Upstream eventDisruption, Microtubule dynamics
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||Moderate|
|Homo sapiens||Homo sapiens||Moderate||NCBI|
Life Stage Applicability
Key Event Relationship Description
Spindle organization and function requires normal microtubule dynamics. When microtubule polymerization is affected (i.e., depolymerization), spindle organization and function is impaired.
Evidence Supporting this KER
The weight of evidence for this KER is moderate. Microtubule polymerization is critical for the appropriate functioning of the spindle. Mitotic and meiotic spindles differ in how they are assembled. In mitotic cells, spindle organization is controlled by centrioles [Walczak and Heald, 2008; Wadsworth et al., 2011; Wittman et al., 2011]. However, centrioles are absent in mammalian oocytes [Manandhar et al., 2005] and meiotic spindle is organized by multiple microtubule organizing centers (MTOCs). Gradually, MTOCs coalesce and surround the chromosomes and subsequently elongate in a typical barrel-shape bipolar spindle [Schuh and Ellenberg, 2007; Clift and Schuh, 2015], similar to the mitotic spindle. Assembly, elongation and function of the spindle requires proper microtubule dynamics. If microtubules become depolymerized, it affects the structural integrity of the spindle resulting in abnormal spindles that are characterized by reduction in microtubule density, loss of barrel shape, mono- or multi-polar spindle, and reduced distance between the poles [Ibanez et al., 2003; Shen et al., 2005; Eichenlaub-Ritter et al., 2007; Xu et al., 2012]. The normal biology underlying the critical role of proper microtubule polymerization for the appropriate structure and function of spindle is well established, and it is widely understood that chemicals that alter microtubule dynamics cause spindle disorganization [Manandhar et al., 2005; Schuh and Ellenberg, 2007].
Several papers have explored the temporal and incidence relationship between microtubule depolymerization and appearance of spindle abnormalities, providing empirical evidence to support this KER in a variety of species. For example, Salmon et al.  showed that within 20 seconds of colchicine or colcemid administration in sea urchin embryos, microtubule depolymerization occurs. This leads to spindle abnormalities at the same concentration that occurs a few minutes later. In Liu et al. , in vitro culturing of human oocytes in the presence of 10 µM colchicine prevented microtubule polymerization and because of this, meiotic spindle did not form (i.e., exposure led to failure to progress to meiosis II supporting temporal concordance). Ibanez et al.  showed that 1 µM colcemid caused microtubule depolymerization that led to smaller spindles and lower microtubule density in mouse oocytes within 15 minutes of exposure, demonstrating temporal and incidence concordance.
Uncertainties and Inconsistencies
There are not a lot of studies that have explored these two events within the same experiment. Thus, the empirical evidence is not based on a large number of papers. However, the papers that are available are of sound experimental design that address both time and incidence relationships, and span three species.
Quantitative Understanding of the Linkage
Limited quantitative understanding. Data from Salmon et al.  established a dose-response relationship for microtubule depolymerization in mitotic sea urchin embryo cells, but similar quantitative data are not available for spindle disorganization.
There are detailed dose-response relationships for microtubule depolymerisation by tubulin binders obtained using acellular tubulin polymerization assays [Zavala et al., 1980; Hamel and Lin, 1981; Verdier-Pinard et al., 1998; Miller and Wilson, 2010]. The rate of depolymerisation has been also measured in whole mitotic cells of sea urchin embryos after microinjection of different doses of colchicine or colcemid, in the range 0.01-5 mM [Salmon et al., 1984]. Comparable data are not available for mammalian oocytes. In addition, no quantitative dose-response relationship has been obtained for spindle disorganization in oocytes treated with tubulin binding chemicals. This lack of data does not allow modelling a response-response relationship between disruption of microtubule dynamics (KEupstream) and spindle disorganization (KEdownstream).
In sea urchin embryo cells microinjected with colchicine concentrations equal to or higher than 0.1 mM, complete depolymerization of non-kinetochore spindle microtubules (KEupstream) is reached in about 20 seconds, corresponding to a depolymerization rate of about 180-992 dimers per second [Salmon et al., 1984]. The order of magnitude of these values corresponds to the fastest rates of tubulin dissociation reported in various acellular systems [Fan’ell et al., 1983]. However, possible modifying factors of the above rates are suggested in the cells (e.g., calcium concentration), conditions that are not reproducible in acellular systems.
In vitro exposure of mouse oocytes to 67 µM nocodazole causes a gradual disorganization of the spindle (KEdownstream), which is completed within 15 min [Xu et al., 2012]. In spite of the limited amount of data on the kinetics of spindle disorganization (KEdownstream) and the further limitation that dysruption of microtubule dynamics (KEupstream) and spindle disorganization (KEdownstream) were not analyzed in the same biological systems, it can be noted that the time-scale in the KEdownstream is coherent with the time-scale of the KEupstream.
Known modulating factors
Due to the heterogeneity of the experimental approaches used to measure dysruption of microtubule dynamics (KEupstream) and spindle disorganization (KEdownstream) it is not feasible to identify modulating factors acting in this KER.
Known Feedforward/Feedback loops influencing this KER
To our knowledge, there are no feedback loops influencing this KER.
Domain of Applicability
Data were produced in sea urchins, mice and human eggs and embryos. This KER should be applicable to any eukaryotic organism.
Clift D, Schuh M. 2015. A three-step MTOC fragmentation mechanism facilitate bipolar spindle assembly in mouse oocytes. Nat Commun 6:7217.
Eichenlaub-Ritter U, Winterscheidt U, Vogt E, Shen Y, Tinneberg HR, Sorensen R. 2007. 2-methoxyestradiol induces spindle aberrations, chromosome congression failure, and nondisjunction in mouse oocytes. Biol Reprod 76:784–793.
Fan'ell KW, Himes RH, Jordon MA,Wilson L. 1983. On the nonlinear relationship between the initial rates of dilution induced microtubule disassembly and the initial free subunit concentration. J Biol Chem 258:14148-14156.
Hamel E, Lin CM. 1981. Stabilization of the colchicine-binding activity of tubulin by organic acids. Biochim Biophys Acta 675:226-231.
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 151:e1–7.
Manandhar G, Schatten H, Sutovsky P. 2005. Centrosome reduction during gametogenesis and its significance. Biol Reprod 72:2-13.
Miller HP, Wilson L. 2010. Chapter 1 - Preparation of Microtubule Protein and Purified Tubulin from Bovine Brain by Cycles of Assembly and Disassembly and Phosphocellulose Chromatography. In: Leslie W, John JC, editors. Methods Cell Biol: Academic Press. p 2-15.
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.
Schuh M, Ellenberg J. 2007. Self-organization of MTOCs replaces centrosome function during acentrosomal spindle assembly in live mouse oocytes. Cell 130:484-498.
Shen Y, Betzendahl I, Sun F, Tinneberg HR, Eichenlaub-Ritter U. 2005. Non-invasive method to assess genotoxicity of nocodazole interfering with spindle formation in mammalian oocytes. Reprod Toxicol 19:459-471.
Verdier-Pinard P, Lai JY, Yoo HD, Yu J, Marquez B, Nagle DG, Nambu M, White JD, Falck JR, Gerwick WH, Day PW, Hamel E. 1998. Structure-activity analysis of the interaction of curacin A, the potent colchicine site antimitotic agent, with tubulin and effects of analogs on the growth of MCF-7 breast cancer cells. Mol Pharmacol 53:62-76.
Wadsworth P, Lee WL, Murata T, Baskin TI. 2011. Variations on a theme: spindle assembly in diverse cells. Protoplasma 248:439-446.
Walczak CE and R Heald. 2008. Mechanisms of mitotic spindle assembly and function. Int. Rev Cytol 265:111-158.
Wittman T, Hyman A, Desai A. 2011. The spindle: a dynamic assembly of microtubules and motors. Nat Cell Biol 3:e28-234.
Xu XL, Ma W, Zhu YB, Wang C, Wang BY, An N, An L, Liu Y, Wu ZH, Tian JH. 2012. The microtubule-associated protein ASPM regulates spindle assembly and meiotic progression in mouse oocytes. PLoS One 7:e49303.
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.