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Event: 720

Key Event Title

A descriptive phrase which defines a discrete biological change that can be measured. More help

Disruption, Microtubule dynamics

Short name

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Biological Context

Structured terms, selected from a drop-down menu, are used to identify the level of biological organization for each KE. More help

Level of Biological Organization
Molecular

Cell term

The location/biological environment in which the event takes place.The biological context describes the location/biological environment in which the event takes place. For molecular/cellular events this would include the cellular context (if known), organ context, and species/life stage/sex for which the event is relevant. For tissue/organ events cellular context is not applicable. For individual/population events, the organ context is not applicable. Further information on Event Components and Biological Context may be viewed on the attached pdf. More help

Cell term
eukaryotic cell

Organ term

Key Event Components

The KE, as defined by a set structured ontology terms consisting of a biological process, object, and action with each term originating from one of 14 biological ontologies (Ives, et al., 2017; https://aopwiki.org/info_pages/2/info_linked_pages/7#List). Biological process describes dynamics of the underlying biological system (e.g., receptor signalling).Biological process describes dynamics of the underlying biological system (e.g., receptor signaling). The biological object is the subject of the perturbation (e.g., a specific biological receptor that is activated or inhibited). Action represents the direction of perturbation of this system (generally increased or decreased; e.g., ‘decreased’ in the case of a receptor that is inhibited to indicate a decrease in the signaling by that receptor). Note that when editing Event Components, clicking an existing Event Component from the Suggestions menu will autopopulate these fields, along with their source ID and description. To clear any fields before submitting the event component, use the 'Clear process,' 'Clear object,' or 'Clear action' buttons. If a desired term does not exist, a new term request may be made via Term Requests. Event components may not be edited; to edit an event component, remove the existing event component and create a new one using the terms that you wish to add. Further information on Event Components and Biological Context may be viewed on the attached pdf. More help

Process	Object	Action
microtubule depolymerization	microtubule	increased

Key Event Overview

AOPs Including This Key Event

All of the AOPs that are linked to this KE will automatically be listed in this subsection. This table can be particularly useful for derivation of AOP networks including the KE.Clicking on the name of the AOP will bring you to the individual page for that AOP. More help

AOP Name	Role of event in AOP	Point of Contact	Author Status	OECD Status
Tubulin binding and aneuploidy	KeyEvent	Francesco Marchetti (send email)	Open for citation & comment	Under Review

Taxonomic Applicability

Latin or common names of a species or broader taxonomic grouping (e.g., class, order, family) that help to define the biological applicability domain of the KE.In many cases, individual species identified in these structured fields will be those for which the strongest evidence used in constructing the AOP was available in relation to this KE. More help

Term	Scientific Term	Evidence	Link
mouse	Mus musculus	High	NCBI
Homo sapiens	Homo sapiens	Moderate	NCBI
Xenopus laevis	Xenopus laevis	Moderate	NCBI

Life Stages

An indication of the the relevant life stage(s) for this KE. More help

Life stage	Evidence
All life stages	High

Sex Applicability

An indication of the the relevant sex for this KE. More help

Term	Evidence
Mixed	High

Key Event Description

A description of the biological state being observed or measured, the biological compartment in which it is measured, and its general role in the biology should be provided. More help

Microtubules are polar structures, and in each filament, subunits are added to one extremity (the plus end) and removed from the other one (the minus end) [reviewed in Marchetti et al. 2016]. Microtubules are dynamic structures characterized by features such as dynamic instability and treadmilling. Dynamic instability defines the ability of microtubules to grow or shorten [Mitchison & Kirschner, 1984; Wade and Hyman, 1997]; the process is based on a multitude of events regulating the assembly/disassembly of the subunits. Treadmilling is the process by which, in the presence of an active loss of subunits (at the minus end) and acquisition of subunits (at the plus end), a steady-state is maintained, and the length of the microtubule remains unchanged [Waterman-Sloter and Salmon, 1997]. Microtubule dynamics can be affected as a result of microtubule depolymerization or microtubule stabilization.

How It Is Measured or Detected

A description of the type(s) of measurements that can be employed to evaluate the KE and the relative level of scientific confidence in those measurements.These can range from citation of specific validated test guidelines, citation of specific methods published in the peer reviewed literature, or outlines of a general protocol or approach (e.g., a protein may be measured by ELISA). Do not provide detailed protocols. More help

Microtubule depolymerization is generally assessed by an acellular tubulin polymerization assay [Salmon et al., 1984; Wilson et al., 1984; Wallin and Hartley-Asp, 1993; Ibanez et al., 2003; Liu et al., 2010]. A reaction mixture containing tubulin and a test agent, after preincubation, is chilled on ice. GTP is added, and turbidity development is followed at 350 nm in a temperature-controlled recording spectrophotometer. The extent of the reaction is then measured and the area under the curve is used to determine the concentration that inhibited tubulin polymerization by 50% (IC50) [Hamel, 2003]. A concentration of 2.5 μM of colchicine is needed to inhibit microtubule polymerization by 50% [Zavala et al., 1980] and the ability of new chemicals to induce this effect is benchmarked against this value (e.g., combretastatin A-4 IC50 is 1.2 μM [Pettit et al., 1998]).

Domain of Applicability

A description of the scientific basis for the indicated domains of applicability and the WoE calls (if provided). More help

Depolymerization of microtubules has been measured in many somatic cell types, in addition to frog and mouse eggs, and in human cells, including eggs, in culture [Salmon et al. 1984; Wilson et al., 1984; Ibanez et al., 2003; Liu et al., 2010]. Quantitative cell-based assays for assessing microtubule activities of compounds are achieved by measuring the indirect effects on cell cycle which result from a disruption of microtubule networks. These methods utilize either fluorescent microscopy or cell cycle analysis. In fluorescent microscopic studies, either the α- or β-tubulin can be labeled directly with a tubulin antibody-conjugated fluorescent probe, or indirectly via a secondary antibody [Zhou et al., 2009]. Tubulin stabilizers and destabilizers cause cell cycle arrest at the G2/M phase [Bhalla, 2003], and therefore measurement of the percentage of cells arrested in G2/M phase is used as a surrogate endpoint for microtubule activity.

References

List of the literature that was cited for this KE description. More help

Bhalla KN. 2003. Microtubule-targeted anticancer agents and apoptosis. Oncogene 22:9075-9086.

Hamel E. 2003. Evaluation of antimitotic agents by quantitative comparisons of their effects on the polymerization of purified tubulin. Cell Biochem Biophys 38:1-22.

Ibanez E, Albertini DF, Overstrom EW. 2003. Demecolcine-induced oocyte enucleation for somatic cell cloning: coordination between cell-cycle egress, kinetics of cortical cytoskeletal interactions, and second polar body extrusion. Biol Reprod 68:1249-1258.

Kops GJ, Weaver BA, Cleveland DW. 2005. On the road to cancer: aneuploidy and the mitotic checkpoint. Nat Rev Cancer 5:773-785.

Lambrus BG, Holland AJ. 2017. A new mode of mitotic surveillance. Trends Cell Biol 27:314-321.

Liu S, Li Y, Feng HL, Yan JH, Li M, Ma SY, Chen ZJ. 2010. Dynamic modulation of cytoskeleton during in vitro maturation in human oocytes. Am J Obstet Gynecol 203:151.e151-157.

Mailhes JB, Carabatsos MJ, Young D, London SN, Bell M, Albertini DF. 1999. Taxol-induced meiotic maturation delay, spindle defects, and aneuploidy in mouse oocytes and zygotes. Mutat Res 423:79-90.

Marchetti F, Massarotti A, Yauk CL, Pacchierotti F, Russo A. 2016. The adverse outcome pathway (AOP) for chemical binding to tubulin in oocytes leading to aneuploid offspring. Environ Mol Mutagen 57:87-113.

Mitchison T, Kirschner M. 1984. Dynamic instability of microtubule growth. Nature. 312:237-242.

Panda D, Jordan MA, Chu KC, Wilson L. 1996. Differential effects of vinblastine on polymerization and dynamics at opposite microtubule ends. J Biol Chem 271:29807-29812.

Pettit GR, Toki B, Herald DL, Verdier-Pinard P, Boyd MR, Hamel E, Pettit RK. 1998. Antineoplastic agents. 379. Synthesis of phenstatin phosphate. J Med Chem 41:1688-1695.

Salmon ED, McKeel M, Hays T. 1984. Rapid rate of tubulin dissociation from microtubules in the mitotic spindle in vivo measured by blocking polymerization with colchicine. J Cell Biol 99:1066-1075.

Stanton RA, Gernert KM, Nettles JH, Aneja R. 2011. Drugs that target dynamic microtubules: a new molecular perspective. Med Res Rev 31:443-481.

Wade RH, Hyman AA. 1997. Microtubule structure and dynamics. Curr Opin Cell Biol 9:12-17.

Wallin M, Hartley-Asp B. 1993. Effects of potential aneuploidy inducing agents on microtubule assembly in vitro. Mutat Res 287:17-22.

Waterman-Sloter CM, Salmon ED. 1997. Microtubule dynamics: treadmilling comes around again. Curr Biol 7:R369-R372.

Wilson L, Miller HP, Pfeffer TA, Sullivan KF, Detrich HW, 3rd. 1984. Colchicine-binding activity distinguishes sea urchin egg and outer doublet tubulins. J Cell Biol 99:37-41.

Zavala F, Guenard D, Robin JP, Brown E. 1980. Structure--antitubulin activity relationship in steganacin congeners and analogues. Inhibition of tubulin polymerization in vitro by (+/-)-isodeoxypodophyllotoxin. J Med Chem 23:546-549.

Zhou YB, Feng X, Wang LN, Du JQ, Zhou YY, Yu HP, Zang Y, Li YJ, Li J. 2009. LGH00031, a novel ortho-quinonoid inhibitor of cell division cycle 25B, inhibits human cancer cells via ROS generation. Acta Pharmacol Sin 30;1359-1368.