Biology subjects

Scott, B.

Publications and source records attributed to Scott, B..

At least 19 recordsLinked to original sources

bioRxiv2026

Diurnal rhythms of choice: a novel state-dependent drift diffusion model uncovers time-dependent changes in rat decision making

2Time-of-day severely impacts human decision-making, with real-world consequences. Studying shifts in decision-making strategy requires controlled, long timescale behavioral measurement and analyses that can extract insight from time-varying behavior. We introduce two complementary advances to address this gap: an autonomous 24-hour training facility for continuous behavioral measurement during decision-making and an interpretable modeling framework that captures non-stationary decision dynamics from reaction times and choices. Rats were trained on a visual evidence accumulation task across months, generating over a half million trials spanning the circadian period. Our model revealed latent behavioral states characterized by distinct evidence accumulation parameters, including differences in drift rate, bias, and decision-commitment time. These states recur across days and align with feeding schedules and the light-dark cycle, producing periodic fluctuations in performance over 24 hours. Together, these results demonstrate how continuous behavioral sampling combined with generative modeling uncovers long-timescale structure in decision-making obscured by stationary analyses. 1 HIGHLIGHTSO_LI24-hour live-in operant system allows autonomous training in cognitive tasks across months C_LIO_LI24-hour measurements reveal that rat performance fluctuates with time of day C_LIO_LINovel DDM-HMM framework identifies reaction time and accuracy shifts across multiple timescales C_LIO_LIDDM-HMM captures serial dependence in decisions that classic models ignore C_LI

animal behavior and cognition↗

bioRxiv2026

Python metabolomics uncovers a conserved postprandial metabolite and gut-brain feeding pathway

Most mammals consume small and frequent meals. By contrast, pythons are ambush predators that exhibit extreme feeding and fasting patterns and provide a unique model for uncovering molecular mediators of the postprandial response1-3. Using untargeted metabolomics, here we show that circulating levels of the metabolite para-tyramine-O-sulfate (pTOS) are increased >1,000-fold in pythons after a single meal. In pythons, pTOS production occurs in a microbiome-dependent manner via sequential decarboxylation and sulfation of dietary tyrosine. In both pythons and mice, pTOS administration activates a neural population in the ventromedial hypothalamus (VMH). In mice, these VMH neurons are required for the anorexigenic effects of pTOS. Chronic administration of pTOS to diet-induced obese male mice suppresses food intake and body weight. pTOS is also present in human blood, where its levels are increased after a meal. Together, these data uncover a conserved postprandial anorexigenic metabolite that links nutrient intake to energy balance.

physiology↗

bioRxiv2025

Songbird connectome reveals tunneling of migratory neurons in the adult striatum

Immature neurons in the adult brain migrate and integrate into existing circuits, where they contribute to plasticity, learning, and complex behaviors. However, how these cells navigate synapse-rich regions of the adult brain remains poorly understood. While prior studies have examined the molecular mechanisms and functional consequences of adult neurogenesis, few have investigated the physical interactions between migrating neurons and their surrounding environment. Here, we use electron microscopy-based connectomics to examine how migrating neurons interact with mature circuit elements in the adult zebra finch striatum. Immature neurons exhibiting migratory features were observed contacting diverse structures in their microenvironment, including the axons, dendrites, synapses, and somas of mature neurons. Surprisingly, these interactions were structurally complex, often involving pronounced deformations of mature somas and the surrounding neuropil. These deformations appeared as "tunnels" made by the migratory neurons as they displaced mature structures along their path. Together, these findings suggest that migrating neurons may physically reshape the mature circuit to reach their targets, revealing an unexpected degree of structural and functional plasticity in the adult brain.

neuroscience↗

bioRxiv2025

Regulation of Decision Threshold by the Locus Coeruleus

A fundamental challenge for decision-making under uncertainty lies in regulating the balance between speed and accuracy. Humans and animals solve this problem by adjusting their decision thresholds -- the criterion that determines how much information is required before committing to a choice. While brain regions associated with this process have been identified, the neural circuits able to directly alter decision thresholds remain to be identified. Here we investigate the role of the locus coeruleus (LC) norepinephrine (NE) system in controlling the balance between speed and accuracy during decision making. Through cell-type specific chemogenetic manipulations, we discovered that LC-NE activation increased decision thresholds. We further demonstrated that this effect is replicated by systemic administration of the 2 adrenergic receptor (2 AR) agonist, clonidine. Notably, 2 AR activation drove changes in decision threshold specifically, without reproducing other LC-NE activation effects such as promoting task engagement. Together, these results suggest that LC-NE regulates decision thresholds via activation of downstream 2 ARs. HighlightsO_LILC-NE activation increases reaction time and accuracy of perceptual decisions C_LIO_LIBehavioral effects are consistent with an elevation of decision thresholds C_LIO_LIHeightened decision thresholds are replicated by selective activation of 2 ARs C_LIO_LIActivation of LC-NE and 2 ARs differently alter task engagement C_LI

animal behavior and cognition↗

bioRxiv2025

Psychedelics Relax Priors and Reshape Orbitofrontal Dynamics

Psychedelics such as psilocybin and ketamine are gaining attention as rapid-acting treatments for psychiatric disorders, yet the mechanisms by which they alter cognition remain unclear. A key hypothesis--the REBUS model--proposes that psychedelics relax high-level priors, allowing bottom-up sensory information to exert greater influence over perception and behavior. Here, we test this model in mice performing a free-response perceptual decision-making task that disambiguates prior-driven and sensory-driven decision strategies. Acute administration of psilocybin or ketamine significantly slowed decision times and improved accuracy. Behavioral modeling that combined drift diffusion and GLM-HMM frameworks revealed that these changes were mediated by increased decision thresholds and a marked shift into sensory-engaged cognitive states. Whole-brain c-Fos mapping identified a distributed decision-making network, with psychedelics selectively modulating cortical and subcortical nodes. Calcium imaging in the orbitofrontal cortex (OFC)--a key region for integrating priors and sensory inputs--revealed preserved decision-related selectivity under psychedelics, while exhibiting reduced neuronal correlations--population-level signatures of weakened top-down influence and relaxed priors. Together, these results provide circuit-level support for the REBUS model, showing that psychedelics reconfigure brain-wide and local dynamics to promote more deliberate, flexible, and sensory-driven decision policies.

neuroscience↗

bioRxiv2024

Blood-tumor barrier organoids recapitulate glioblastoma microenvironment and enable high-throughput modeling of therapeutic delivery

The blood-brain barrier (BBB) is a highly specialized system that is critical for regulating transport between the blood and the central nervous system. In brain tumors, the vasculature system is compromised, and is referred to as the blood-tumor barrier (BTB). The ability to precisely model the unique physiological properties of the BTB is essential to decipher its role in tumor pathophysiology and for the rational design of efficacious therapeutics. Here, we introduce a robust and high-throughput in vitro 3D human BTB organoid model that recapitulates various key features of the BTB observed in vivo and in clinical GBM samples. The organoids are composed of patient-derived glioblastoma stem cells (GSCs), human brain endothelial cells (EC), astrocytes and pericytes, which are formed through self-assembly. Transcriptomic and functional analyses reveal that the GSCs in the BTB organoids exhibit enhanced level of stemness, mesenchymal signature, invasiveness and angiogenesis, and this is further confirmed in in vivo studies. We demonstrate the ability of the BTB organoids to model therapeutic delivery and drug efficacy on brain tumor cells. Collectively, our findings show that the BTB organoid model has broad utility as a clinically representative system for studying the BTB and evaluating brain tumor therapies.

bioengineering↗

bioRxiv2024

Danicamtiv reduces myosin's working stroke but enhances contraction by activating the thin filament

Heart failure is a leading cause of death worldwide, and even with current treatments, the 5-year transplant-free survival rate is only [~]50-70%. As such, there is a need to develop new treatments for patients that improve survival and quality of life. Recently, there have been efforts to develop small molecules for heart failure that directly target components of the sarcomere, including cardiac myosin. One such molecule, danicamtiv, recently entered phase II clinical trials; however, its mechanism of action and direct effects on myosins mechanics and kinetics are not well understood. Using optical trapping techniques, stopped flow transient kinetics, and in vitro reconstitution assays, we found that danicamtiv reduces the size of cardiac myosins working stroke, and in contrast to studies in muscle fibers, we found that it does not affect actomyosin detachment kinetics at the level of individual crossbridges. We demonstrate that danicamtiv accelerates actomyosin association kinetics, leading to increased recruitment of myosin crossbridges and subsequent thin filament activation at physiologically-relevant calcium concentrations. Finally, we computationally model how the observed changes in mechanics and kinetics at the level of single crossbridges contribute to increased cardiac contraction and improved diastolic function compared to the related myotrope, omecamtiv mecarbil. Taken together, our results have important implications for the design of new sarcomeric-targeting compounds for heart failure. SIGNIFICANCE STATEMENTHeart failure is a leading cause of death worldwide, and there is a need to develop new treatments that improve outcomes for patients. Recently, the myosin-binding small molecule danicamtiv entered clinical trials for heart failure; however, its mechanism at the level of single myosin crossbridges is not well understood. We determined the molecular mechanism of danicamtiv and showed how drug-induced molecular changes can mechanistically increase heart contraction. Moreover, we demonstrate fundamental differences between danicamtiv and the related myosin-binding small molecule omecamtiv mecarbil that explain the improved diastolic function seen with danicamtiv. Our results have important implications for the design of new therapeutics for heart failure.

biophysics↗

bioRxiv2024

A cross-species framework for investigating perceptual evidence accumulation

Cross-species studies are important for a comprehensive understanding of brain functions. However, direct quantitative comparison of behaviors across species presents a significant challenge. To enable such comparisons in perceptual decision-making, we developed a synchronized evidence accumulation task for human and non-human animals, by aligning mechanics, stimuli, and training. The task was readily learned by rats, mice and humans, with each species exhibiting qualitatively similar performance. Quantitative model comparison revealed that all three species employed an evidence accumulation strategy, but differed in speed, accuracy, and key decision parameters. Human performance prioritized accuracy, whereas rodent performance was limited by internal time-pressure. Rats optimized reward rate, while mice appeared to switch between evidence accumulation and other strategies trial-to-trial. Together, these results reveal striking similarities and species-specific priorities in decision-making. Furthermore, the synchronized behavioral framework we present may facilitate future studies involving cross-species comparisons, such as evaluating the face validity of animal models of neuropsychiatric disorders. HighlightsO_LIDevelopment of an evidence accumulation task for rats and mice C_LIO_LISynchronized video game allows direct comparisons with humans C_LIO_LIRat, mouse and human behavior are well fit by the same decision models C_LIO_LIModel parameters reveal species-specific priorities in accumulation strategy C_LI

animal behavior and cognition↗

bioRxiv2023

Modeling Thick Filament Activation Suggests a Molecular Basis for Force Depression

Multiscale models aiming to connect muscles molecular and cellular function have been difficult to develop, in part, due to a lack of self-consistent multiscale data. To address this gap, we measured the force response from single skinned rabbit psoas muscle fibers to ramp shortenings and step stretches performed on the plateau region of the force-length relationship. We isolated myosin from the same muscles and, under similar conditions, performed single molecule and ensemble measurements of myosins ATP-dependent interaction with actin using laser trapping and in vitro motility assays. We fit the fiber data by developing a partial differential equation model that includes thick filament activation, whereby an increase in force on the thick filament pulls myosin out of an inhibited state. The model also includes a series elastic element and a parallel elastic element. This parallel elastic element models a titin-actin interaction proposed to account for the increase in isometric force following stretch (residual force enhancement). By optimizing the model fit to a subset of our fiber measurements, we specified seven unknown parameters. The model then successfully predicted the remainder of our fiber measurements and also our molecular measurements from the laser trap and in vitro motility. The success of the model suggests that our multiscale data are self-consistent and can serve as a testbed for other multiscale models. Moreover, the model captures the decrease in isometric force observed in our muscle fibers after active shortening (force depression), suggesting a molecular mechanism for force depression, whereby a parallel elastic element combines with thick filament activation to decrease the number of cycling cross-bridges. SIGNIFICANCEConnecting the molecular and cellular scales of muscle contraction would assist in, e.g., the treatment of genetic muscle diseases, the development of heart drugs, and the design of prostheses. The history dependence of muscle contraction, having no clear molecular basis, has remained an obstacle in making this connection for the seventy years since its discovery. We measured the force- and motion-generating capacity of rabbit psoas muscle from the scale of single molecules to single cells. We developed a mathematical model that, when fit to some of the cellular measurements, predicted the remaining cellular measurements and also the molecular measurements. The models ability to capture muscles history dependence suggests a unified description of muscle contraction from the molecular to cellular scale.

biophysics↗

bioRxiv2023

Single Molecule Mechanics and Kinetics of Cardiac Myosin Interacting with Regulated Thin Filaments

The cardiac cycle is a tightly regulated process wherein the heart generates force to pump blood to the body during systole and then relaxes during diastole. Disruption of this finely tuned cycle can lead to a range of diseases including cardiomyopathies and heart failure. Cardiac contraction is driven by the molecular motor myosin, which pulls regulated thin filaments in a calcium-dependent manner. In some muscle and non-muscle myosins, regulatory proteins on actin tune the kinetics, mechanics, and load dependence of the myosin working stroke; however, it is not well understood whether or how thin filament regulatory proteins tune the mechanics of the cardiac myosin motor. To address this critical gap in knowledge, we used single-molecule techniques to measure the kinetics and mechanics of the substeps of the cardiac myosin working stroke in the presence and absence of thin filament regulatory proteins. We found that regulatory proteins gate the calcium-dependent interactions between myosin and the thin filament. At physiologically relevant ATP concentrations, cardiac myosins mechanics and unloaded kinetics are not affected by thin filament regulatory proteins. We also measured the load-dependent kinetics of cardiac myosin at physiologically relevant ATP concentrations using an isometric optical clamp, and we found that thin filament regulatory proteins do not affect either the identity or magnitude of myosins primary load-dependent transition. Interestingly, at low ATP concentrations, thin filament regulatory proteins have a small effect on actomyosin dissociation kinetics, suggesting a mechanism beyond simple steric blocking. These results have important implications for both disease modeling and computational models of muscle contraction. Significance StatementHuman heart contraction is powered by the molecular motor {beta}-cardiac myosin, which pulls on thin filaments consisting of actin and the regulatory proteins troponin and tropomyosin. In some muscle and non-muscle systems, these regulatory proteins tune the kinetics, mechanics, and load dependence of the myosin working stroke. Despite having a central role in health and disease, it is not well understood whether the mechanics or kinetics of {beta}-cardiac myosin are affected by regulatory proteins. We show that regulatory proteins do not affect the mechanics or load-dependent kinetics of the working stroke at physiologically relevant ATP concentrations; however, they can affect the kinetics at low ATP concentrations, suggesting a mechanism beyond simple steric blocking. This has important implications for modeling of cardiac physiology and diseases.

biophysics↗

bioRxiv2022

Performance of the cobas(R) HBV RNA Automated Investigational Assay for the Detection and Quantification of Circulating HBV RNA in Chronic HBV Patients

BackgroundThe amount of HBV RNA in peripheral blood may reflect HBV covalently closed circular DNA (cccDNA) transcriptional activity within infected hepatocytes. Quantification of circulating HBV RNA (cirB-RNA) is thus a promising biomarker for monitoring antiviral treatment. ObjectivesWe evaluated the performance of an automated, prototype quantitative HBV RNA assay for use on the Roche cobas(R) 6800/8800 systems. Study DesignThe sensitivity, specificity, linearity, and potential interference by HBV DNA of the cobas(R) HBV RNA assay were assessed using synthetic HBV armored RNA and clinical specimens. Resultscobas(R) HBV RNA results were linear between 10 and 107 copies/mL in clinical samples of several HBV genotypes, and up to 109 copies/mL with synthetic RNA. Precision and reproducibility were excellent, with standard deviation below 0.15 log10 copies/mL and coefficients of variation below 5% throughout the linear range. The presence of HBV DNA had minimal (<0.3 log10 copies/mL) impact on HBV RNA quantification at DNA:RNA ratios of up to approximately one million. In a panel of 36 untreated patient samples, cirB-RNA concentrations were approximately 200-fold lower than HBV DNA. cirB-RNA was detected in all 13 HBeAg-positive patients (mean 6.0 log10 copies/mL), and in 20 of 23 HBeAg-negative patients (mean of quantifiable samples 2.2 log10 copies/mL). Finally, cirB-RNA was detected in 12 of 20 nucleoside analog-treated patients (mean of quantifiable samples 3.4 log10 copies/mL). ConclusionsThe cobas(R) 6800/8800 investigational HBV RNA assay is a high throughput, sensitive and inclusive assay to evaluate the clinical relevance of cirB-RNA quantification in patients with chronic hepatitis B.

microbiology↗

bioRxiv2021

A Hypoxia Sensitive to Hypoxia Resistant Transformation in Long-Lived Germline Mutants

Signals from the germline play a significant role in determining longevity in numerous animal models. In C. elegans, ablation of the germline leads to long life span and various other types of stress resistance. It has been reported that mutations that block oogenesis or an upstream step in germline development confer strong resistance to hypoxia. We report here that the hypoxia resistance of sterile mutants is dependent on developmental stage and age. In just a 12-hour period, sterile animals transform from hypoxia sensitive L4 larvae into highly hypoxia resistant adults. Since this transformation occurs in animals with no germline, the physiological programs that determine hypoxia sensitivity must occur independently of germline signals and instead rely on developmental signals from somatic tissues. Furthermore, we found two distinct mechanisms of hypoxia resistance in long-lived germline deficient animals. First, a DAF-16/FoxO independent mechanism that occurs in all hypoxia resistant sterile adults and, second, a DAF-16/FoxO dependent mechanism that confers an added layer of resistance, or "super-resistance", to animals with no germline as they age past day 1 of adulthood. RNAseq data showed that nearly all genes involved in both cytosolic and mitochondrial protein translation, as well as in mitochondrial protein import, are repressed in germline deficient adults and further repressed as they age. The hypoxia super-resistance of aging germline deficient animals was suppressed by dual mutation of ncl-1 and larp-1, two regulators of nucleolar biology and protein translation, demonstrating that the hypoxia super-resistance mechanism involves reduced protein translation. These studies provide novel insight into a profound physiological transformation that takes place in germline mutants during development, showing that some of the unique physiological properties of these long-lived animals are dependent on developmental repression of genes involved in protein translation, which operate independently of germline signals. AUTHOR SUMMARYIn addition to being extremely long lived, germline deficient animals have other extraordinary properties, such as robust resistance to oxygen deprivation. Here we provide new insight into the mechanisms of hypoxia resistance in germline deficient animals. We demonstrate that, in just a 12-hour period, germline mutants transform from hypoxia sensitive larvae into highly hypoxia resistant adults. Therefore, hypoxia resistance is not a general property of germline ablated animals, but is instead "switched on" only in adult animals. We have found two distinct mechanisms of hypoxia resistance in germline deficient animals and both mechanisms are mediated by signals from somatic tissues and do not require the germline. We have determined that reduced transcription of genes involved in protein translation is one of the mechanisms of hypoxia resistance. Like hypoxia resistance, repression of protein translation genes only occurs in adults. Our findings establish that the unique physiological properties of germline-deficient animals are "switched" on in adults and therefore must be mediated by developmental signals from somatic tissues. We conclude that the L4/adult developmental switch in germline ablated animals presents an excellent system for investigating the longevity and hypoxia resistance of germline deficient animals.

genetics↗

bioRxiv2021

Expression of extracellular Hsp90 is a molecular signature of T cell activation, providing a means to image and target T Cell activation in autoimmune disease

Heat shock protein 90 (Hsp90) maintains cellular proteostasis during stress and has been under investigation as a therapeutic target in cancer for over two decades. We and others have identified an extracellularly expressed form of Hsp90, eHsp90, that previously appeared to be restricted to rapidly proliferating cells exhibiting a metastatic phenotype. Here, we used HS-131, a fluor-tethered eHsp90 inhibitor, to quantify the effect of T cell activation on the expression of eHsp90 in human and mouse T cells. In cell based assays, stimulation of human T cells induced a 20-fold increase in eHsp90 expression at the plasma membrane, suggesting trafficking of eHsp90 is acutely regulated by TCR and inflammatory mediated signaling. Following injection of HS-131 in mouse models of human rheumatoid arthritis and inflammatory bowel disease, we detected localization of the probe at sites of active disease, consistent with immune cell invasion. Moreover, despite rapid hepatobiliary clearance, HS-131 demonstrated efficacy in delaying the onset and progression of disease in the arthritis model. Our results suggest eHsp90 expression at the plasma membrane of T cells is a molecular marker of autoimmune induced activation and potentially a therapeutic target for chronic diseases such as rheumatoid arthritis and inflammatory bowel disease. PerspectiveT cells mediate many maladaptive disease pathologies such as autoimmune disorders. Many immunosuppressants in clinical use act by preventing the activation of T cells. However, this approach can lead to increased rates of opportunistic infection and malignancy. Here, we show for the first time that eHsp90 is upregulated during T cell activation in response to exogenous ligands. Therefore, pharmacological inhibition of eHsp90 may provide a means to target only activated T cells for immunosuppression. Selective targeting of only activated T cells could be efficacious against autoimmune disease without increasing the incidence of undesirable outcomes. Additionally, the presence of eHsp90 on the surface of activated T cells could be diagnostically useful to track the status and progression of autoimmune disease in patients.

pharmacology and toxicology↗

bioRxiv2021

GraFT: Graph Filtered Temporal Dictionary Learning for Functional Neural Imaging

Optical imaging of calcium signals in the brain has enabled researchers to observe the activity of hundreds-to-thousands of individual neurons simultaneously. Current methods predominantly focus on matrix factorization and aim at detecting neurons in the imaged field-of-view, and then inferring the corresponding time-traces. The explicit locality constraints on the cell shapes additionally limits the applicability to optical imaging at different scales (i.e., dendritic or widefield data). Here we present a new method that frames the problem of isolating independent fluorescing components as a dictionary learning problem. Specifically, we focus on the time-traces, which are the main quantity used in scientific discovery, and learn the dictionary of time traces with the spatial maps acting as the presence coefficients encoding which pixels the time traces are active in. Furthermore, we present a novel graph filtering model which redefines connectivity between pixels in terms of their shared temporal activity, rather than spatial proximity. This model greatly eases the ability of our method to handle data with complex non-local spatial structure, such as dendritic imaging. We demonstrate important properties of our method, such as robustness to initialization, implicitly inferring number of neurons and simultaneously detecting different neuronal types, on both synthetic data and real data examples. Specifically, we demonstrate applications of our method to calcium imaging both at the dendritic, somatic, and widefield scales.

neuroscience↗

bioRxiv2020

Epichloe festucae in mutualistic association with Lolium perenne suppresses host apoplastic cysteine protease activity

Plants secrete various defence-related proteins into the apoplast, including proteases. Papain-like cysteine proteases (PLCPs) are central components of the plant immune system. To overcome plant immunity and successfully colonise their hosts, several plant pathogens secrete effector proteins inhibiting plant PLCPs. We hypothesized that not only pathogens but also mutualistic microorganisms interfere with PLCP-meditated plant defences to maintain endophytic colonisation with their hosts. Epichloe festucae forms mutualist associations with cool season grasses and produces a range of secondary metabolites that protect the host against herbivores. In this study, we performed a genome wide identification of Lolium perenne PLCPs, analysed their evolutionary relationship and classified them into nine PLCP subfamilies. Using activity-based protein profiling, we identified four active PLCPs in the apoplast of L. perenne leaves that are inhibited during endophyte interactions. We characterized the L. perenne cystatin LpCys1 for its inhibitory capacity against ryegrass PLCPs. LpCys1 inhibits LpCP2, indicating that LpCys1 might play a role in the suppression of PLCP activity during the interaction with E. festucae. However, since the activity of other L. perenne PLCPs is not sensitive to LpCys1 we propose that additional inhibitors are involved in the suppression of apoplastic PLCPs during E. festucae infection.

plant biology↗

bioRxiv2020

A homologue of the mammalian tumour suppressor protein PTEN is a functional lipid phosphatase and required for chemotaxis in filamentous fungi

Phosphoinositides (PI) are essential components of eukaryotic membranes and function in a large number of signalling processes. While lipid second messengers are well studied in mammals and yeast, their role in filamentous fungi is poorly understood. We used fluorescent PI-binding molecular probes to localise the phosphorylated phosphatidylinositol species PI[3]P, PI[3,5]P2, PI[4]P and PI[4,5]P2 in hyphae of the endophyte Epichloe festucae in axenic culture and during interaction with its grass host Lolium perenne. We also analysed the roles of the phosphatidylinositol-4-phosphate 5-kinase MssD and the predicted phosphatidylinositol-3,4,5-triphosphate 3-phosphatase TepA, a homologue of the mammalian tumour suppressor protein PTEN. Deletion of tepA in E. festucae and in the root-infecting tomato pathogen Fusarium oxysporum had no impact on growth in culture or the host interaction phenotype. However, this mutation did uncover the presence of PI[3,4,5]P3 in septa of E. festucae and showed that TepA is required for chemotropism in F. oxysporum. The identification of PI[3,4,5]P3 in septa of{Delta} tepA strains suggests that filamentous fungi are able to generate PI[3,4,5]P3 using an alternative biosynthetic pathway and that fungal PTEN homologues are functional lipid phosphatases. The F. oxysporum chemotropism defect demonstrates a conserved role of PTEN homologues in chemotaxis across protists, fungi and mammals.

cell biology↗

bioRxiv2020

Host infection by the grass-symbiotic fungus Epichloë festucaerequires catalytically active H3K9 and H3K36 methyl transferases

Recent studies have identified key genes in Epichloe festucae that control the symbiotic interaction of this filamentous fungus with its grass host. Here we report on the identification of specific fungal genes that determine its ability to infect and colonize the host. Deletion of setB, which encodes a homolog of the H3K36 histone methyltransferase Set2/KMT3, specifically reduced histone H3K36 trimethylation and led to severe defects in colony growth and hyphal development. The E. festucae {Delta}clrD mutant, which lacks the gene encoding the homolog of the H3K9 methyltransferase KMT1, displays similar developmental defects. Both mutants are completely defective in their ability to infect the host grass, and mutational studies of key residues in the catalytic SET domains from these proteins show that these phenotypes are dependent on the methyltransferase activities of SetB and ClrD. A comparison of the differences in the host transcriptome between seedlings inoculated with wild-type versus mutants suggests that the inability of these mutants to infect the host was not due to an aberrant host defense response. Co-inoculation of either {Delta}setB or {Delta}clrD with the wild-type strain enables these mutants to colonize the host. However, successful colonization by the mutants resulted in death or stunting of the host plant. Transcriptome analysis at the early infection stage identified four fungal candidate genes, three of which encode small-secreted proteins, that are differentially regulated in these mutants compared to wild-type. Deletion of crbA, which encodes a putative carbohydrate binding protein, resulted in significantly reduced host infection rates by E. festucae. Author SummaryThe filamentous fungus Epichloe festucae is an endophyte that forms highly regulated symbiotic interactions with the perennial ryegrass. Proper maintenance of such interactions is known to involve several signalling pathways, but much less is understood about the infection capability of this fungus in the host. In this study, we uncovered two epigenetic marks and their respective histone methyltransferases that are required for E. festucae to infect perennial ryegrass. Null mutants of the histone H3 lysine 9 and lysine 36 methyltransferases are completely defective in colonizing the host intercellular space, and these defects are dependent on the methyltransferase activities of these enzymes. Importantly, we observed no evidence for increased host defense response to these mutants that can account for their non-infection. Rather, these infection defects can be rescued by the wild-type strain in co-inoculation experiments, suggesting that failure of the mutants to infect is due to altered expression of genes encoding infection factors that are under the control of the above epigenetic marks that can be supplied by the wild-type strain. Among genes differentially expressed in the mutants at the early infection stage is a putative small-secreted protein with a carbohydrate binding function, which deletion in E. festucae severely reduced infection efficiency.

genetics↗

bioRxiv2019

Positional isomers of a non-nucleoside substrate differentially affect myosin function

Molecular motors have evolved to transduce chemical energy from adenosine triphosphate into mechanical work to drive essential cellular processes, from muscle contraction to vesicular transport. Dysfunction of these motors is a root cause of many pathologies necessitating the need for intrinsic control over molecular motor function. Herein, we demonstrate that positional isomerism can be used as a simple and powerful tool to control the molecular motor of muscle, myosin. Using three isomers of a synthetic non-nucleoside triphosphate we demonstrate that myosins force and motion generating capacity can be dramatically altered at both the ensemble and single molecule levels. By correlating our experimental results with computation, we show that each isomer exerts intrinsic control by affecting distinct steps in myosins mechano-chemical cycle. Our studies demonstrate that subtle variations in the structure of an abiotic energy source can be used to control the force and motility of myosin without altering myosins structure. Statement of SignificanceMolecular motors transduce chemical energy from ATP into the mechanical work inside a cell, powering everything from muscle contraction to vesicular transport. While ATP is the preferred source of energy, there is growing interest in developing alternative sources of energy to gain control over molecular motors. We synthesized a series of synthetic compounds to serve as alternative energy sources for muscle myosin. Myosin was able to use this energy source to generate force and velocity. And by using different isomers of this compound we were able to modulate, and even inhibit, the activity of myosin. This suggests that changing the isomer of the substrate could provide a simple, yet powerful, approach to gain control over molecular motor function.

biophysics↗