Microtome-integrated microscope system for high sensitivity tracking of in-resin fluorescence in blocks and ultrathin sections for correlative microscopy

Many areas of biological research demand the combined use of different imaging modalities to cover a wide range of magnifications and measurements or to place fluorescent patterns into an ultrastructural context. A technically difficult problem is the efficient specimen transfer between different imaging modalities without losing the coordinates of the regions-of-interest (ROI). Here, we report a new and highly sensitive integrated system that combines a custom designed microscope with an ultramicrotome for in-resin-fluorescence detection in blocks, ribbons and sections on EM-grids. Although operating with long-distance lenses, this system achieves a very high light sensitivity. Our instrumental set-up and operating workflow are designed to investigate rare events in large tissue volumes. Applications range from studies of individual immune, stem and cancer cells to the investigation of non-uniform subcellular processes. As a use case, we present the ultrastructure of a single membrane repair patch on a muscle fiber in intact muscle in a whole animal context

Zebrafish biosensor for toxicant induced muscle hyperactivity

Robust and sensitive detection systems are a crucial asset for risk management of chemicals, which are produced in increasing number and diversity. To establish an in vivo biosensor system with quantitative readout for potential toxicant effects on motor function, we generated a transgenic zebrafish line TgBAC(hspb11:GFP) which expresses a GFP reporter under the control of regulatory elements of the small heat shock protein hspb11. Spatiotemporal hspb11 transgene expression in the musculature and the notochord matched closely that of endogenous hspb11 expression. Exposure to substances that interfere with motor function induced a dose-dependent increase of GFP intensity beginning at sub-micromolar concentrations, while washout of the chemicals reduced the level of hspb11 transgene expression. Simultaneously, these toxicants induced muscle hyperactivity with increased calcium spike height and frequency. The hspb11 transgene up-regulation induced by either chemicals or heat shock was eliminated after co-application of the anaesthetic MS-222. TgBAC(hspb11:GFP) zebrafish embryos provide a quantitative measure of muscle hyperactivity and represent a robust whole organism system for detecting chemicals that affect motor function

Evaluating the climate impact of aviation emission scenarios towards the Paris agreement including COVID-19 effects

Aviation is an important contributor to the global economy, satisfying society’s mobility needs. It contributes to climate change through CO2 and non-CO2 effects, including contrail-cirrus and ozone formation. There is currently significant interest in policies, regulations and research aiming to reduce aviation’s climate impact. Here we model the effect of these measures on global warming and perform a bottom-up analysis of potential technical improvements, challenging the assumptions of the targets for the sector with a number of scenarios up to 2100. We show that although the emissions targets for aviation are in line with the overall goals of the Paris Agreement, there is a high likelihood that the climate impact of aviation will not meet these goals. Our assessment includes feasible technological advancements and the availability of sustainable aviation fuels. This conclusion is robust for several COVID-19 recovery scenarios, including changes in travel behaviour

Mandipropamid as a chemical inducer of proximity for in vivo applications

Direct control of protein interactions by chemically induced protein proximity holds great potential for both cell and synthetic biology as well as therapeutic applications. Low toxicity, orthogonality and excellent cell permeability are important criteria for chemical inducers of proximity (CIPs), in particular for in vivo applications. Here, we present the use of the agrochemical mandipropamid (Mandi) as a highly efficient CIP in cell culture systems and living organisms. Mandi specifically induces complex formation between a sixfold mutant of the plant hormone receptor pyrabactin resistance 1 (PYR1) and abscisic acid insensitive (ABI). It is orthogonal to other plant hormone-based CIPs and rapamycin-based CIP systems. We demonstrate the applicability of the Mandi system for rapid and efficient protein translocation in mammalian cells and zebrafish embryos, protein network shuttling and manipulation of endogenous proteins

The Mesoderm Specification Factor Twist in the Life Cycle of Jellyfish

AbstractThe basic helix-loop-helix (bHLH) transcription factor Twist is highly conserved from Drosophila to vertebrates and plays a major role in mesoderm specification of triploblasts. The presence of a Twist homologue in diploblasts such as the cnidarian Podocoryne carnea raises questions on the evolution of mesoderm, the third cell layer characteristic for triploblasts. Podocoryne Twist is expressed in the early embryo until the myoepithelial cells of the larva differentiate and then again during medusa development. There, the gene is detected first when the myoepithelial cells of the polyp dedifferentiate to form the medusa bud and later Twist is found transiently in the entocodon, a mesoderm-like cell layer which differentiates into the smooth muscle and striated muscle of the bell. On the other hand, in later bud stages and the medusa, expression is seen where non-muscle tissues differentiate. Experimental analysis of in vitro transdifferentiation and regeneration demonstrates that Twist activity is not needed when isolated striated muscle regenerate medusa organs. Developmental roles of Twist are discussed with respect to early animal evolution from a common ancestor of cnidarians and bilaterians

Distinct amino acid motifs carrying multiple positive charges regulate membrane targeting of dysferlin and MG53

<div><p>Dysferlin (Dysf) and mitsugumin53 (MG53) are two key proteins involved in membrane repair of muscle cells which are efficiently recruited to the sarcolemma upon lesioning. Plasma membrane localization and recruitment of a Dysf fragment to membrane lesions in zebrafish myofibers relies on the presence of a short, polybasic amino acid motif, WRRFK. Here we show that the positive charges carried by this motif are responsible for this function. In mouse MG53, we have identified a similar motif with multiple basic residues, WKKMFR. A single amino acid replacement, K279A, leads to severe aggregation of MG53 in inclusion bodies in HeLa cells. This result is due to the loss of positive charge, as shown by studying the effects of other neutral amino acids at position 279. Consequently, our data suggest that positively charged amino acid stretches play an essential role in the localization and function of Dysf and MG53.</p></div

Confocal images of C2C12 myoblasts expressing turboGFP:MG53 and mutants K278A, R282A, K279A.

<p>Fluorescence was excited with a 488-nm laser. As in HeLa cells, K279A shows substantial vesicular localization in myoblasts. Scale bars, 10 μm.</p

The cellular distribution of smDysf depends on the positive charge of the WRRFK motif.

<p>The cytoplasmic fraction is significantly increased upon reducing the net charge but not by polypeptide truncation on the C-terminal side of smDysf. For this plot, the cytoplasmic fluorescence of 9–15 myofibers was measured and averaged for each mutant. All data are referenced to the wildtype set to 100% and plotted as mean ± SD. The original data are provides as <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0202052#pone.0202052.s001" target="_blank">S1 Dataset</a>. Zebrafish embryos expressing these ten variants were injected, treated and imaged under identical conditions. The mutant sequences are grouped according to the net charge (1+– 3+) of the different motifs under physiological conditions. Significance was tested against the wildtype control smDysf (WRRFK-TM-C) by Student’s t-test (** <i>p</i> < 0.01, *** <i>p</i> < 0.001).</p