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The extremophile Alvinella pompejana, an annelid worm living on the edge of hydrothermal vents in the Pacific Ocean, is an excellent model system for studying factors that govern protein stability. Low intrinsic stability is a crucial factor for the susceptibility of the transcription factor p53 to inactivating mutations in human cancer. Understanding its molecular basis may facilitate the design of novel therapeutic strategies targeting mutant p53. By analyzing expressed sequence tag (EST) data, we discovered a p53 family gene in A. pompejana. Protein crystallography and biophysical studies showed that it has a p53/p63-like DNA-binding domain (DBD) that is more thermostable than all vertebrate p53 DBDs tested so far, but not as stable as that of human p63. We also identified features associated with its increased thermostability. In addition, the A. pompejana homolog shares DNA-binding properties with human p53 family DBDs, despite its evolutionary distance, consistent with a potential role in maintaining genome integrity. Through extensive structural and phylogenetic analyses, we could further trace key evolutionary events that shaped the structure, stability, and function of the p53 family DBD over time, leading to a potent but vulnerable tumor suppressor in humans.
Cryo-electron tomography provides a snapshot of the cellular proteome. With template matching, the spatial positions of various macromolecular complexes within their native cellular context can be detected. However, the growing awareness of the reference bias introduced by the cross-correlation based approaches, and more importantly the lack of a reliable confidence measurement in the selection of these macromolecular complexes, has restricted the use of these applications. Here we propose a heuristic, in which the reference bias is measured in real space in an analogous way to the R-free value in X-ray crystallography. We measure the reference bias within the mask used to outline the area of the template, and do not modify the template itself. The heuristic works by splitting the mask into a working and a testing area in a volume ratio of 9:1. While the working area is used during the calculation of the cross-correlation function, the information from both areas is explored to calculate the M-free score. We show using artificial data, that the M-free score gives a reliable measure for the reference bias. The heuristic can be applied in template matching and in sub-tomogram averaging. We further test the applicability of the heuristic in tomograms of purified macromolecules, and tomograms of whole Mycoplasma cells.
The Kinase Chemogenomic Set (KCGS): An open science resource for kinase vulnerability identification
(2019)
We describe the assembly and annotation of a chemogenomic set of protein kinase inhibitors as an open science resource for studying kinase biology. The set only includes inhibitors that show potent kinase inhibition and a narrow spectrum of activity when screened across a large panel of kinase biochemical assays. Currently, the set contains 187 inhibitors that cover 215 human kinases. The kinase chemogenomic set (KCGS) is the most highly annotated set of selective kinase inhibitors available to researchers for use in cell-based screens.
Macrophages exert the primary cellular immune response. Pathogen components like bacterial lipopolysaccharides (LPS) stimulate macrophage migration, phagocytotic activity and cytokine expression. Previously, we identified the poly(A)+ RNA interactome of RAW 264.7 macrophages. Of the 402 RNA-binding proteins (RBPs), 32 were classified as unique in macrophages, including nineteen not reported to interact with nucleic acids before. Remarkably, P23 a HSP90 co-chaperone, also known as cytosolic prostaglandin E2 synthase (PTGES3), exhibited differential poly(A)+ RNA binding in untreated and LPS-induced macrophages. To identify mRNAs bound by P23 and to elucidate potential regulatory RBP functions in macrophages, we immunoprecipitated P23 from cytoplasmic extracts of cross-linked untreated and LPS-induced cells. RNAseq revealed that enrichment of 44 mRNAs was reduced in response to LPS. Kif15 mRNA, which encodes kinesin family member 15 (KIF15), a motor protein implicated in cytoskeletal reorganization and cell mobility was selected for further analysis. Noteworthy, phagocytic activity of LPS-induced macrophages was enhanced by P23 depletion. Specifically, in untreated RAW 264.7 macrophages, decreased P23 results in Kif15 mRNA destabilization, diminished KIF15 expression and accelerated macrophage migration. We show that the unexpected RBP function of P23 contributes to the regulation of macrophage phagocytotic activity and migration.
Optogenetic manipulation of neuronal activity through excitatory and inhibitory opsins has become an indispensable experimental strategy in neuroscience research. For many applications bidirectional control of neuronal activity allowing both excitation and inhibition of the same neurons in a single experiment is desired. This requires low spectral overlap between the excitatory and inhibitory opsin, matched photocurrent amplitudes and a fixed expression ratio. Moreover, independent activation of two distinct neuronal populations with different optogenetic actuators is still challenging due to blue-light sensitivity of all opsins. Here we report BiPOLES, an optogenetic tool for potent neuronal excitation and inhibition with light of two different wavelengths. BiPOLES enables sensitive, reliable dual-color neuronal spiking and silencing with single- or two-photon excitation, optical tuning of the membrane voltage, and independent optogenetic control of two neuronal populations using a second, blue-light sensitive opsin. The utility of BiPOLES is demonstrated in worms, flies, mice and ferrets.
MicroRNAs (miRNAs) are critical post-transcriptional regulators in many biological processes. They act by guiding RNA-induced silencing complexes to miRNA response elements (MREs) in target mRNAs, inducing translational inhibition and/or mRNA degradation. Functional MREs are expected to predominantly occur in the 3' untranslated region and involve perfect base-pairing of the miRNA seed. Here, we generate a high-resolution map of miR-181a/b-1 (miR-181) MREs to define the targeting rules of miR-181 in developing murine T-cells. By combining a multi-omics approach with computational high-resolution analyses, we uncover novel miR-181 targets and demonstrate that miR-181 acts predominantly through RNA destabilization. Importantly, we discover an alternative seed match and identify a distinct set of targets with repeat elements in the coding sequence which are targeted by miR-181 and mediate translational inhibition. In conclusion, deep profiling of MREs in primary cells is critical to expand physiologically relevant targetomes and establish context-dependent miRNA targeting rules.
MicroRNAs (miRNAs) are critical post-transcriptional regulators in many biological processes. They act by guiding RNA-induced silencing complexes to miRNA response elements (MREs) in target mRNAs, inducing translational inhibition and/or mRNA degradation. Functional MREs are expected to predominantly occur in the 3’ untranslated region and involve perfect base-pairing of the miRNA seed. Here, we generate a high-resolution map of miR-181a/b-1 (miR-181) MREs to define the targeting rules of miR-181 in developing murine T-cells. By combining a multi-omics approach with computational high-resolution analyses, we uncover novel miR-181 targets and demonstrate that miR-181 acts predominantly through RNA destabilization. Importantly, we discover an alternative seed match and identify a distinct set of targets with repeat elements in the coding sequence which are targeted by miR-181 and mediate translational inhibition. In conclusion, deep profiling of MREs in primary cells is critical to expand physiologically relevant targetomes and establish context-dependent miRNA targeting rules.
Key Points:
* Deep profiling identifies novel targets of miR-181 associated with global gene regulation.
* miR-181 MREs in repeat elements in the coding sequence act through translational inhibition.
* High-resolution analysis reveals an alternative seed match in functional MREs.
MicroRNAs (miRNAs) are critical post-transcriptional regulators in many biological processes. They act by guiding RNA-induced silencing complexes to miRNA response elements (MREs) in target mRNAs, inducing translational inhibition and/or mRNA degradation. Functional MREs are expected to predominantly occur in the 3' untranslated region and involve perfect base-pairing of the miRNA seed. Here, we generate a high-resolution map of miR-181a/b-1 (miR-181) MREs to define the targeting rules of miR-181 in developing murine T-cells. By combining a multi-omics approach with computational high-resolution analyses, we uncover novel miR-181 targets and demonstrate that miR-181 acts predominantly through RNA destabilization. Importantly, we discover an alternative seed match and identify a distinct set of targets with repeat elements in the coding sequence which are targeted by miR-181 and mediate translational inhibition. In conclusion, deep profiling of MREs in primary cells is critical to expand physiologically relevant targetomes and establish context-dependent miRNA targeting rules.
mRNA localization to subcellular compartments has been reported across all kingdoms of life and it is generally believed to promote asymmetric protein synthesis and localization. In striking contrast to previous observations, we show that in S. cerevisiae the B-type cyclin CLB2 mRNA is localized and translated in the yeast bud, while the Clb2 protein, a key regulator of mitosis progression, is concentrated in the mother nucleus. Using single-molecule RNA imaging in fixed (smFISH) and living cells (MS2 system), we show that the CLB2 mRNA is transported to the yeast bud by the She2-She3 complex, via an mRNA ZIP-code situated in the coding sequence. In CLB2 mRNA localization mutants, Clb2 protein synthesis in the bud is decreased resulting in changes in cell cycle distribution and genetic instability. Altogether, we propose that CLB2 mRNA localization acts as a sensor for bud development to couple cell growth and cell cycle progression, revealing a novel function for mRNA localization.
Legionella pneumophila, a Gram-negative intracellular bacterium, is one of the major causes of Legionnaires’ disease, a specific type of atypical pneumonia. Despite intensive research efforts that elucidated many relevant structural, molecular and medical insights into Legionella’s pathogenicity, Legionnaires’ disease continues to present an ongoing public health concern. Legionella’s virulence is based on its ability to simultaneously hijack multiple molecular pathways of the host cell to ensure its fast replication and dissemination. Legionella usurps the host ubiquitin system through multiple effector proteins, using the advantage of both conventional and unconventional (phosphoribosyl-linked) ubiquitination, thus providing optimal conditions for its replication. In this review, we summarize the current understanding of L. pneumophila from medical, biochemical and molecular perspectives. We describe the clinical disease presentation, its diagnostics and treatment, as well as host-pathogen interactions, with the emphasis on the ability of Legionella to target the host ubiquitin system upon infection. Furthermore, the interdisciplinary use of innovative technologies enables better insights into the pathogenesis of Legionnaires’ disease and provides new opportunities for its treatment and prevention.
Phenotypical screening is a widely used approach in drug discovery for the identification of small molecules with cellular activities. However, functional annotation of identified hits often poses a challenge. The development of small molecules with narrow or exclusive target selectivity such as chemical probes and chemogenomic (CG) libraries, greatly diminishes this challenge, but non-specific effects caused by compound toxicity or interference with basic cellular functions still pose a problem to associate phenotypic readouts with molecular targets. Hence, each compound should ideally be comprehensively characterized regarding its effects on general cell functions. Here, we report an optimized live-cell multiplexed assay that classifies cells based on nuclear morphology, presenting an excellent indicator for cellular responses such as early apoptosis and necrosis. This basic readout in combination with the detection of other general cell damaging activities of small molecules such as changes in cytoskeletal morphology, cell cycle and mitochondrial health provides a comprehensive time-dependent characterization of the effect of small molecules on cellular health in a single experiment. The developed high-content assay offers multi-dimensional comprehensive characterization that can be used to delineate generic effects regarding cell functions and cell viability, allowing an assessment of compound suitability for subsequent detailed phenotypic and mechanistic studies.
Acinetobacter baumannii is a highly antibiotic resistant Gram-negative bacterium that causes life-threatening infections in humans with a very high mortality rate. A. baumannii is an extracellular pathogen with poorly understood virulence mechanisms. Here we report that A. baumannii employs the release of outer membrane vesicles (OMVs) containing the outer membrane protein A (OmpAAb) to promote bacterial pathogenesis and dissemination. OMVs containing OmpAAb are taken up by mammalian cells where they activate the host GTPase dynamin-related protein 1 (DRP1). OmpAAb mediated activation of DRP1 enhances its accumulation on mitochondria that causes mitochondrial fragmentation, elevation in reactive oxygen species (ROS) production and cell death. Loss of DRP1 rescues these phenotypes. Our data show that OmpAAb is sufficient to induce mitochondrial fragmentation and cytotoxicity since its expression in E. coli transfers its pathogenic properties to E. coli. A. baumannii infection in mice also induces mitochondrial damage in alveolar macrophages in an OmpAAb dependent manner. We finally show that OmpAAb is also required for systemic dissemination in the mouse lung infection model. In this study we uncover the mechanism of OmpAAb as a virulence factor in A. baumannii infections and further establish the host cell factor required for its pathogenic effects.
Salt-inducible kinases (SIKs) are key metabolic regulators. Imbalance of SIK function is associated with the development of diverse cancers, including breast, gastric and ovarian cancer. Chemical tools to clarify the roles of SIK in different diseases are, however, sparse and are generally characterized by poor kinome-wide selectivity. Here, we have adapted the pyrido[2,3-d]pyrimidin-7-one-based PAK inhibitor G-5555 for the targeting of SIK, by exploiting differences in the back-pocket region of these kinases. Optimization was supported by high-resolution crystal structures of G-5555 bound to the known off-targets MST3 and MST4, leading to a chemical probe, MRIA9, with dual SIK/PAK activity and excellent selectivity over other kinases. Furthermore, we show that MRIA9 sensitizes ovarian cancer cells to treatment with the mitotic agent paclitaxel, confirming earlier data from genetic knockdown studies and suggesting a combination therapy with SIK inhibitors and paclitaxel for the treatment of paclitaxel-resistant ovarian cancer.
Cryo electron tomography (cryo-ET) combined with subtomogram averaging (StA) enables structural determination of macromolecules in their native context. A few structures were reported by StA at resolution higher than 4.5 Å, however all of these are from viral structural proteins or vesicle coats. Reaching high resolution for a broader range of samples is uncommon due to beam-induced sample drift, poor signal-to-noise ratio (SNR) of images, challenges in CTF correction, limited number of particles. Here we propose a strategy to address these issues, which consists of a tomographic data collection scheme and a processing workflow. Tilt series are collected with higher electron dose at zero-degree tilt in order to increase SNR. Next, after performing StA conventionally, we extract 2D projections of the particles of interest from the higher SNR images and use the single particle analysis tools to refine the particle alignment and generate a reconstruction. We benchmarked our proposed hybrid StA (hStA) workflow and improved the resolution for tobacco mosaic virus from 7.2 to 5.2 Å and the resolution for the ion channel RyR1 in crowded native membranes from 12.9 to 9.1 Å. We demonstrate that hStA can improve the resolution obtained by conventional StA and promises to be a useful tool for StA projects aiming at subnanometer resolution or higher.
Cryo-electron tomography combined with subtomogram averaging (StA) has yielded high-resolution structures of macromolecules in their native context. However, high-resolution StA is not commonplace due to beam-induced sample drift, images with poor signal-to-noise ratios (SNR), challenges in CTF correction, and limited particle number. Here we address these issues by collecting tilt series with a higher electron dose at the zero-degree tilt. Particles of interest are then located within reconstructed tomograms, processed by conventional StA, and then re-extracted from the high-dose images in 2D. Single particle analysis tools are then applied to refine the 2D particle alignment and generate a reconstruction. Use of our hybrid StA (hStA) workflow improved the resolution for tobacco mosaic virus from 7.2 to 4.4 Å and for the ion channel RyR1 in crowded native membranes from 12.9 to 9.1 Å. These resolution gains make hStA a promising approach for other StA projects aimed at achieving subnanometer resolution.
We present a deterministic workflow for genotyping single and double transgenic individuals directly upon nascence that prevents overproduction and reduces wasted animals by two-thirds. In our vector concepts, transgenes are accompanied by two of four clearly distinguishable transformation markers that are embedded in interweaved, but incompatible Lox site pairs. Following Cre-mediated recombination, the genotypes of single and double transgenic individuals were successfully identified by specific marker combinations in 461 scorings.
The original version of this Article contained errors where Table S5 and Table S6 were incorrectly cited. As the result, in the Methods section, under the subheading ‘Germline transformation, crossing setups and insertion junction sequencing’, “Progeny were scored for transformation marker presence during either the larval, pupal and adult stage by using a fluorescence stereo microscope (SteREO Discovery.V8, Zeiss) with appropriate filter sets (Table S4).” now reads: “Progeny were scored for transformation marker presence during either the larval, pupal and adult stage by using a fluorescence stereo microscope (SteREO Discovery.V8, Zeiss) with appropriate filter sets (Table S5).” And, under the subheading ‘Light sheet-based fluorescence microscopy’, “Metadata for the three datasets are provided in Table S5.” now reads: “Metadata for the three datasets are provided in Table S6.” In Data availability section, “Microscopy data can be accessed as described in Table S5.” now reads: “Microscopy data can be accessed as described in Table S6.” Additionally, in the Supplementary Information 8 file, the “Data Access” row was omitted in Table S6. The “Data Access” row now reads: Dataset (DS) DS0001 DS0002 DS0003 Dataset Access DOI: 10.5281/zenodo.4892363 DOI: 10.5281/zenodo.4892373 DOI: 10.5281/zenodo.4892381 The original Supplementary Information 8 file is provided below. Finally, the Supplementary Information 1 and 5 files published with this Article contained tracked changes, these have now been removed. The original Article and accompanying Supplementary Information files have been corrected.
The Mediterranean fruit fly (medfly), Ceratitis capitata, is an important model organism in biology and agricultural research with high economic relevance. However, information about its embryonic development is still sparse. We share nine long-term live imaging datasets acquired with light sheet fluorescence microscopy (484.5 h total recording time, 373 995 images, 256 Gb) with the scientific community. Six datasets show the embryonic development in toto for about 60 hours at 30 minutes intervals along four directions in three spatial dimensions, covering approximately 97% of the entire embryonic development period. Three datasets focus on germ cell formation and head involution. All imaged embryos hatched morphologically intact. Based on these data, we suggest a two-level staging system that functions as a morphogenetic framework for upcoming studies on medfly. Our data supports research on wild-type or aberrant morphogenesis, quantitative analyses, comparative approaches to insect development as well as studies related to pest control. Further, they can be used to test advanced image processing approaches or to train machine learning algorithms and/or neuronal networks.
Natural products can contribute to abiotic stress tolerance in plants and fungi. We hypothesize that biosynthetic gene clusters (BGCs), the genomic elements that underlie natural product biosynthesis, display structured differences along elevation gradients. We analysed biosynthetic gene variation in natural populations of the lichen-forming fungus Umbilicaria pustulata. We collected a total of 600 individuals from the Mediterranean and cold-temperate climates. Population genomic analyses indicate that U. pustulata contains three clusters that are highly differentiated between the Mediterranean and cold-temperate populations. One entire cluster is exclusively present in cold-temperate populations, and a second cluster is putatively dysfunctional in all cold-temperate populations. In the third cluster variation is fixed in all cold-temperate populations due to hitchhiking. In these two clusters the presence of consistent allele frequency differences among replicate populations/gradients suggests that selection rather than drift is driving the pattern. We advocate that the landscape of fungal biosynthetic genes is shaped by both positive and hitchhiking selection. We demonstrate, for the first time, the presence of climate-associated BGCs and BGC variations in lichen-forming fungi. While the associated secondary metabolites of the candidate clusters are presently unknown, our study paves the way for targeted discovery of natural products with ecological significance.
During animal development, it is crucial that cells can sense and adapt to mechanical forces from their environment. Ultimately, these forces are transduced through the actomyosin cortex. How the cortex can simultaneously respond to and create forces during cytokinesis is not well understood. Here we show that under mechanical stress, cortical actomyosin flow switches its polarization during cytokinesis in the C. elegans embryo. In unstressed embryos, longitudinal cortical flows contribute to contractile ring formation, while rotational cortical flow is additionally induced in uniaxially loaded embryos. Rotational cortical flow is required for the redistribution of the actomyosin cortex in loaded embryos. Rupture of longitudinally aligned cortical fibers during cortex rotation releases tension, initiates orthogonal longitudinal flow and thereby contributes to furrowing in loaded embryos. A targeted screen for factors required for rotational flow revealed that actomyosin regulators involved in RhoA regulation, cortical polarity and chirality are all required for rotational flow and become essential for cytokinesis under mechanical stress. In sum, our findings extend the current framework of mechanical stress response during cell division and show scaling of orthogonal cortical flows to the amount of mechanical stress.