Affinity purification of label-free tubulins from xenopus egg extracts

© The Author(s), 2020. This article is distributed under the terms of the Creative Commons Attribution License. The definitive version was published in Reusch, S., Biswas, A., Hirst, W. G., & Reber, S. Affinity purification of label-free tubulins from xenopus egg extracts. STAR Protocols, 1(3), (2020): 100151, doi:10.1016/j.xpro.2020.100151.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).This article was prompted by our stay at the Marine Biological Laboratory (MBL), Woods Hole, MA, in the summer of 2016 funded by the Princeton-Humboldt Strategic Partnership Grant together with the lab of Sabine Petry (Princeton University). We are grateful to the National Xenopus Resource (NXR) for supplying frogs. For mass spectrometry, we would like to acknowledge the assistance of Benno Kuropka and Chris Weise from the Core Facility BioSupraMol supported by the Deutsche Forschungsgemeinschaft (DFG). We thank the Protein Expression Purification and Characterization (PEPC) facility at the MPI-CBG; in particular, we thank Aliona Bogdanova and Barbara Borgonovo. We thank all former and current members of the Reber lab for discussions and helpful advice, in particular Christoph Hentschel and Soma Zsoter for technical assistance. S.R. acknowledges funding from the IRI Life Sciences (Humboldt-Universität zu Berlin, Excellence Initiative/DFG). W.H. was supported by the Alliance Berlin Canberra co-funded by a grant from the Deutsche Forschungsgemeinschaft (DFG) for the International Research Training Group (IRTG) 2290 and the Australian National University

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

© The Author(s), 2020. This article is distributed under the terms of the Creative Commons Attribution License. The definitive version was published in Hirst, W. G., Biswas, A., Mahalingan, K. K., & Reber, S. Differences in intrinsic tubulin dynamic properties contribute to spindle length control in Xenopus species. Current Biology, 30(11), (2020): 2184-2190.e5, doi: 10.1016/j.cub.2020.03.067.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.This article was prompted by our stay at the Marine Biological Laboratory (MBL), Woods Hole, MA in the summer of 2016 funded by the Princeton-Humboldt Strategic Partnership Grant together with the lab of Sabine Petry (Princeton University). We thank Jeff Woodruff (UT Southwestern), David Drechsel (IMP), and Marcus J. Taylor (MPI IB) for constructive criticism and comments on the manuscript and Helena Jambor for constructive comments on figure design. We thank the AMBIO imaging facility (Charité, Berlin) and Nikon at MBL for imaging support, Aliona Bogdanova and Barbara Borgonovo (MPI CBG) for their help with protein purification, and Francois Nedelec (University of Cambridge) for help with Cytosim. We are grateful to the Görlich lab (MPI BPC), in particular Bastian Hülsmann and Jens Krull, and the NXR for supply with X. tropicalis frogs. We thank Antonina Roll-Mecak (National Institute of Neurological Disorders and Stroke) for help with mass spectrometry analysis and discussions and Duck-Yeon Lee in the Biochemistry Core (National Heart, Lung and Blood Institute) for access to mass spectrometers. For mass spectrometry, we would like to acknowledge the assistance of Benno Kuropka and Chris Weise from the Core Facility BioSupraMol supported by the Deutsche Forschungsgemeinschaft (DFG). We thank all former and current members of the Reber lab for discussion and helpful advice, in particular, Christoph Hentschel and Soma Zsoter for technical assistance and Sebastian Reusch for help with tubulin purification. S.R. acknowledges funding from the IRI Life Sciences (Humboldt-Universität zu Berlin, Excellence Initiative/DFG). W.G.H. was supported by the Alliance Berlin Canberra co-funded by a grant from the Deutsche Forschungsgemeinschaft (DFG) for the International Research Training Group (IRTG) 2290 and the Australian National University. K.K.M. was supported by funds in the Roll-Mecak lab, intramural program of the National Institute of Neurological Disorders and Stroke

In Vitro reconstitution and imaging of microtubule dynamics by fluorescence and label-free microscopy

© The Author(s), 2020. This article is distributed under the terms of the Creative Commons Attribution License. The definitive version was published in Hirst, W. G., Kiefer, C., Abdosamadi, M. K., Schäffer, E., & Reber, S. In Vitro reconstitution and imaging of microtubule dynamics by fluorescence and label-free microscopy. STAR Protocols, 1(3), (2020): 100177, doi:10.1016/j.xpro.2020.100177.Dynamic microtubules are essential for many processes in the lives of eukaryotic cells. To study and understand the mechanisms of microtubule dynamics and regulation, in vitro reconstitution with purified components has proven a vital approach. Imaging microtubule dynamics can be instructive for a given species, isoform composition, or biochemical modification. Here, we describe two methods that visualize microtubule dynamics at high speed and high contrast: (1) total internal reflection fluorescence microscopy and (2) label-free interference reflection microscopy.We thank the AMBIO imaging facility (Charité, Berlin) and Nikon at MBL for imaging support. We thank all former and current members of the Reber lab for discussion and helpful advice, in particular Christoph Hentschel and Soma Zsoter for technical assistance. S.R. acknowledges funding by the IRI Life Sciences (Humboldt-Universität zu Berlin, Excellence Initiative/DFG). W.H. was supported by the Alliance Berlin Canberra co-funded by a grant from the Deutsche Forschungsgemeinschaft (DFG) for the International Research Training Group (IRTG) 2290 and the Australian National University. C.K. thanks the Deutsche Forschungsgesellschaft (DFG, JA 2589/1-1). C.K. and M.A. thank Steve Simmert and Tobias Jachowski former and current members of the Schäffer lab

The Third wave in globalization theory

This essay examines a proposition made in the literature that there are three waves in globalization theory—the globalist, skeptical, and postskeptical or transformational waves—and argues that this division requires a new look. The essay is a critique of the third of these waves and its relationship with the second wave. Contributors to the third wave not only defend the idea of globalization from criticism by the skeptics but also try to construct a more complex and qualified theory of globalization than provided by first-wave accounts. The argument made here is that third-wave authors come to conclusions that try to defend globalization yet include qualifications that in practice reaffirm skeptical claims. This feature of the literature has been overlooked in debates and the aim of this essay is to revisit the literature and identify as well as discuss this problem. Such a presentation has political implications. Third wavers propose globalist cosmopolitan democracy when the substance of their arguments does more in practice to bolster the skeptical view of politics based on inequality and conflict, nation-states and regional blocs, and alliances of common interest or ideology rather than cosmopolitan global structures

‘Dominant ethnicity’ and the ‘ethnic-civic’ dichotomy in the work of A. D. Smith

This article considers the way in which the work of Anthony Smith has helped to structure debates surrounding the role of ethnicity in present-day nations. Two major lines of enquiry are evident here. First, the contemporary role of dominant ethnic groups within 'their' nations and second, the interplay between ethnic and civic elements in nationalist argument. The two processes are related, but maintain elements of distinctiveness. Smith's major contribution to the dominant ethnicity debate has been to disembed ethnicity from the ideologically-charged and/or anglo-centric discourse of ethnic relations and to place it in historical context, thereby opening up space for dominant group ethnicity to be considered as a distinct phenomenon. This said, Smith's work does not adequately account for the vicissitudes of dominant ethnicity in the contemporary West. Building on the classical works of Hans Kohn and Friedrich Meinecke, Anthony Smith has also made a seminal contribution to the debate on civic and ethnic forms of national identity and nationalist ideology. As well as freeing this debate from the strong normative overtones which it has often carried, he has continued to insist that the terms civic and ethnic should be treated as an ideal-typical distinction rather than a scheme of classification

On the Formation of Collective Memories: The Role of a Dominant Narrator.

To test our hypothesis that conversations can contribute to the formation of collective memory, we asked participants to study stories and to recall them individually (pregroup recollection), then as a group (group recounting), and then once again individually (postgroup recollection). One way that postgroup collective memories can be formed under these circumstances is if unshared pregroup recollections in the group recounting influences others\u27 postgroup recollections. In the present research, we explored (using tests of recall and recognition) whether the presence of a dominant narrator can facilitate the emergence of unshared pregroup recollections in a group recounting and whether this emergence is associated with changes in postgroup recollections. We argue that the formation of a collective memory through conversation is not inevitable but is limited by cognitive factors, such as conditions for social contagion, and by situational factors, such as the presence of a narrator

Enantioselective component selection in multicomponent supramolecular gels

We investigate a two-component acid-amine gelation system in which chirality plays a vital role. A carboxylic acid based on a second generation l-lysine dendron interacts with chiral amines and subsequently assembles into supramolecular gel fibers. The chirality of the amine controls the assembly of the resulting diastereomeric complexes, even if this chirality is relatively "poor quality". Importantly, the selective incorporation of one enantiomer of an amine over the other into the gel network has been demonstrated, with the R amine that forms complexes which assemble into the most stable gel being primarily selected for incorporation. Thermodynamic control has been proven by forming a gel exclusively with an S amine, allowing the R enantiomer to diffuse through the gel network, and displacing it from the "solidlike" fibers, demonstrating that these gels adapt and evolve in response to chemical stimuli to which they are exposed. Excess amine, which remains unincorporated within the solidlike gel fiber network, can diffuse out and be reacted with an isocyanate, allowing us to quantify the enantioselectivity of component selection but also demonstrating how gels can act as selective reservoirs of potential reagents, releasing them on demand to undergo further reactions; hence, component-selective gel assembly can be coupled with controlled reactivity

Neptune to the Common-wealth of England (1652): the republican Britannia and the continuity of interests

In the seventeenth century, John Kerrigan reminds us, “models of empire did not always turn on monarchy”. In this essay, I trace a vision of “Neptune’s empire” shared by royalists and republicans, binding English national interest to British overseas expansion. I take as my text a poem entitled “Neptune to the Common-wealth of England”, prefixed to Marchamont Nedham’s 1652 English translation of Mare Clausum (1635), John Selden’s response to Mare Liberum (1609) by Hugo Grotius. This minor work is read alongside some equally obscure and more familiar texts in order to point up the ways in which it speaks to persistent cultural and political interests. I trace the afterlife of this verse, its critical reception and its unique status as a fragment that exemplifies the crossover between colonial republic and imperial monarchy at a crucial moment in British history, a moment that, with Brexit, remains resonant