Biswas Abin

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Biswas
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Abin
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Affinity purification of label-free tubulins from xenopus egg extracts

2020-12-18 , Reusch, Sebastian , Biswas, Abin , Hirst, William G. , Reber, Simone

Cytoplasmic extracts from unfertilized Xenopus eggs have made important contributions to our understanding of microtubule dynamics, spindle assembly, and scaling. Until recently, these in vitro studies relied on the use of heterologous tubulin. This protocol allows for the purification of physiologically relevant Xenopus tubulins in milligram yield, which are a complex mixture of isoforms with various post-translational modifications. The protocol is applicable to any cell or tissue of interest. For complete details on the use and execution of this protocol, please refer to Hirst et al. (2020).

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Differences in intrinsic tubulin dynamic properties contribute to spindle length control in Xenopus species

2020-06-08 , Hirst, William G. , Biswas, Abin , Mahalingan, Kishore K. , Reber, Simone

The function of cellular organelles relates not only to their molecular composition but also to their size. However, how the size of dynamic mesoscale structures is established and maintained remains poorly understood [1, 2, 3]. Mitotic spindle length, for example, varies several-fold among cell types and among different organisms [4]. Although most studies on spindle size control focus on changes in proteins that regulate microtubule dynamics [5, 6, 7, 8], the contribution of the spindle’s main building block, the αβ-tubulin heterodimer, has yet to be studied. Apart from microtubule-associated proteins and motors, two factors have been shown to contribute to the heterogeneity of microtubule dynamics: tubulin isoform composition [9, 10] and post-translational modifications [11]. In the past, studying the contribution of tubulin and microtubules to spindle assembly has been limited by the fact that physiologically relevant tubulins were not available. Here, we show that tubulins purified from two closely related frogs, Xenopus laevis and Xenopus tropicalis, have surprisingly different microtubule dynamics in vitro. X. laevis microtubules combine very fast growth and infrequent catastrophes. In contrast, X. tropicalis microtubules grow slower and catastrophe more frequently. We show that spindle length and microtubule mass can be controlled by titrating the ratios of the tubulins from the two frog species. Furthermore, we combine our in vitro reconstitution assay and egg extract experiments with computational modeling to show that differences in intrinsic properties of different tubulins contribute to the control of microtubule mass and therefore set steady-state spindle length.