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 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 depolymerisation, 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. In addition, 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].
Include consideration of temporal concordance here
The stoichiometry of colchicine binding to tubulin dimers is well established. Empirical evidence indicates that approximately 50% inhibition of microtubule assembly occurs when half of the tubulin dimers are bound by colchine [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. 1991 (Mutagenesis, 6, 65-70) tested the effects of 10 chemicals that binding to tubulin on their ability to induce aneuploidy. These chemicals were all compared relative to colchine as a measure of potency in tubulin binding. FRANCESCO TO INSERT TEXT TO DESCRIBE HOW MUCH COLCH binding leads to HOW MUCH depolymerization.
Salmon et al. (1984) showed that in sea urchins, injection of colchicine or colcemid at final intracellular concentrations of 0.1-3.0 mM leads to a rapid decrease in 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.
Quantitative Understanding of the Linkage
Is it known how much change in the first event is needed to impact the second? Are there known modulators of the response-response relationships? Are there models or extrapolation approaches that help describe those relationships?
As described above, the quantitative relationship is well established for colchicine, and other chemicals are benchmarked against this chemicals. Microtubule assembly is inhibited by approximately 50% when half of the tubulin dimers are bound by colchine [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.