Severing of stable microtubules by a mitotically activated protein in xenopus egg extracts

doi:10.1016/0092-8674(91)90511-V

Cell

Volume 64, Issue 4, 22 February 1991, Pages 827-839

https://doi.org/10.1016/0092-8674(91)90511-V Get rights and content

Abstract

Eukaryotic cells disassemble and reorganize their cytoskeleton during the cell cycle and in response to environmental cues. Disassembly of the actin cytoskeleton is aided by proteins that sever filamentous actin, but microtubule-severing proteins thus far have not been identified. Here, we describe an activity in extracts from Xenopus eggs that rapidly severs stable microtubules along their length. Severing is elicited by a protein(s) whose activity is greatly stimulated during mitosis through a posttranslational mechanism. The microtubule-severing factor may be involved in disassembling the interphase microtubule network prior to constructing the mitotic spindle.

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Microtubule-based cytoskeletal structures aid in cell motility, cell polarization, and intracellular transport. These functions require a coordinated effort of regulatory proteins which interact with microtubule cytoskeleton distinctively. In-vitro experiments have shown that free tubulin can repair nanoscale damages of microtubules created by severing proteins. Based on this observation, we theoretically analyze microtubule severing as a competition between the processes of damage spreading and tubulin-induced repair. We demonstrate that this model is in quantitative agreement with in-vitro experiments and predict the existence of a critical tubulin concentration above which severing becomes rare, fast, and sensitive to concentration of free tubulin. We show that this sensitivity leads to a dramatic increase in the dynamic range of steady-state microtubule lengths when the free tubulin concentration is varied, and microtubule lengths are controlled by severing. Our work demonstrates how synergy between tubulin and microtubule-associated proteins can bring about specific dynamical properties of microtubules.
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Modifications concentrate on the intrinsically disordered C-terminal tails of tubulin, which form a dense lawn on the microtubule surface. Katanins are a subfamily of microtubule-severing enzymes,25,26 important in cellular processes ranging from cilia biogenesis and axonal elongation to phototropism. They require tubulin C-terminal tails for activity (reviewed by Lynn et al.27 and McNally et al.28).
Microtubules have spatiotemporally complex posttranslational modification patterns. How cells interpret this tubulin modification code is largely unknown. We show that C. elegans katanin, a microtubule severing AAA ATPase mutated in microcephaly and critical for cell division, axonal elongation, and cilia biogenesis, responds precisely, differentially, and combinatorially to three chemically distinct tubulin modifications—glycylation, glutamylation, and tyrosination—but is insensitive to acetylation. Glutamylation and glycylation are antagonistic rheostats with glycylation protecting microtubules from severing. Katanin exhibits graded and divergent responses to glutamylation on the α- and β-tubulin tails, and these act combinatorially. The katanin hexamer central pore constrains the polyglutamate chain patterns on β-tails recognized productively. Elements distal to the katanin AAA core sense α-tubulin tyrosination, and detyrosination downregulates severing. The multivalent microtubule recognition that enables katanin to read multiple tubulin modification inputs explains in vivo observations and illustrates how effectors can integrate tubulin code signals to produce diverse functional outcomes.
Cutting, Amplifying, and Aligning Microtubules with Severing Enzymes
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Microtubule-severing enzymes – katanin, spastin, fidgetin – are related AAA-ATPases that cut microtubules into shorter filaments. These proteins, also called severases, are involved in a wide range of cellular processes including cell division, neuronal development, and tissue morphogenesis. Paradoxically, severases can amplify the microtubule cytoskeleton and not just destroy it. Recent work on spastin and katanin has partially resolved this paradox by showing that these enzymes are strong promoters of microtubule growth. Here, we review recent structural and biophysical advances in understanding the molecular mechanisms of severing and growth promotion that provide insight into how severing enzymes shape microtubule networks.
The Tubulin Code in Microtubule Dynamics and Information Encoding
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Microtubules are non-covalent mesoscale polymers central to the eukaryotic cytoskeleton. Microtubule structure, dynamics, and mechanics are modulated by a cell’s choice of tubulin isoforms and post-translational modifications, a “tubulin code,” which is thought to support the diverse morphology and dynamics of microtubule arrays across various cell types, cell cycle, and developmental stages. We give a brief historical overview of research into tubulin diversity and highlight recent progress toward uncovering the mechanistic underpinnings of the tubulin code. As a large number of essential pathways converge upon the microtubule cytoskeleton, understanding how cells utilize tubulin diversity is crucial to understanding cellular physiology and disease.
Katanin Grips the β-Tubulin Tail through an Electropositive Double Spiral to Sever Microtubules
2020, Developmental Cell
Citation Excerpt :
Microtubule severing enzymes break microtubules in the middle and remodel the microtubule lattice by promoting the exchange of tubulin subunits with the soluble tubulin pool (reviewed in McNally and Roll-Mecak, 2018). Katanin was the first microtubule severing enzyme discovered (McNally and Vale, 1993; Vale, 1991). Its activity is critical for the assembly and disassembly of cilia and flagella (Casanova et al., 2009; Hu et al., 2014; Sharma et al., 2007), spindle formation, maintenance and size regulation (Loughlin et al., 2011; McNally et al., 2006; McNally et al., 2014; Mishra-Gorur et al., 2014), chromosome dynamics (Zhang et al., 2007), neuronal morphogenesis (Ahmad et al., 1999; Karabay et al., 2004; Mishra-Gorur et al., 2014; Yu et al., 2008), and plant phototropism (Lindeboom et al., 2013; Zhang et al., 2013).
The AAA ATPase katanin severs microtubules. It is critical in cell division, centriole biogenesis, and neuronal morphogenesis. Its mutation causes microcephaly. The microtubule templates katanin hexamerization and activates its ATPase. The structural basis for these activities and how they lead to severing is unknown. Here, we show that β-tubulin tails are necessary and sufficient for severing. Cryoelectron microscopy (cryo-EM) structures reveal the essential tubulin tail glutamates gripped by a double spiral of electropositive loops lining the katanin central pore. Each spiral couples allosterically to the ATPase and binds alternating, successive substrate residues, with consecutive residues coordinated by adjacent protomers. This tightly couples tail binding, hexamerization, and ATPase activation. Hexamer structures in different states suggest an ATPase-driven, ratchet-like translocation of the tubulin tail through the pore. A disordered region outside the AAA core anchors katanin to the microtubule while the AAA motor exerts the forces that extract tubulin dimers and sever the microtubule.
Predicted Effects of Severing Enzymes on the Length Distribution and Total Mass of Microtubules
2019, Biophysical Journal
Microtubules are dynamic cytoskeletal polymers whose growth and shrinkage are highly regulated as eukaryotic cells change shape, move, and divide. One family of microtubule regulators includes the ATP-hydrolyzing enzymes spastin, katanin, and fidgetin, which sever microtubule polymers into shorter fragments. Paradoxically, severases can increase microtubule number and mass in cells. Recent work with purified spastin and katanin accounts for this phenotype by showing that, in addition to severing, these enzymes modulate microtubule dynamics by accelerating the conversion of microtubules from their shrinking to their growing states and thereby promoting their regrowth. This leads to the observed exponential increase in microtubule mass. Spastin also influences the steady-state distribution of microtubule lengths, changing it from an exponential, as predicted by models of microtubule dynamic instability, to a peaked distribution. This effect of severing and regrowth by spastin on the microtubule length distribution has not been explained theoretically. To solve this problem, we formulated and solved a master equation for the time evolution of microtubule lengths in the presence of severing and microtubule dynamic instability. We then obtained numerical solutions to the steady-state length distribution and showed that the rate of severing and the speed of microtubule growth are the dominant parameters determining the steady-state length distribution. Furthermore, we found that the amplification rate is predicted to increase with severing, which is, to our knowledge, a new result. Our results establish a theoretical basis for how severing and dynamics together can serve to nucleate new microtubules, constituting a versatile mechanism to regulate microtubule length and mass.

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Cell

ArticleSevering of stable microtubules by a mitotically activated protein in xenopus egg extracts

Abstract

J. Mol. Biol.

Cell

J. Biol. Chem.

J. Biol. Chem.

J. Biol. Chem.

Cell

J. Biol. Chem.

Neuron

Cell

Cell

Meth. Enzymol.

Cell

J. Biol. Chem.

Tropomyosin binding to F-actin protects the F-actin from dissembly by brain actin-depolymerizing factor (ADF)

Cell Motil.

Real-time observations of microtubule dynamic instability in living cells

J. Cell Biol.

Organization, nucleation, acetylation of microtubules in Xenopus laevis oocytes: a study by confocal immunofluorescence microscopy

Dev. Biol.

A polymer-dependent increase in phosphorylation of beta-tubulin accompanies differentiation of a mouse neuroblastoma cell line

J. Cell Biol.

A microtubule-associated protein from Xenopus eggs that specifically promotes assembly at the plus-end

J. Cell Biol.

Microtubule assembly in cytoplasmic extracts of Xenopus oocytes and eggs

J. Cell Biol.

Ca2+-dependent binding of severin to actin: a one-to-one complex is formed

J. Cell Biol.

Cyclin is degraded by the ubiquitin pathway

Nature

Postpolymerization detyrosination of alpha-tubulin: a mechanism for subcellular differentiation of microtubules

J. Cell Biol.

Cloning and sequencing of the cyclin-related cdc13+ gene and a cytological study of its role in fission yeast mitosis

J. Cell Sci.

Fractionation of brain microtubule-associated proteins

Eur. J. Biochem.

Visualization of the dynamic instability of individual microtubules by dark-field microscopy

Nature

Movement of microtubules by single kinesin molecules

Nature

Preparation of modified tubulins

Meth. Enzymol.

Interactions of gelsolin and gelsolin-actin complexes with actin. Effects of calcium on actin nucleation, filament severing, and end blocking

Biochemistry

Enhanced stability of microtubules enriched in detyrosinated tubulin is not a direct function of detyrosination level

J. Cell Biol.

Force measurements by manipulation of a single actin filament

Nature

Reorganization of microtubules during mitosis in Dictyostelium: dissociation from MTOC and selective assembly/disassembly in situ

Cell Motil. Cytoskel.

Article
Severing of stable microtubules by a mitotically activated protein in xenopus egg extracts

Ca²⁺-dependent binding of severin to actin: a one-to-one complex is formed

Cloning and sequencing of the cyclin-related cdc13⁺ gene and a cytological study of its role in fission yeast mitosis