<p>by Anubhav Dhar, Sucheta Dey, Sanjana Mullick, Nishant Kumar Suman, Maxime C. van Zwam, Nishaant Kumar Palani Balaji, Angana Ghosh, Deepak Nair, Koen van den Dries, Sudarshan Gadadhar, Saravanan Palani</p>
Expansion microscopy (ExM) has revolutionized super-resolution imaging in cell biology due to its simple and inexpensive workflow. The use of ExM has revealed several novel insights into the nanoscale architectures of cellular protein complexes, especially the microtubule cytoskeleton in model and non-model systems. Despite tremendous progress in expansion microscopy protocols that preserve cellular ultrastructure (U-ExM), compatible probes for imaging actin isoforms with U-ExM are still lacking and have hindered the study of diverse actin isoforms and networks across model systems. Here, we use IntAct, an internally tagged actin that incorporates into cellular actin networks, to develop and optimize U-ExM for diverse actin structures in yeast, mammalian cells, and primary neurons. Using ALFA-tagged IntAct variants, we achieve robust visualization of actin patches, cables, and rings in yeast, as well as diverse actin architectures including the cortex, stress fibers, filopodia, and lamellipodia in mammalian cells at improved resolution. In primary hippocampal neurons, IntAct efficiently labels actin throughout the soma and neuronal projections, revealing strong enrichment at dendritic spines and synaptic boutons. Notably, we observe a periodic organization of F-actin along axons consistent with the membrane-associated periodic cytoskeleton, thereby resolving the periodic, sub-diffraction actin ring organization. We also detect transient nuclear actin filaments using IntAct-U-ExM underscoring the advantages offered by our approach to image understudied actin structures. Overall, we demonstrate the effectiveness of IntAct-U-ExM for performing super-resolution imaging of various actin structures in an isoform-specific manner and highlight the potential of IntA…
Science Journals
Phytoplankton serve as a source of nutrients for bacteria in the marine environment. The interactions between algae and bacteria are known to include mutualism, commensalism, competition, or antagonism. This occurs in the microenvironment surrounding phytoplankton cells, the phycosphere, an interface rich in nutrients and organic molecules exuded by the cells. Here, based on <i>in situ</i> observations and on an <i>in vitro</i> interaction study, we report on a novel form of starvation-induced hunting that the cells of selected Vibrio species exert on dinoflagellates. The results showed that <i>Vibrio atlanticus</i> was capable of attacking and killing the dinoflagellate <i>Alexandrium pacificum</i> ACT03. Briefly, the observed mechanism of algal-killing consists of first, the ‘immobilization stage’ involving the secretion of algicidal metabolites that disrupt the flagella of the algae. In the ‘attack stage’, Vibrios simultaneously surround algal cells at high density for a brief period without invading them. Finally, the ‘killing stage’ in which the lysis and consumption of the dinoflagellates occur. By using a combination of biochemical, proteomic, molecular, and fluorescence microscopy approaches, we showed that this relationship is not related to the decomposition of algal organic matter, <i>Vibrio</i> quorum-sensing pathways, toxicity of the algae, or pathogenicity of the bacterium but is conditioned by nutrient stress, iron availability, and linked to the iron-vibrioferrin transport system of <i>V. atlanticus</i>. This is the first evidence of a new mechanism that could be involved in regulating <i>Alexandrium</i> spp. blooms and giving Vibrio a competitive advantage in obtaining nutrients from the environment. The interaction model we propose here suggests that Vibrio could play a role in regulating the proliferation of <i>Alexandrium</i> spp., giving it a competitive advantage in obtaining nutrients from the environment.
Traumatic brain injury (TBI) often results in persistent learning and memory deficits, likely due to disrupted hippocampal circuitry underlying these processes. Precise temporal control of hippocampal neuronal activity is thought to be important for memory encoding and retrieval and is supported by oscillations that dynamically organize single-unit firing. Using high-density laminar electrophysiology, we found a loss of oscillatory power across CA1 lamina, with a profound, layer-specific reduction in theta-gamma phase-amplitude coupling in injured rats. Interneurons from injured animals were less strongly entrained to theta and gamma oscillations, but both interneurons and pyramidal cells from injured animals became more strongly entrained to theta during periods of high theta power. During quiet immobility, sharp-wave ripple amplitudes were lower in injured animals compared to shams. These results reveal physiological deficits across brain states that may contribute to TBI-associated learning and memory impairments and elucidate potential targets for future neuromodulation therapies.
Chromosomes must efficiently and properly interact with the mitotic spindle during prometaphase for correct segregation in anaphase. Chromosomes at the nuclear periphery or behind the spindle poles interact less efficiently with the mitotic spindle, increasing the risk of missegregation. The mechanisms that mitigate such risks in unperturbed cells are unknown. An actomyosin network (PANEM) forms around the nucleus during prophase. While the myosin-II-dependent PANEM contraction immediately after nuclear envelope breakdown (NEBD) facilitates chromosome interaction with the mitotic spindle, the mechanism by which it does so remains unclear. Here, using human cell lines, we show that immediately after NEBD, PANEM contraction directly pushes chromosomes at the nuclear periphery or behind spindle poles toward the center of cells. Detailed tracking of kinetochore movements following light-induced activation of a myosin II inhibitor reveals that this inward movement of chromosomes facilitates kinetochores’ initial interaction with spindle microtubules. It also promotes the onset of kinetochores’ congression toward the spindle mid-plane, but not congression itself once it starts. Thus, PANEM contraction ensures high-fidelity chromosome segregation by relocating chromosomes from unfavorable locations. Since some chromosomally unstable cancer cells fail to establish PANEM during early mitosis, the absence of PANEM may contribute to numerical chromosomal instability in these cells.
Touch plays a key role in our perception of our body and shapes our interactions with the world, from the objects we manipulate to the people we touch. While the tactile sensibility of the hand has been extensively characterized, much less is known about touch on other parts of the body. Despite the important role of the breast in lactation, as well as in affective and sexual touch, relatively little is known about its sensory properties. To fill this gap, we investigated the ability of women to locate touches on the breast and compared it to that of the hand and back, body regions that span the range of tactile discriminative capabilities. First, we found that the tactile precision of the breast was even lower than that of the back, heretofore the paragon of poor precision. Second, precision was lower for breasts that had undergone greater expansion, consistent with the hypothesis that innervation capacity does not scale with body size. Third, touches to different regions of the nipple were largely indistinguishable, suggesting sparse innervation density. Fourth, localization errors were systematically biased toward the nipple.
<p>by Yagmur Ozturk, Solal Pirelli, Reese A. K. Richardson</p>
Investigating the integrity of published scientific papers is key to the scientific process, but the necessary knowledge is in short supply. We present COSIG, an open collection of meta-scientific guides enabling anyone to perform forensic peer review.
Investigating the integrity of published scientific papers is key to the scientific process, but the necessary knowledge is in short supply. This Community Page presents COSIG, an open collection of meta-scientific guides enabling anyone to perform forensic peer review.
<p>by Mingzi Xu, Lata Kalra</p>
How does the tempo of animal communication signals evolve? This Primer explores two recent reports published in PLOS Biology, identifying a hotspot of signal tempos between 0.5-4 Hz and independently hypothesizing that delta waves in the receivers’ brains may drive the evolution of this pattern.
<p>by Thi D. Nguyen, Mia J. Konjikusic, Lorenzo M. Del Castillo, Roshanak Irannejad, Jeremy F. Reiter</p>
Hedgehog (HH) signaling in vertebrates is dependent on the primary cilium, an organelle that scaffolds signal transduction. HH signals induce ciliary enrichment of Smoothened (SMO) and ciliary departure of the G protein-coupled receptor (GPCR) GPR161 to trigger GLI activation of the HH transcriptional program. Recently, SMO has been shown to inhibit protein kinase A (PKA). To test the hypothesis that SMO inhibits PKA at cilia to activate the HH signal transduction pathway, we developed a ciliary PKA reporter. Ciliary PKA activity was graded during zebrafish development. Activation of the HH signal transduction pathway by either Sonic hedgehog (SHH) or SMO agonist (SAG) inhibited ciliary PKA activity. Blocking SMO phosphorylation by GRK2/3 prevented ciliary SMO from inhibiting ciliary PKA activity. The SMO C-terminal PKA pseudosubstrate site was critical for SMO-mediated inhibition of ciliary PKA activity. A ciliary GPCR, SSTR3, activated ciliary PKA and induced HH transcriptional responses in NIH-3T3 cells via a different mechanism: activation of Gα<sub>i/o</sub>. A different ciliary GPCR, GPR161, possesses an A-Kinase Anchoring Protein (AKAP), which we found was critical for the ciliary localization of the catalytic subunit of PKA (PKA-C) to promote ciliary PKA activity. We propose that HH signal transduction is inhibited by GPR161-mediated ciliary enrichment of PKA-C, and activated by GRK2/3-phosphorylated SMO inhibition of ciliary PKA activity.
Deep mutational scanning reveals pharmacologically relevant insights into TYK2 signaling and disease
Tyrosine kinase 2 (TYK2) is a genetically defined target for autoimmune disease, with first-generation inhibitors showing clinical success in some but not all associated indications. A deeper understanding of TYK2 structure-function relationships, protein-ligand interactions, and the impact of human variants could inform next-generation therapeutics. Here, we applied deep mutational scanning (DMS) to assess >23,000 amino acid substitutions across two TYK2 functions: interferon alpha (IFN-α) signaling and protein abundance. This enabled high-resolution structure-function mapping and the identification of novel allosteric sites. By coupling DMS with inhibitor treatment, we uncovered variants that modulate compound potency. We also show that human variants – both common and rare – that are protective against autoimmune phenotypes reduce TYK2 protein abundance. Together, these findings demonstrate that DMS can prospectively reveal novel druggable sites, clarify structure-activity relationships (SAR), and highlight TYK2 degradation as a potential therapeutic strategy in autoimmunity.
Snake venoms contain diverse mixtures of toxins that evolved to incapacitate prey, but in humans, they cause extensive pathology following snakebite envenomation. In viper venom, some of the most potent toxins are the haemorrhagic and coagulopathic snake venom metalloproteinases (SVMPs). Because venoms contain an SVMP cocktail and due to their cytotoxicity, SVMP characterisations have been hampered by the lack of purified enzymes. By incorporating their prodomain, which blocks the active SVMP site, we overcame their cytotoxicity and enabled recombinant production of zymogens from all three structurally variable SVMP classes (PI, PII, and PIII) using our baculovirus/insect cell expression system. Zymogens were auto-activated by incubation with Zn<sup>2+</sup> ions, resulting in prodomain cleavage, PII disintegrin cleavage and PIII prodomain proteolysis. Auto-activated SVMPs were characterised using protein substrate degradation, platelet aggregation and blood coagulation assays, benchmarked to native venom-purified SVMP. Our recombinant zymogen production protocol is generically applicable for the expression of SVMPs, unlocking biomedical use in haematology and discovery of novel snakebite therapeutics.
Size is a fundamental property of cells that influences many aspects of their physiology. This is because cell size sets the scale for all subcellular components and drives changes in the composition of the proteome. Given that large and small cells differ in their biochemical composition, we hypothesized that they should also differ in how they respond to signals and make decisions. Here, we investigated how cell size affects the susceptibility of human cells to cell death. We found that large cells are more resistant to ferroptosis caused by system x<sub>c</sub><sup>-</sup> inhibition. Ferroptosis is a type of cell death characterized by the iron-dependent accumulation of toxic lipid peroxides. This process is opposed by cysteine-dependent lipid peroxide detoxification mechanisms. We found that larger cells exhibit higher concentrations of the cysteine-containing metabolite glutathione and lower concentrations of membrane lipid peroxides. Mechanistically, this can be explained by the fact that larger cells had lower concentrations of an enzyme that enriches cellular membranes with peroxidation-prone polyunsaturated fatty acids, ACSL4, and increased concentrations of the glutathione-producing enzymes glutamate-cysteine ligase and glutathione synthetase, the iron-chelating protein ferritin, and the lysosomal protease cathepsin B, which can catabolize cysteine-rich extracellular proteins to produce additional cystine for fueling the synthesis of glutathione. Taken together, our results highlight the significant impact of cell size on cellular function and survival, revealing a size-dependent vulnerability to ferroptosis that could influence therapeutic strategies based on this cell death pathway.
Altered sensory perception is a hallmark of autism and shapes how individuals engage with their environment, with tactile perception playing a critical role in daily functioning and for social interactions. While sensory alterations are thought to contribute to cognitive differences in autism, the impact of cognition on sensory perception remains unclear. Here, we investigated how cognitive processes modulate tactile perception in the <i>Fmr1-</i>KO genetic mouse model of autism through a translational perceptual decision-making task. Our results revealed salience-dependent cognitive alterations that influenced sensory performance. During training, <i>Fmr1</i><sup>-/y</sup> male mice distinguishing between a high- and a low-salience stimulus exhibited an increased choice consistency bias in low-salience trials. When tested across a continuum of intermediate stimulus intensities, these mice demonstrated enhanced tactile discrimination of low-salience stimuli but reduced discrimination facilitation for stimuli crossing category boundaries. These effects were accompanied by diminished integration of sensory history and were dissociable from the attention deficits that emerged under high cognitive load. Together, our findings reveal that tactile perceptual alterations reflect context-dependent weighting and integration of sensory information during decision-making rather than uniform sensory deficits or enhancements, supporting a shift beyond traditional sensory–cognitive dichotomies.
S1–S8 Figs were uploaded incorrectly. Please see the correct S1–S8 Figs below.
Supporting information
S1 Fig. Behavior plots for both rules.
A: Cartoon showing the organization of the AM pure tones in each rule for ferret B. “T” is a go stimulus, “R” is a reference stimulus. Ferret A would be the same, but with T and R locations reversed. B: Measured psychometric curves for ferret A, for each rule. C: Same as B, but for ferret B.
(EPS)
S2 Fig. Demonstration of the method used for judging anatomical correspondences.
In order to judge the anatomical location of any particular micromanipulator position, information from all anatomical images was taken into account. The images for each ferret were taken at anterio-posterior steps of 200 micrometers. Images on the left are anterior, and on the right are posterior. The AP position estimates can be used for comparison to pages 86, 92, 98, 104, and 110 of the Radtke-Schuller ferret atlas [17]. Particularly important landmarks in this case include the cruciate sulcus, which branches out from the interhemispheric fissure in a Y formation. Moving anterior from the branch point, it becomes more and more lateral. Also important and reliable as an anatomical anchor is the corpus callosum: when it is present, the large blood vessel in the intrahemispheric fissure has a “gap” in the image. Moving anterior, the corpus callosum is no longer present. This transition, according to the atlas, happens in between 26.1 and 25.5 mm anterior to the occipital crest. The example slice in the text was ferret A at −25.9. Green boxes surround the locations used for repeated recordings; an arrow at the bottom points to the recording location used as an example throughout the text.
(EPS)
S3 Fig. Replication of example figures from Figs 1, 2, and 3 for all slices and both ferrets.
The ferret and estimated slice AP position are noted on the left of each row. The example slice from the main text is in the second row. A, E, I, M: Percents correctly …
<p>by Theophane Piette, Chundra Cathcart, Chiaria Barbieri, Keesha Martin Ming, Didier Grandjean, Balthasar Bickel, Eloïse Déaux, Anne-Lise Giraud</p>
Acoustic communication is crucial for survival across the animal kingdom, with acoustic signals being shaped by the interaction of producer and receiver selective pressures. While spectral features’ variation reflects species-specific selection, the evolutionary history of acoustic communication rhythms, i.e., the rhythmic modulations of acoustic signals, remains unknown. Using data from 98 species spanning primarily mammals and birds, with additional representation from amphibians, reptiles, fishes, and insects, we investigate the origins of acoustic communication rhythms, notably whether they are shaped by the producer’s anatomical characteristics, environmental constraints, or social complexity. Regression models which controlled for phylogenetic relatedness did not support an influence of these species-specific selective forces; instead, explicit phylogenetic models of trait evolution showed that most species’ rhythms are conserved around an evolutionary optimum of 2.7 Hz that falls within the neural delta range (1–4 Hz) and predates mammalian divergence. Given the known conserved brain oscillations across species and delta involvement in active sensing, we propose that, unlike spectral features, acoustic rhythm could be governed by a universal neural mechanism facilitating effective intra and interspecific communication via a shared channel that has persisted through evolutionary times.
<p>by Mckenna Eklund, Edan Foley</p>
Mutations in the innate immune receptor NOD2 are the greatest single genetic risk factor for Crohn’s disease, yet the mechanisms by which NOD2 regulates intestinal homeostasis remain unclear. We used a CRISPR-generated zebrafish model to determine the impacts of NOD2 deficiency on intestinal health. In cellular, molecular, and transcriptomic studies, we uncovered substantial effects of NOD2 deficiency on epithelial and immune compartments, including deregulated expression of developmental pathways that establish and maintain the gut epithelium, and an unexpected increase in the expression of multiple estrogen-response genes. In functional assays, we uncovered a mechanistic link between estrogenic signals and NOD2-deficiency phenotypes, whereby exposure to estrogen alone replicated the effects of NOD2-deficiency, and treatment with the estrogen receptor modulator tamoxifen reverted the epithelial defects observed in <i>nod2</i> mutants. Our findings identify a NOD2-estrogen regulatory axis that supports intestinal homeostasis and suggest that hormonal signaling may contribute to sex-specific aspects of Crohn’s disease.
This cohort study examines whether combining clinical history, imaging, and genetic and blood markers could predict risk of adverse events in patients with hypertrophic cardiomyopathy, including sudden cardiac death and heart failure.
This JAMA Insights discusses the epidemiology, clinical presentation, and diagnostic evaluation of benign paroxysmal positional vertigo.
Listen to the JAMA Editor’s Summary for an overview and discussion of the important articles appearing in JAMA.
The World Health Organization (WHO) prequalified the first malaria treatment developed specifically for newborns and young infants.
This Medical News article discusses the critical response to a proposal to split obesity into preclinical and clinical categories and diagnose it using more than just body mass index (BMI).
M. Pinson Neal, M.D., Columbia, Mo.
This Medical News article discusses the US Food and Drug Administration’s plans to increase the availability of unapproved peptides for which there is little evidence of safety and efficacy.
It’s self on self. What a war cry when it is me I crucify. Victor and vanquished are both me. Plumb the depths to send up a plea to me the receiver most high.
Less than half of patients undergoing intrauterine device (IUD) insertion at a large academic health system received a paracervical block, according to a study published in JAMA Network Open.