Upstream eventBinding, Tubulin
Key Event Relationship Overview
AOPs Referencing Relationship
|AOP Name||Adjacency||Weight of Evidence||Quantitative Understanding|
|Chemical binding to tubulin in oocytes leading to aneuploid offspring||adjacent||High|
|Homo sapiens||Homo sapiens||Not Specified||NCBI|
|Xenopus laevis||Xenopus laevis||Low||NCBI|
Life Stage Applicability
Key Event Relationship Description
Chemicals that bind to tubulin directly interfere with the addition of new tubulin dimers to the microtubules. The result of this process is a net loss of microtubules (i.e., microtubule depolymerization).
Evidence Supporting this KER
Strong based on biological plausibility and available empirical data. There is no uncertainty.
The weight of evidence for this KER is strong. The majority of work for this KER has been derived from research on the prototypical chemical colchicine; however, information is also available for other chemicals such as podophillotoxin, vinblastin, and colcemid. There is high biological plausibility for the binding of colchicine to tubulin leading to microtubule depolymerization, which is one of the most studied chemical interactions with a biological molecule [Margolis and Wilson, 1977; Garland, 1978; Ravelli et al., 2004]. There is extensive understanding of the chemistry of both the binding interactions and the subsequent interference with microtubule dynamics. Depolymerization following colchicine exposure has been measured in 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].
The stoichiometry of colchicine binding to tubulin dimers is well established. Empirical evidence shows that approximately 50% inhibition of microtubule assembly occurs when half of the tubulin dimers are bound by colchicine [Margolis et al., 1980] indicating concordance in the response between the number of bound dimers and degree of depolymerization. Colchicine binding in vitro occurs within minutes of exposure and this timing is concordant with microtubule depolymerization [Margolis and Wilson, 1977]. Colchicine binds to tubulin with an average affinity constant of 10E-6 to 10E-7/M at 37 degrees Celsius. The half-life of the binding site is up to 7.5 hours [Hastie 1991; Bhattacharyya et al. 2008] and is concordant with effects on depolymerization and recovery [Margolis and Wilson, 1977].
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. For example, the IC50 of the tubulin-binding chemical combretastatin A-4 is 1.2 μM [Pettit et al., 1998]. Specificity for tubulin binding is also measured by incubation of tubulin extract with the test chemical in the presence and absence of colchicine. For example, co-incubation of colchicine and the potent microtubule inhibitor podophyllotoxin yields a Ki of 3.3 x 10-06 M [Margolis et al., 1980]. Binding constants and resulting effects of depolymerization have been established for at least 20 agents.
Brunner et al.  tested the effects of 10 chemicals on brain tubulin assembly and disassembly in vitro. Tubulin disassembly is monitored for 30-40 minutes following chemical exposure. The 10 chemicals were all compared relative to colchicine as a measure of potency in tubulin binding. Five chemicals (colchicine, vinblastine, thimerosal, thiabendazole, and chloral hydrate) inhibited tubulin polymerization, however, effective concentrations varied by a factor of 30,000 among them. In fact, the concentrations needed to reduce by 30% the steady state of tubulin polymerization were: 0.002 mM for colchicine and vinblastine, 0.03 mM for thimerosal, 0.05 mM for thiabendazole and 60 mM for chloral hydrate. Furthermore, except for chloral hydrate, these compounds also induced a dose-dependent decline in polymerization velocity. Wallin and Hartley-Asp  tested the same 10 chemicals for effects on the assembly of bovine microtubules and reported similar results to those of Brunner et al  although some differences in potencies were also noted.
Salmon et al.  showed that in sea urchins, injection of colchicine or colcemid at final intracellular concentrations of 0.1-3.0 mM leads to a rapid microtubule depolymerization throughout the central spindle and aster. Microtubule concentration in the central half-spindle decreased exponentially to 10% of its initial value within ~20 s. For both colchicine and colcemid, the rate of microtubule depolymerization below 0.1 mM was concentration dependent. In addition, they show that increasing doses of colchicine lead to reductions in the amount of time necessary to detect microtubule depolymerization. As a control, lumicolchicine (which does not bind to tubulin with high affinity) had no effect on microtubule polymerization at intracellular concentrations of 0.5 mM.
Uncertainties and Inconsistencies
No apparent uncertainties or inconsistencies. This KER is biologically plausible and broadly accepted. Indeed, in vitro assays to measure tubulin depolymerization are well standardized and represent the gold standard to determine whether a chemical is binding to tubulin.
Quantitative Understanding of the Linkage
As described above, the quantitative relationship is well established for colchicine, and other chemicals are benchmarked against this chemical [Brunner et al., 1991]. Microtubule assembly is inhibited by approximately 50% when half of the tubulin dimers are bound by colchicine [Margolis et al., 1980], and a concentration of 2.5 μM of colchicine is needed to inhibit microtubule polymerization by 50% [Zavala et al., 1980].
Known modulating factors
Known Feedforward/Feedback loops influencing this KER
Domain of Applicability
This KER has been demonstrated in multiple species including sea urchins, frogs, mice, rats, cows, and human cells in culture.
Bhattacharyya B, Panda D, Gupta S, Banerjee M. 2008. Anti-mitotic activity of colchicine and the structural basis for its interaction with tubulin. Med Res Rev 28:155-183.
Brunner M, Albertini S, Würgler FE. 1991. Effects of 10 known or suspected spindle poisons in the in vitro porcine brain tubulin assembly assay. Mutagen 6:65-70.
Garland DL. 1978. Kinetics and mechanism of colchicine binding to tubulin: Evidence for ligand-induced conformational change. Biochemistry 17:4266–4272.
Hastie SB. 1991. Interaction of colchicine with tubulin. Pharmacol Ther 51:377-401.
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.
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.
Margolis RL, Wilson L. 1977. Addition of colchicine-tubulin complex to microtubule ends: the mechanism of substoichiometric colchicine poisoning. Proc Natl Acad Sci U S A 74:3466-3470.
Margolis RL, Rauch CT, Wilson L. 1980. Mechanism of colchicine-dimer addition to microtubule polymerization mechanism. Biochemistry 19:5550-5557.
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.
Ravelli RB, Gigant B, Curmi PA, Jourdain I, Lachkar S, Sobel A, Knossow M. 2004. Insight into tubulin regulation from a complex with colchicine and a stathmin-like domain. Nature 428:198–202.
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.
Wallin M, Hartley-Asp B. 1993. Effects of potential aneuploidy inducing agents on microtubule assembly in vitro. Mutat Res 287:17-22.
Wilson L, Miller HP, Pfeffer TA, Sullivan KF, Detrich HW,3. 1984. Colchicine-binding activity distinguishes sea urchin egg and outer doublet tubulins. J Cell Biol 99:37–41.