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

MTrack: Automated Detection, Tracking, and Analysis of Dynamic Microtubules

Microtubules are polar, dynamic filaments fundamental to many cellular processes. In vitro reconstitution approaches with purified tubulin are essential to elucidate different aspects of microtubule behavior. To date, deriving data from fluorescence microscopy images by manually creating and analyzing kymographs is still commonplace. Here, we present MTrack, implemented as a plug-in for the open-source platform Fiji, which automatically identifies and tracks dynamic microtubules with sub-pixel resolution using advanced objection recognition. MTrack provides automatic data interpretation yielding relevant parameters of microtubule dynamic instability together with population statistics. The application of our software produces unbiased and comparable quantitative datasets in a fully automated fashion. This helps the experimentalist to achieve higher reproducibility at higher throughput on a user-friendly platform. We use simulated data and real data to benchmark our algorithm and show that it reliably detects, tracks, and analyzes dynamic microtubules and achieves sub-pixel precision even at low signal-to-noise ratios.V.K. was supported by the IRI Life Sciences postdoc fellowship in the labs of S.R. and S.P. C.H. and S.R. acknowledge funding by the IRI Life Sciences (Humboldt-Universität zu Berlin, Excellence Initiative/DFG). W.H. was supported by the Alliance Berlin Canberra “Crossing Boundaries: Molecular Interactions in Malaria”, which is co-funded by a grant from the Deutsche Forschungsgemeinschaf (DFG) for the International Research Training Group (IRTG) 2290 and the Australian National University. S.P. was supported by the MDC Berlin

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

Predicting disordered regions driving phase separation of proteins under variable salt concentration

We determine the intrinsically disordered regions (IDRs) of phase separating proteins and investigate their impact on liquid-liquid phase separation (LLPS) with a random-phase approx- imation (RPA) that accounts for variable salt concentration. We focus on two proteins, PGL-3 and FUS, known to undergo LLPS. For PGL-3 we predict that an IDR near the C-terminus pro- motes LLPS, which we validate through direct comparison with in vitro experimental results. For the structurally more complex protein FUS the role of the low complexity (LC) domain in LLPS is not as well understood. Apart from the LC domain we here identify two IDRs, one near the N-terminus and another near the C-terminus. Our RPA analysis of these domains predict that, surprisingly, the IDR at the N-terminus (aa 1-285) and not the LC domain promotes LLPS of FUS by comparison to in vitro experiments under physiological temperature and salt conditions

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

No Evidence for Immune Priming in Ants Exposed to a Fungal Pathogen

There is accumulating evidence that invertebrates can acquire long-term protection against pathogens through immune priming. However, the range of pathogens eliciting immune priming and the specificity of the response remain unclear. Here, we tested if the exposure to a natural fungal pathogen elicited immune priming in ants. We found no evidence for immune priming in Formica selysi workers exposed to Beauveria bassiana. The initial exposure of ants to the fungus did not alter their resistance in a subsequent challenge with the same fungus. There was no sign of priming when using homologous and heterologous combinations of fungal strains for exposure and subsequent challenges at two time intervals. Hence, within the range of conditions tested, the immune response of this social insect to the fungal pathogen appears to lack memory and strain-specificity. These results show that immune priming is not ubiquitous across pathogens, hosts and conditions, possibly because of immune evasion by the pathogen or efficient social defences by the host

Quantitative analysis of conditional gene inactivation using rationally designed, tetracycline-controlled miRNAs

The combination of RNA interference (RNAi) with the tetracycline-controlled transcription activation (tet) system promises to become a powerful method for conditional gene inactivation in cultured cells and in whole organisms. Here, we tested critical sequence elements that originated from miRNA mR-30 for optimal efficiency of RNAi-based gene knockdown in mammalian cells. Rationally designed miRNAs, expressed conditionally via the tet system, led to an efficient knockdown of the expression of both reporter genes and the endogenous mitotic spindle protein TPX2 in HeLa cells. Quantitative studies of the tet-controlled gene inactivation revealed that the residual expression of the target gene is an intrinsic attribute of all cells that cannot be eliminated either by increasing the miRNA to target mRNA ratio or by simultaneous expression of miRNAs targeting different sequences within the transcript. The kinetic analysis of the reversibility of the miRNA mediated knockdown suggests that the recovery of target gene expression is primarily driven by cell division. Our miRNA design provides a useful tool for conditional gene inactivation in combination with the RNA-polymerase II based tet system. The identified characteristics of the conditional RNAi-mediated knockdown need to be considered for its application in cell culture or in vivo

Evaluation of changes in the epidemiology of leptospirosis in dogs after introduction of a quadrivalent antileptospiral vaccine in a highly endemic area

Background - Since 2003, a marked increase in leptospirosis serogroup Australis has been observed in dogs in Switzerland. In 2013, a new quadrivalent antileptospiral vaccine (L4) was introduced, adding serogroups Australis and Grippotyphosa to Canicola and Icterohaemorrhagiae of the previous bivalent vaccines (L2). Objective - To examine whether introduction of L4 was associated with decreased incidence of leptospirosis and decreased odds for dogs with acute kidney injury (AKI) to be diagnosed with leptospirosis. Animals - Four hundred and sixty‐nine dogs with AKI presented to a referral hospital, including 269 dogs with leptospirosis and 200 controls with other causes. Methods - Descriptive section: disease incidence was evaluated for 3 consecutive periods: before (PRE, 2011‐2012), transition (TRANS, 2013‐2014), and after introduction of L4 (POST, 2015‐2017). Analytical section: variables associated with a diagnosis of leptospirosis were investigated in a case‐control study using multivariable logistic regression, and focusing on vaccination. Results - The number of dogs diagnosed with leptospirosis (AKI‐L) decreased from 56.5 (PRE) to 15.7 (POST) cases/year while controls increased from 16.5 to 38.0 cases/year. Control dogs (AKI‐nL) showed a decrease in L2 vaccination (100% to 26%) and an increase in L4 vaccination (0% to 70%). The odds ratio for vaccinated dogs to be diagnosed with leptospirosis was 0.11 (95% confidence interval [CI], 0.06‐0.22; P < .001) for L4 and 2.08 (0.58‐7.42; P = .26) for L2. Conclusions and Clinical Importance - The introduction of L4 was associated with a marked decrease in dogs with leptospirosis and AKI in Switzerland. Use of the L4 vaccine was associated with significantly decreased odds of disease