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

A quantitative analysis of the optical and material properties of metaphase spindles

Die Metaphasenspindel ist eine selbstorganisierende molekulare Maschine, die die entscheidende Funktion erfüllt, das Genom während der Zellteilung gleichmäßig zu trennen. Spindellänge und -form sind emergente Eigenschaften, die durch komplexe Wechselwirkungsnetzwerke zwischen Molekülen hervorgerufen werden. Obwohl erhebliche Fortschritte beim Verständnis der einzelnen molekularen Akteure erzielt wurden, die ihre Länge und Form beeinflussen, haben wir erst kürzlich damit begonnen, die Zusammenhänge zwischen Spindelmorphologie, Dynamik und Materialeigenschaften zu untersuchen. In dieser Arbeit untersuchte ich zunächst quantitativ die Rolle zweier molekularer Kraftgeneratoren - Kinesin-5 und Dynein - bei der Regulierung der Spindelform von Xenopus-Eiextrakt. Eine Störung ihrer Aktivität veränderte die Spindelmorphologie, ohne die Gesamtmasse der Mikrotubuli zu beeinflussen. Um die Spindelform physikalisch zu stören, wurde ein Optical Stretcher (OS) -Aufbau entwickelt. Obwohl das OS Vesikel in Extrakten verformen könnte, konnte keine Kraft auf Spindeln ausgeübt werden. Die Untersuchung des Brechungsindex der Struktur mittels optischer Beugungstomographie (ODT) ergab, dass es keinen Unterschied zwischen Spindel und Zytoplasma gab. Korrelative Fluoreszenz- und ODT-Bildgebung zeigten, wie sich die Materialeigenschaften innerhalb verschiedener Biomoleküle räumlich unterschieden. Die Gesamttrockenmasse der Spindel skalierte mit der Länge, während die Gesamtdichte konstant blieb. Interessanterweise waren die Spindeln in HeLa-Zellen dichter als das Zytoplasma. Schließlich deckte eine störende Mikrotubulusdichte auf, wie die Gesamttubulinkonzentration die Spindelgröße, die Gesamtmasse und die Materialeigenschaften regulierte. Insgesamt bietet diese Studie eine grundlegende Charakterisierung der physikalischen Eigenschaften der Spindel und hilft dabei, Zusammenhänge zwischen der Biochemie und der Biophysik einer aktiven Form weicher Materie zu beleuchten.The metaphase spindle is a self-organising molecular machine that performs the critical function of segregating the genome equally during cell division. Spindle length and shape are emergent properties brought about by complex networks of interactions between molecules. Although significant progress has been made in understanding the individual molecular players influencing its length and shape, we have only recently started exploring the links between spindle morphology, dynamics, and material properties. A thorough analysis of spindle material properties is essential if we are to comprehend how such a dynamic structure responds to forces, and maintains its steady-state length and shape. In this work, I first quantitatively investigated the role of two molecular force generators– Kinesin-5 and Dynein in regulating Xenopus egg extract spindle shape. Perturbing their activity altered spindle morphology without impacting total microtubule mass. To physically perturb spindle shape, an Optical Stretcher (OS) setup was developed. Although the OS could deform vesicles in extracts, force could not be exerted on spindles. Investigating the structure’s refractive index using Optical Diffraction Tomography (ODT) revealed that there was no difference between the spindle and cytoplasm. Correlative fluorescence and ODT imaging revealed how material properties varied spatially within different biomolecules. Additionally, spindle mass density and the microtubule density were correlated. The total dry mass of the spindle scaled with length while overall density remained constant. Interestingly, spindles in HeLa cells were denser than the cytoplasm. Finally, perturbing microtubule density uncovered how total tubulin concentration regulated spindle size, overall mass and material properties. Overall, this study provides a fundamental characterisation of the spindle’s physical properties and helps illuminate links between the biochemistry and biophysics of an active form of soft matter

Elucidation of the Clustered Nano-Architecture of Radiation-Induced DNA Damage Sites and Surrounding Chromatin in Cancer Cells: A Single Molecule Localization Microscopy Approach

In cancer therapy, the application of (fractionated) harsh radiation treatment is state of the art for many types of tumors. However, ionizing radiation is a “double-edged sword”—it can kill the tumor but can also promote the selection of radioresistant tumor cell clones or even initiate carcinogenesis in the normal irradiated tissue. Individualized radiotherapy would reduce these risks and boost the treatment, but its development requires a deep understanding of DNA damage and repair processes and the corresponding control mechanisms. DNA double strand breaks (DSBs) and their repair play a critical role in the cellular response to radiation. In previous years, it has become apparent that, beyond genetic and epigenetic determinants, the structural aspects of damaged chromatin (i.e., not only of DSBs themselves but also of the whole damage-surrounding chromatin domains) form another layer of complex DSB regulation. In the present article, we summarize the application of super-resolution single molecule localization microscopy (SMLM) for investigations of these structural aspects with emphasis on the relationship between the nano-architecture of radiation-induced repair foci (IRIFs), represented here by γH2AX foci, and their chromatin environment. Using irradiated HeLa cell cultures as an example, we show repair-dependent rearrangements of damaged chromatin and analyze the architecture of γH2AX repair clusters according to topological similarities. Although HeLa cells are known to have highly aberrant genomes, the topological similarity of γH2AX was high, indicating a functional, presumptively genome type-independent relevance of structural aspects in DSB repair. Remarkably, nano-scaled chromatin rearrangements during repair depended both on the chromatin domain type and the treatment. Based on these results, we demonstrate how the nano-architecture and topology of IRIFs and chromatin can be determined, point to the methodological relevance of SMLM, and discuss the consequences of the observed phenomena for the DSB repair network regulation or, for instance, radiation treatment outcomes