function. Finally, we demonstrate that Katanin can bind to filaments that lack the CTT, contrary to previous reports. The results of our work indicate that microtubule depolymerization likely involves a mechanism in which binding, but not enzymatic activity, is required for tubulin dimer removal from the filament ends.
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Creation and testing of a new, local microtubule-disruption tool based on the microtubule-severing enzyme, Katanin p60.
Cytoskeleton (Hoboken N.J.), 2018Co-Authors: Siddheshwari Advani, Thomas J. Maresca, Jennifer L. RossAbstract:Current methods to disrupt the microtubule cytoskeleton do not easily provide rapid, local control with standard cell manipulation reagents. Here, we develop a new microtubule-disruption tool based on Katanin p60 severing activity and demonstrate proof-of-principle by targeting it to kinetochores in Drosophila melanogaster S2 cells. Specifically, we show that human Katanin p60 can remove microtubule polymer mass in S2 cells and an increase in misaligned chromosomes when globally overexpressed. When Katanin p60 was targeted to the kinetochores via Mis12, we were able to recapitulate the misalignment only when using a phosphorylation-resistant mutant Katanin p60. Our results demonstrate that targeting an active version of Katanin p60 to the kinetochore can reduce the fidelity of achieving full chromosome alignment in metaphase and could serve as a microtubule disruption tool for the future.
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Modifications of Alpha and Beta Carboxy-Terminal Tails Regulate Microtubule Severing by Katanin
Biophysical Journal, 2017Co-Authors: Madison Tyler, Corey E. Reed, Dan L. Sackett, Jennifer L. RossAbstract: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.
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Katanin Severing and Binding Microtubules Are Inhibited by Tubulin Carboxy Tails
Biophysical journal, 2015Co-Authors: Megan E. Bailey, Dan L. Sackett, Jennifer L. RossAbstract: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.
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Katanin Activity is Regulated by Common Cellular Components
Biophysical Journal, 2015Co-Authors: Megan E. Bailey, Jennifer L. RossAbstract: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.
Peter W. Baas - One of the best experts on this subject based on the ideXlab platform.
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USP47 and C Terminus of Hsp70-Interacting Protein (CHIP) Antagonistically Regulate Katanin-p60-Mediated Axonal Growth
The Journal of neuroscience : the official journal of the Society for Neuroscience, 2013Co-Authors: Seung Wook Yang, Esther Park, Hyun Min Chang, Jung Mi Park, Min Woo Seong, Woo Keun Song, Dong Eun Park, Peter W. BaasAbstract: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.
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Strategies for diminishing Katanin-based loss of microtubules in tauopathic neurodegenerative diseases
Human molecular genetics, 2010Co-Authors: Haruka Sudo, Peter W. BaasAbstract:It is commonly stated that microtubules gradually disintegrate as tau becomes dissociated from them in tauopathies such as Alzheimer's disease. However, there has been no compelling evidence to date that such disintegration is due to depolymerization of microtubules from their ends. In recent studies, we have shown that neurons contain sufficient levels of the microtubule-severing protein termed Katanin to completely break down the axonal microtubule array if not somehow attenuated. The presence of tau on axonal microtubules renders them notably less sensitive to Katanin, prompting us to posit that microtubule disintegration in tauopathies may result from elevated severing of the microtubules as they lose tau. In support of this hypothesis, we demonstrate here that pathogenic tau mutants that bind less strongly to microtubules than wild-type tau provide correspondingly less protection against Katanin-based severing. Using cultured rat hippocampal neurons, we pursued two potential therapies for fortifying axonal microtubules against excess severing by Katanin, under conditions of tau depletion. We found that either deacetylating the microtubules via overexpression of HDAC6 or treating the neurons with NAP, a microtubule-interacting neuroprotective peptide, resulted in notable protection of the microtubules against Katanin-based loss. In both cases, we found that these treatments also diminished the characteristic increase in axonal branching that normally accompanies tau depletion, an effect that is also known to be directly related to the severing of microtubules. These observations may be useful in developing therapeutic regimes for preserving microtubules against loss in the axons of patients suffering from tauopathies.
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Acetylation of Microtubules Influences Their Sensitivity to Severing by Katanin in Neurons and Fibroblasts
The Journal of neuroscience : the official journal of the Society for Neuroscience, 2010Co-Authors: Haruka Sudo, Peter W. BaasAbstract:Here we investigated whether the sensitivity of microtubules to severing by Katanin is regulated by acetylation of the microtubules. During interphase, fibroblasts display long microtubules with discrete regions rich in acetylated tubulin. Overexpression of Katanin for short periods of time produced breaks preferentially in these regions. In fibroblasts with experimentally enhanced or diminished microtubule acetylation, the sensitivity of the microtubules to severing by Katanin was increased or decreased, respectively. In neurons, microtubules are notably more acetylated in axons than in dendrites. Experimental manipulation of microtubule acetylation in neurons yielded similar results on dendrites as observed on fibroblasts. However, under these experimental conditions, axonal microtubules were not appreciably altered with regard to their sensitivity to Katanin. We hypothesized that this may be attributable to the effects of tau on the axonal microtubules, and this was validated by studies in which overexpression of tau caused microtubules in dendrites and fibroblasts to be more resistant to severing by Katanin in a manner that was not dependent on the acetylation state of the microtubules. Interestingly, none of these various findings apply to spastin, because the severing of microtubules by spastin does not appear to be strongly influenced by either the acetylation state of the microtubules or tau. We conclude that sensitivity to microtubule severing by Katanin is regulated by a balance of factors, including the acetylation state of the microtubules and the binding of tau to the microtubules. In the neuron, this contributes to regional differences in the microtubule arrays of axons and dendrites.
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the cul3 klhdc5 e3 ligase regulates p60 Katanin and is required for normal mitosis in mammalian cells
Journal of Biological Chemistry, 2009Co-Authors: Cristina M. Cummings, Peter W. Baas, Cornelia A. Bentley, Sarah A. Perdue, Jeffrey D. SingerAbstract:The proper regulation of factors involved in mitosis is crucial to ensure normal cell division. Levels and activities of proteins are regulated in many ways, one of which is ubiquitin-mediated protein degradation. E3 ubiquitin ligases are involved in targeting specific substrates for degradation by facilitating their ubiquitination. In seeking to elucidate additional biological roles for Cul3 we performed a two-hybrid screen and identified Ctb9/KLHDC5 as a Cul3-interacting protein. Overexpression of Ctb9/KLHDC5 resulted in an increase in microtubule density as well as persistent microtubule bridges between post-mitotic cells. Conversely, down-regulation of Ctb9/KLHDC5 showed a pronounced reduction in microtubule density. Based on these observations, we examined the interactions between Cul3, Ctb9/KLHDC5, and the microtubule-severing protein, p60/Katanin. Here we show that p60/Katanin interacts with a complex consisting of Cul3 and Ctb9/KLHDC5, which results in ubiquitin laddering of p60/Katanin. Also, Cul3-deficient cells or Ctb9/KLHDC5-deficient cells show an increase in p60/Katanin levels, indicating that Cul3/Ctb9/KLHDC5 is required for efficient p60/Katanin removal. We demonstrate a novel regulatory mechanism for p60/Katanin that occurs at the level of targeted proteolysis to allow normal mitotic progression in mammalian cells.
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The Cul3/Klhdc5 E3 Ligase Regulates p60/Katanin and Is Required for Normal Mitosis in Mammalian Cells
The Journal of biological chemistry, 2009Co-Authors: Cristina M. Cummings, Peter W. Baas, Cornelia A. Bentley, Sarah A. Perdue, Jeffrey D. SingerAbstract:The proper regulation of factors involved in mitosis is crucial to ensure normal cell division. Levels and activities of proteins are regulated in many ways, one of which is ubiquitin-mediated protein degradation. E3 ubiquitin ligases are involved in targeting specific substrates for degradation by facilitating their ubiquitination. In seeking to elucidate additional biological roles for Cul3 we performed a two-hybrid screen and identified Ctb9/KLHDC5 as a Cul3-interacting protein. Overexpression of Ctb9/KLHDC5 resulted in an increase in microtubule density as well as persistent microtubule bridges between post-mitotic cells. Conversely, down-regulation of Ctb9/KLHDC5 showed a pronounced reduction in microtubule density. Based on these observations, we examined the interactions between Cul3, Ctb9/KLHDC5, and the microtubule-severing protein, p60/Katanin. Here we show that p60/Katanin interacts with a complex consisting of Cul3 and Ctb9/KLHDC5, which results in ubiquitin laddering of p60/Katanin. Also, Cul3-deficient cells or Ctb9/KLHDC5-deficient cells show an increase in p60/Katanin levels, indicating that Cul3/Ctb9/KLHDC5 is required for efficient p60/Katanin removal. We demonstrate a novel regulatory mechanism for p60/Katanin that occurs at the level of targeted proteolysis to allow normal mitotic progression in mammalian cells.
Yoshiro Maru - One of the best experts on this subject based on the ideXlab platform.
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The tumor suppressor LZTS2 functions through the cellular samurai Katanin
Central European Journal of Biology, 2009Co-Authors: Yoshiro MaruAbstract:The leucine zipper putative tumor suppressor (LZTS) 2 is frequently and specifically found in LOH (loss of heterozygosity) analysis in cancer. Different from other LZTS family members, it regulates the microtubule-severing protein Katanin by binding the p80 regulatory subunit of Katanin and inhibiting its interaction with microtubules. At specific phases of the cell cycle, LZTS2 suppresses cell migration and establishes proper central spindle assembly for cytokinesis. Importantly, those biological effects are mediated by the inhibitory activity of LZTS2 on Katanin. LZTS2 binding to Katanin also plays a role in Katanin transport to the midbody to control proper abscission. Therapeutic applications of the interaction between LZTS2 and Katanin in tumor cells are a potential area for future research.
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lapser1 lzts2 a pluripotent tumor suppressor linked to the inhibition of Katanin mediated microtubule severing
Human Molecular Genetics, 2008Co-Authors: Haruka Sudo, Yoshiro MaruAbstract:Human chromosome region 10q23-24 is one of the most frequently found regions that show loss of heterozygosity in prostate cancers. A candidate tumor suppressor LAPSER1/LZTS2 (LAPSER1) is located in 10q24.3 that has been reported to be deleted as frequently as the neighboring PTEN locus. We previously reported that LAPSER1 binds p80 Katanin, a subunit of the Katanin heterodimer. In this report, we show that the LAPSER1 C terminal domain inhibits Katanin-mediated microtubule severing in vitro and we detected this inhibition at centrosomes by tracing the nucleated de novo, severed, and transported microtubules in cells. This functional association is also supported by the intracellular localization. Centrosomal localization of LAPSER1 was independent of microtubules and was preferential to mother centrioles. In primary cultured neurons, LAPSER1 also colocalizes with p80 Katanin. LAPSER1 alters cell proliferation by regulating cytokinesis. As subcellular mechanisms that underlie the tumor suppressive activity, exogenous LAPSER1 expression inhibited central spindle formation by abrogating microtubule transportation and a similar mode of inhibition was found in axogenesis. Katanin knockdown and dominant negative inhibitor of Katanin provided similar phenotypes. Prophase LAPSER1 inhibited centrosomal γ-tubulin accumulation, which resulted in retardation of mitotic entry. Furthermore, interphase inhibition of Katanin by LAPSER1 expression resulted in prevention of cell motility that was accompanied by the increased acetylated microtubules. LAPSER1 knockdown increased cell migration that was inhibited by the expression of ninein, a microtubule release inhibitor. These results indicate that microtubule severing at centrosomes is a novel tumor-associated molecular subcircuit in cells, in which LAPSER1 is a regulator.
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LAPSER1/LZTS2: a pluripotent tumor suppressor linked to the inhibition of Katanin-mediated microtubule severing.
Human molecular genetics, 2008Co-Authors: Haruka Sudo, Yoshiro MaruAbstract:Human chromosome region 10q23-24 is one of the most frequently found regions that show loss of heterozygosity in prostate cancers. A candidate tumor suppressor LAPSER1/LZTS2 (LAPSER1) is located in 10q24.3 that has been reported to be deleted as frequently as the neighboring PTEN locus. We previously reported that LAPSER1 binds p80 Katanin, a subunit of the Katanin heterodimer. In this report, we show that the LAPSER1 C terminal domain inhibits Katanin-mediated microtubule severing in vitro and we detected this inhibition at centrosomes by tracing the nucleated de novo, severed, and transported microtubules in cells. This functional association is also supported by the intracellular localization. Centrosomal localization of LAPSER1 was independent of microtubules and was preferential to mother centrioles. In primary cultured neurons, LAPSER1 also colocalizes with p80 Katanin. LAPSER1 alters cell proliferation by regulating cytokinesis. As subcellular mechanisms that underlie the tumor suppressive activity, exogenous LAPSER1 expression inhibited central spindle formation by abrogating microtubule transportation and a similar mode of inhibition was found in axogenesis. Katanin knockdown and dominant negative inhibitor of Katanin provided similar phenotypes. Prophase LAPSER1 inhibited centrosomal γ-tubulin accumulation, which resulted in retardation of mitotic entry. Furthermore, interphase inhibition of Katanin by LAPSER1 expression resulted in prevention of cell motility that was accompanied by the increased acetylated microtubules. LAPSER1 knockdown increased cell migration that was inhibited by the expression of ninein, a microtubule release inhibitor. These results indicate that microtubule severing at centrosomes is a novel tumor-associated molecular subcircuit in cells, in which LAPSER1 is a regulator.
Karen Mcnally - One of the best experts on this subject based on the ideXlab platform.
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The spindle assembly function of Caenorhabditis elegans Katanin does not require microtubule-severing activity.
Molecular biology of the cell, 2011Co-Authors: Karen Mcnally, Francis J. McnallyAbstract: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.
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Katanin controls mitotic and meiotic spindle length
Journal of Cell Biology, 2006Co-Authors: Karen Mcnally, Anjon Audhya, Karen Oegema, Francis J. McnallyAbstract: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.
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Katanin-mediated microtubule severing can be regulated by multiple mechanisms.
Cell motility and the cytoskeleton, 2002Co-Authors: Karen Mcnally, Dan Buster, Francis J. McnallyAbstract: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. Cell Motil. Cytoskeleton 53:337–349, 2002. © 2002 Wiley-Liss, Inc.
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Katanin inhibition prevents the redistribution of γ-tubulin at mitosis
Journal of Cell Science, 2002Co-Authors: Dan Buster, Karen Mcnally, Francis J. McnallyAbstract:Katanin is a microtubule-severing protein that is concentrated at mitotic spindle poles but Katanin9s function in the mitotic spindle has not been previously reported. Inhibition of Katanin with either of two dominant-negative proteins or a subunit-specific antibody prevented the redistribution of γ-tubulin from the centrosome to the spindle in prometaphase CV-1 cells as assayed by immunofluorescence microscopy. Becauseγ -tubulin complexes can bind to pre-existing microtubule minus ends, these results could be explained by a model in which the broad distribution ofγ -tubulin in the mitotic spindle is in part due to cytosolicγ -tubulin ring complexes binding to microtubule minus ends generated by Katanin-mediated microtubule severing. Because microtubules depolymerize at their ends, we hypothesized that a greater number of microtubule ends generated by severing in the spindle would result in an increased rate of spindle disassembly when polymerization is blocked with nocodazole. Indeed, Katanin inhibition slowed the rate of spindle microtubule disassembly in the presence of nocodazole. However, Katanin inhibition did not affect the rate of exchange between polymerized and unpolymerized tubulin as assayed by fluorescence recovery after photobleaching. These results support a model in which Katanin activity regulates the number of microtubule ends in the spindle.
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Katanin inhibition prevents the redistribution of gamma-tubulin at mitosis.
Journal of cell science, 2002Co-Authors: Dan Buster, Karen Mcnally, Francis J. McnallyAbstract:Katanin is a microtubule-severing protein that is concentrated at mitotic spindle poles but Katanin's function in the mitotic spindle has not been previously reported. Inhibition of Katanin with either of two dominant-negative proteins or a subunit-specific antibody prevented the redistribution of gamma-tubulin from the centrosome to the spindle in prometaphase CV-1 cells as assayed by immunofluorescence microscopy. Because gamma-tubulin complexes can bind to pre-existing microtubule minus ends, these results could be explained by a model in which the broad distribution of gamma-tubulin in the mitotic spindle is in part due to cytosolic gamma-tubulin ring complexes binding to microtubule minus ends generated by Katanin-mediated microtubule severing. Because microtubules depolymerize at their ends, we hypothesized that a greater number of microtubule ends generated by severing in the spindle would result in an increased rate of spindle disassembly when polymerization is blocked with nocodazole. Indeed, Katanin inhibition slowed the rate of spindle microtubule disassembly in the presence of nocodazole. However, Katanin inhibition did not affect the rate of exchange between polymerized and unpolymerized tubulin as assayed by fluorescence recovery after photobleaching. These results support a model in which Katanin activity regulates the number of microtubule ends in the spindle.