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

Currency, Exchange, and Inheritance in the Evolution of Symbiosis

Highlights: Inspired by the evolution of eukaryotic organelles, we propose a conceptual framework to study the evolutionary and ecological drivers of symbiosis, including three main elements: a currency, mechanisms of currency exchange, and inheritance. Currency in symbiosis is the type resources that species in a beneficial symbiosis gain from their partner. Currency exchange is a complex process that requires molecular adaptations in one or both partners. We identify two distinct but not mutually exclusive initial evolutionary imperatives for the establishment of symbiosis, termed currency first, in which the initial interaction stems from a common currency exchange between the interacting partners to complement their environmental requirements, and transmission first, in which stable transgenerational transmission precedes the evolution of currency exchange. Symbiotic interactions between eukaryotes and prokaryotes are widespread in nature. Here we offer a conceptual framework to study the evolutionary origins and ecological circumstances of species in beneficial symbiosis. We posit that mutual symbiotic interactions are well described by three elements: a currency, the mechanism of currency exchange, and mechanisms of symbiont inheritance. Each of these elements may be at the origin of symbiosis, with the other elements developing with time. The identity of currency in symbiosis depends on the ecological context of the symbiosis, while the specificity of the exchange mechanism underlies molecular adaptations for the symbiosis. The inheritance regime determines the degree of partner dependency and the symbiosis evolutionary trajectory. Focusing on these three elements, we review examples and open questions in the research on symbiosis

Endothelial Cell and Platelet Bioenergetics: Effect of Glucose and Nutrient Composition

It has been suggested that cells that are independent of insulin for glucose uptake, when exposed to high glucose or other nutrient concentrations, manifest enhanced mitochondrial substrate oxidation with consequent enhanced potential and generation of reactive oxygen species (ROS); a paradigm that could predispose to vascular complications of diabetes. Here we exposed bovine aortic endothelial (BAE) cells and human platelets to variable glucose and fatty acid concentrations. We then examined oxygen consumption and acidification rates using recently available technology in the form of an extracellular oxygen and proton flux analyzer. Acute or overnight exposure of confluent BAE cells to glucose concentrations from 5.5 to 25 mM did not enhance or change the rate of oxygen consumption (OCR) under basal conditions, during ATP synthesis, or under uncoupled conditions. Glucose also did not alter OCR in sub-confluent cells, in cells exposed to low serum, or in cells treated with added pyruvate. Likewise, overnight exposure to fatty acids of varying saturation had no such effects. Overnight exposure of BAE cells to low glucose concentration decreased maximal uncoupled respiration, but not basal or ATP related oxygen consumption. Labeled glucose oxidation to CO2 increased, but only marginally after high glucose exposure while oleate oxidation to CO2 decreased. Overnight exposure to linolenic acid, but not oleic or linoleic acid increased extracellular acidification consistent with enhanced glycolytic metabolism. We were unable to detect an increase in production of reactive oxygen species (ROS) from BAE cells exposed to high medium glucose. Like BAE cells, exposure of human platelets to glucose did not increase oxygen consumption. As opposed to BAE cells, platelet mitochondria demonstrate less respiratory reserve capacity (beyond that needed for basal metabolism). Our data do not support the concept that exposure to high glucose or fatty acids accelerates mitochondrial oxidative metabolism in endothelial cells or platelets

Population Genomics of Parallel Adaptation in Threespine Stickleback using Sequenced RAD Tags

Next-generation sequencing technology provides novel opportunities for gathering genome-scale sequence data in natural populations, laying the empirical foundation for the evolving field of population genomics. Here we conducted a genome scan of nucleotide diversity and differentiation in natural populations of threespine stickleback (Gasterosteus aculeatus). We used Illumina-sequenced RAD tags to identify and type over 45,000 single nucleotide polymorphisms (SNPs) in each of 100 individuals from two oceanic and three freshwater populations. Overall estimates of genetic diversity and differentiation among populations confirm the biogeographic hypothesis that large panmictic oceanic populations have repeatedly given rise to phenotypically divergent freshwater populations. Genomic regions exhibiting signatures of both balancing and divergent selection were remarkably consistent across multiple, independently derived populations, indicating that replicate parallel phenotypic evolution in stickleback may be occurring through extensive, parallel genetic evolution at a genome-wide scale. Some of these genomic regions co-localize with previously identified QTL for stickleback phenotypic variation identified using laboratory mapping crosses. In addition, we have identified several novel regions showing parallel differentiation across independent populations. Annotation of these regions revealed numerous genes that are candidates for stickleback phenotypic evolution and will form the basis of future genetic analyses in this and other organisms. This study represents the first high-density SNP–based genome scan of genetic diversity and differentiation for populations of threespine stickleback in the wild. These data illustrate the complementary nature of laboratory crosses and population genomic scans by confirming the adaptive significance of previously identified genomic regions, elucidating the particular evolutionary and demographic history of such regions in natural populations, and identifying new genomic regions and candidate genes of evolutionary significance

Multi-messenger searches via IceCube’s high-energy neutrinos and gravitational-wave detections of LIGO/Virgo

We summarize initial results for high-energy neutrino counterpart searches coinciding with gravitational-wave events in LIGO/Virgo\u27s GWTC-2 catalog using IceCube\u27s neutrino triggers. We did not find any statistically significant high-energy neutrino counterpart and derived upper limits on the time-integrated neutrino emission on Earth as well as the isotropic equivalent energy emitted in high-energy neutrinos for each event

In-situ estimation of ice crystal properties at the South Pole using LED calibration data from the IceCube Neutrino Observatory

The IceCube Neutrino Observatory instruments about 1 km3 of deep, glacial ice at the geographic South Pole using 5160 photomultipliers to detect Cherenkov light emitted by charged relativistic particles. A unexpected light propagation effect observed by the experiment is an anisotropic attenuation, which is aligned with the local flow direction of the ice. Birefringent light propagation has been examined as a possible explanation for this effect. The predictions of a first-principles birefringence model developed for this purpose, in particular curved light trajectories resulting from asymmetric diffusion, provide a qualitatively good match to the main features of the data. This in turn allows us to deduce ice crystal properties. Since the wavelength of the detected light is short compared to the crystal size, these crystal properties do not only include the crystal orientation fabric, but also the average crystal size and shape, as a function of depth. By adding small empirical corrections to this first-principles model, a quantitatively accurate description of the optical properties of the IceCube glacial ice is obtained. In this paper, we present the experimental signature of ice optical anisotropy observed in IceCube LED calibration data, the theory and parametrization of the birefringence effect, the fitting procedures of these parameterizations to experimental data as well as the inferred crystal properties.</p