Biology subjects

Green, L.

Publications and source records attributed to Green, L..

18 recordsLinked to original sources

bioRxiv2026

A disinhibition-based model of CA1 place field sequences

Because of the paucity of pattern-generating recurrent excitation in the CA1 hippocampal region, place field sequences are assumed to be inherited from upstream regions. Alternatively, disinhibition may play a role in shaping pyramidal cell activity. We propose a model of CA1 activity using one layer of excitatory cells and one layer of inhibitory cells, with local excitatory-to-inhibitory connectivity, broad inhibitory-to-excitatory connectivity without recurrent excitatory connections, and broad inhibitory-inhibitory connections. The layers need not follow a topographic organization; instead they can represent sequences of place cells structured by local inhibition. Our mathematical model of CA1 neurons with data-inspired connectivity demonstrates place fields sharpened by disinhibition, mimicking a recently discovered decrease in inhibitory input within place fields and matching in vivo patterns of disinhibition. Mathematical analysis of our model suggests the importance of inhibitory-to-excitatory connections in control of place cell firing rate and sparsity. We also demonstrate that a disinhibition-based mechanism of pattern generation is more flexible without recurrent excitation than the recurrent excitation-rich CA3-like model. Finally, we demonstrate disinhibition as an efficient mechanism for internally-generated sequential activity, where the speed of sequential change depends on both input strength and inhibitory-to-excitatory connection strength. Overall, our model suggests that theta time-scale disinhibition is a foundational mechanism of self-organized sequential activity in the CA1 region, and, possibly, in other networks.

neuroscience↗

bioRxiv2026

Gradients of local circuit architecture shape stability and information integration across the cortical hierarchy

The mammalian neocortex is a complex structure with many interacting regions and cell types. Multiple efforts have recently generated large datasets from multiple cortical regions to determine generalizing principles of cortical organization and activity. One such generalizing principle is the concept of a cortical hierarchy - low-dimensional macroscopic gradients of biological properties across cortical areas and a hierarchy of timescales for information processing. Such gradients in properties along a cortical hierarchy may explain how different cortical regions serve different computational roles (i.e. sensory processing versus working memory). We set out to investigate such principles using biologically realistic simulations. Starting from our previously published model of the mouse primary visual cortex (VISp) with updated connectivity reflecting recent large-scale connectomics and synaptic physiology datasets, we generated multiple versions of the cortical circuit model with increased connectivity as measured along the cortical hierarchy. We then measure the resulting neural activity in response to a simulated single-cell photostimulation paradigm and thalamocortical inputs. We found that overall circuit stability increased across the cortical hierarchy, due to both increases in connection density and increases in the relative proportion of SST cells. The results of this study link cortical cell types to population-level activity, generating insight into the function of local circuits across cortex.

neuroscience↗

bioRxiv2026

Functional characterization of hypertriglyceridemia-associated mutations in Lipase Maturation Factor 1 by a second-generation activity assay

Lipase maturation factor 1 (LMF1) is a chaperone for lipoprotein lipase (LPL) and critically required for the enzyme to attain lipase activity. LMF1 has been identified as a canonical gene affected in Familial Chylomicronemia Syndrome (FCS) and it has also been extensively analyzed in Multifactorial Chylomicronemia Syndrome (MCS), the polygenic form of severe hypertriglyceridemia (hTG). While recent genetic studies resulted in an explosion in the number of hTG-associated LMF1 variants in different populations, their functional significance remains largely unknown. Here, we present a second-generation LMF1 activity assay allowing the streamlined quantitative functional analysis of LMF1 variants. The assay is based on the reconstitution of lipase maturation in transfected LMF1-deficient cells and fluorescence-based measurement of LPL activity in the culture medium. The use of Gaussia luciferase (GLuc)-LMF1 fusion constructs allows the assessment of LMF1 protein expression and the calculation of LMF1 specific activity. Several variants previously reported as neutral resulted in loss of LMF1 function in the new assay demonstrating increased sensitivity to detect variants of modest effect sizes. Furthermore, the functional analysis of 14 hTG-associated missense mutations identified 12 loss-of-function (LOF) variants. To gain initial insights into the structural determinants of LMF1 function, we analyzed the domain distribution of 73 hTG-associated missense variants and identified Loop C as a critical region in lipase maturation. In conclusion, we developed a simple, non-radioactive and sensitive quantitative assay of LMF1 activity to facilitate the functional analysis of hyperlipidemia-associated genetic variants and structure-function studies.

biochemistry↗

bioRxiv2026

NATURAL VARIATION IN RADIOTOLERANCE IS ASSOCIATED WITH DIFFERENCES IN INNATE IMMUNE ACTIVITY AND S-PHASE TRANSCRIPTION IN DROSOPHILA MELANOGASTER

Organismal tolerance of ionizing radiation is a complex trait whose genetic basis has been studied extensively, in large part due to its significance to human health and technological advancement. Conventional mutant screens in model organisms have revealed the paramount role of DNA damage response (DDR) and repair pathways in determining tolerance to ionizing radiation. However, uncovering natural genetic variation in radiotolerance is also of critical importance, as individual differences are associated with the differential susceptibility to cancer as well as differential response to radiation treatment. Genetic variation that underlies phenotype differences in natural populations often occurs in distinct genes and pathways as compared to the genes of major effect revealed by mutant screens, owing to the impact of natural selection on the former. We therefore sought to isolate natural variation in radiotolerance of Drosophila melanogaster by performing extreme QTL mapping. We generated a large genetically diverse multiparental population and exposed 3rd instar larvae to a semi-lethal dose or ionizing radiation. By sequencing surviving adults and comparing their haplotypes to unexposed controls from the same population, we identified a single major effect QTL spanning the 3rd chromosome centromere. The QTL contains 34 genes, none of which are previously implicated in radiotolerance. We interrogated the impact of these genes on radiotolerance through forward genetic analysis and RNA-seq. Our findings implicate diverse processes in radiotolerance including cell-cycle regulation and innate immune function.

genetics↗

bioRxiv2024

MYBPC3 D389V Variant Induces Hypercontractility in Cardiac Organoids

BACKGROUNDMYBPC3, encoding cardiac myosin binding protein-C (cMyBP-C), is the most mutated gene known to cause hypertrophic cardiomyopathy (HCM). However, since little is known about the underlying etiology, additional in vitro studies are crucial to defining the underlying molecular mechanisms. Accordingly, this study aimed to investigate the molecular mechanisms underlying the pathogenesis of HCM associated with a polymorphic variant (D389V) in MYBPC3 by using human-induced pluripotent stem cell (hiPSC)-derived cardiac organoids (hCOs). METHODSThe hiPSC-derived cardiomyocytes (hiPSC-CMs) and hCOs were generated from human subjects to define the molecular, cellular, and functional changes caused by the MYBPC3D389V variant. This variant is associated with increased fractional shortening and is highly prevalent in South Asian descendants. Recombinant C0-C2, N-region of cMyBP-C (wildtype and D389V), and myosin S2 proteins were also utilized to perform binding and motility assays in vitro. RESULTSConfocal and electron microscopic analyses of hCOs generated from noncarriers (NC) and carriers of the MYBPC3D389V variant revealed the presence of highly organized sarcomeres. Furthermore, functional experiments showed hypercontractility with increased contraction velocity, faster calcium cycling, and faster contractile kinetics in hCOs expressing MYBPC3D389V than NC hCOs. Interestingly, significantly increased cMyBP-C phosphorylation in MYBPC3D389V hCOs was observed, but without changes in total protein levels, in addition to higher oxidative stress and lower mitochondrial membrane potential ({Delta}{Psi}m). Next, spatial mapping revealed the presence of endothelial cells, fibroblasts, macrophages, immune cells, and cardiomyocytes in the hCOs. The hypercontractile function was significantly improved after treatment with the myosin inhibitor mavacamten (CAMZYOS(R)) in MYBPC3D389V hCOs. Lastly, various in vitro binding assays revealed a significant loss of affinity in the presence of MYBPC3D389V with myosin S2 region as a likely mechanism for hypercontraction. CONCLUSIONSConceptually, we showed the feasibility of assessing the functional and molecular mechanisms of HCM using highly translatable hCOs through pragmatic experiments that led to determining the MYBPC3D389V hypercontractile phenotype, which was rescued by administration of a myosin inhibitor. Novelty and SignificanceO_ST_ABSWhat Is Known?C_ST_ABSO_LIMYBPC3 mutations have been implicated in hypertrophic cardiomyopathy. C_LIO_LID389V is a polymorphic variant of MYBPC3 predicted to be present in 53000 US South Asians owing to the founder effect. D389V carriers have shown evidence of hyperdynamic heart, and human-induced pluripotent stem cells (hiPSC)-derived cardiomyocytes with D389V show cellular hypertrophy and irregular calcium transients. C_LIO_LIThe molecular mechanism by which the D389V variant develops pathological cardiac dysfunction remains to be conclusively determined. C_LI What New Information Does This Article Contribute?O_LIThe authors leveraged a highly translational cardiac organoid model to explore the role of altered cardiac calcium handling and cardiac contractility as a common pathway leading to pathophysiological phenotypes in patients with early HCM. C_LIO_LIThe MYBPC3D389V-mediated pathological pathway is first studied here by comparing functional properties using three-dimensional cardiac organoids differentiated from hiPSC and determining the presence of hypercontraction. C_LIO_LIOur data demonstrate that faster sarcomere kinetics resulting from lower binding affinity between D389V-mutated cMyBP-C protein and myosin S2, as evidenced by in vitro studies, could cause hypercontractility which was rescued by administration of mavacamten (CAMZYOS(R)), a myosin inhibitor. C_LIO_LIIn addition, hypercontractility causes secondary mitochondrial defects such as higher oxidative stress and lower mitochondrial membrane potential ({Delta}{Psi}m), highlighting a possible early adaptive response to primary sarcomeric changes. C_LIO_LIEarly treatment of MYBPC3D389V carriers with mavacamten may prevent or reduce early HCM-related pathology. C_LI GRAPHICAL ABSTRACTA graphical abstract is available for this article. O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=132 SRC="FIGDIR/small/596463v1_ufig1.gif" ALT="Figure 1"> View larger version (30K): org.highwire.dtl.DTLVardef@1489301org.highwire.dtl.DTLVardef@1ab60c1org.highwire.dtl.DTLVardef@5d49d1org.highwire.dtl.DTLVardef@9944d6_HPS_FORMAT_FIGEXP M_FIG C_FIG

cell biology↗

bioRxiv2024

Monitoring of the invasive round goby in an estuarine seascape based on eDNA

0.0Non-indigenous species (NIS) is one of the five most global concerns when it comes to ecosystem services and threats to native biodiversity. This is especially true in aquatic environments which are harder to monitor than terrestrial environments, and NIS are often found first when they are fully established and basically impossible to eradicate. In marine environments, this is further complicated due to the connectivity and difficulty of eradication. The development and implementation of effective monitoring methods for marine NIS are therefore crucial to enable early detection useful for management strategies. In this study, we develop and evaluate environmental DNA monitoring using quantitative (q)PCR as a means to assess presence of the euryhaline round goby fish (Neogobius melanostomus) in a seascape environment close to Scandinavias largest shipping port. We developed a dPCR assay for the species, targeting a region of 12S gene, and verified its specificity compared to other common species from the gobiid-family in the region. We also experimentally determined the decaying rate of round goby DNA in water to a half-life of 9.8 hours in 15 PSU and 15{degrees}C with live fish in captivity. Finally, we sampled 10 sites within a 400 km2 area for eDNA and presence of the species using fyke-nets and baited remote video to validate the accuracy of the water samples to predict presence, and abundance. We found that the number of DNA copies extracted from the water samples varied strongly at sites where round gobies were caught in nets or on video, but that the average value from four water samples significantly correlated with an average value from four video samples, and also with the total catch at each site. The eDNA assay also detected signals from the species at sites where no fish were caught by fishing or on video. These results show that this method is highly sensitive for the species even in low abundance, and with sufficient amounts of replicates, it can be possible to determine the relative abundance between sites.

molecular biology↗

bioRxiv2023

Detailed Survey of an in-vitro Intestinal Epithelium Model by Single-Cell Transcriptomics

The gut plays a critical role in maintaining human health by facilitating the absorption of nutrients, regulating metabolism, and interacting with the immune system and gut microbiota. The co-culture of two human colorectal cancer cell lines, Caco-2 and HT29, on Transwell is commonly used as an in vitro gut mimic in studies of intestinal absorption pharmacokinetics, gut mechanics, and gut-microbe interplay given the similar morphology, expression of transporters and enzymes, and barrier function. However, to sufficiently evaluate the translatability of insights from such a system to human physiological contexts, a detailed survey of cell type heterogeneity in the system and a holistic comparison with human physiology are needed to be conducted rather than by the presence of a few well-studied proteins. Single-cell RNA sequencing provides high-resolution expression profiles of cells in the co-culture, enabling the heterogeneity to be characterized and the similarity to human epithelial cells to be evaluated. Transcriptional profiles of 16019 genes in 13784 cells were acquired and compared to human epithelial cells (GSE185224). We identified the intestinal stem cell-, transit amplifying-, enterocyte-, goblet cell-, and enteroendocrine-like cells together with differentiating HT29 cells in the system based on the expression of canonical markers in healthy adult human epithelial cells. The epithelium-like co-culture was fetal intestine-like, with less variety of gene expression compared to the human gut. Transporters for major types of substance (lipid, amino acid, ion, water, etc.) were found transcribed in the majority of the enterocytes-like cells in the system. However, some of the well-studied transporters such as FATP4 and GLUT2 were absent. Toll-like receptors were not highly expressed in the sample, yet the treatment of lipopolysaccharide still caused a mild change in trans-epithelial electrical resistance and gene expression, possibly by the interaction with CD14, the co-receptor for TLRs. Overall, the Caco-2/HT29 co-culture is a cost-effective epithelium model for drug permeability testing or mechanical simulation, but its phenotypic discrepancy with the real epithelium is not negligible. As a result, its response to biological factors might not provide transferrable knowledge to the study of human gut physiology, especially the innate immune aspect.

cell biology↗

bioRxiv2023

Model-guided Design of Biological Controller for Septic Wound Healing Regulation

Immune response is critical in septic wound healing. The aberrant and imbalanced signaling dynamics primarily cause a dysfunctional innate immune response, exacerbating pathogen invasion of injured tissue and further stalling the healing process. To design biological controllers that regulate the critical divergence of the immune response during septicemia, we need to understand the intricate differences in immune cell dynamics and coordinated molecular signals of healthy and sepsis injury. Here, we deployed an ordinary differential equation (ODE)-based model to capture the hyper and hypo-inflammatory phases of sepsis wound healing. Our results indicate that impaired macrophage polarization leads to a high abundance of monocytes, M1, and M2 macrophage phenotypes, resulting in immune paralysis. Using a model-based analysis framework, we designed a biological controller which successfully regulates macrophage dysregulation observed in septic wounds. Our model describes a systems biology approach to predict and explore critical parameters as potential therapeutic targets capable of transitioning septic wound inflammation toward a healthy, wound-healing state.

systems biology↗

bioRxiv2022

Exploring the potential of using simulation games for engaging with sheep farmers about lameness recognition

IntroductionComputer simulation games are increasingly being used in agriculture as a promising tool to study, support and influence real-life farming practices. We explored the potential of using simulation games to engage with sheep farmers on the ongoing challenge of reducing lameness. Working with UK stakeholders, we developed a game in which players are challenged with identifying all the lame sheep in a simulated flock. Here, we evaluate the games potential to act as a tool for to help assess, train and understand farmers ability to recognise the early signs of lameness. MethodsParticipants in the UK were invited to play the game in an online study, sharing with us their in-game scores alongside information relating to their real-life farming experience, how they played the game, and feedback on the game. Mixed methods were used to analyse this information in order to evaluate the game. Quantitative analyses consisted of linear modelling to test for statistical relationships between participants in-game recall (% of the total number of lame sheep that were marked as lame), and the additional information they provided. Qualitative analyses of participants feedback on the game consisted of thematic analysis and a Likert Scale questionnaire to contextualise the quantitative results and identify additional insights from the study. ResultsQuantitative analyses identified no relationships between participants (n = 63) recall scores and their real life farming experience, or the lameness signs they looked for when playing the game. The only relationship identified was a relationship between participants recall score and time spent playing the game. Qualitative analyses identified that participants did not find the game sufficiently realistic or engaging, though several enjoyed playing it and saw potential for future development. Qualitative analyses also identified several interesting and less-expected insights about real-life lameness recognition practices that participants shared after playing the game. DiscussionSimulation games have potential as a tool in livestock husbandry education and research, but achieving the desired levels of realism and/or engagingness may be an obstacle to realising this. Future research should explore this potential further, aided by larger budgets and closer collaboration with farmers, stockpeople and veterinarians.

scientific communication and education↗

bioRxiv2022

Basal and stress-induced expression changes consistent with water loss reduction explain desiccation tolerance of natural Drosophila melanogaster populations

BackgroundClimate change is one of the main factors shaping the distribution and biodiversity of organisms, among others by greatly altering water availability, thus exposing species and ecosystems to harsh desiccation conditions. Insects are especially threatened by these challenging dry environments, because of their small size and thus large surface area to volume ratio. Integrating transcriptomics and physiology is key to advancing our knowledge on how species cope with desiccation stress, and these studies are still best accomplished in model organisms. ResultsHere, we characterized the natural variation of European D. melanogaster populations across climate zones and found that strains from arid regions were similar or more tolerant to desiccation compared with strains from temperate regions. Tolerant and sensitive strains differed not only in their transcriptomic response to stress but also in their basal expression levels. We further showed that gene expression changes in tolerant strains correlated with their physiological response to desiccation stress and with their cuticular hydrocarbon composition. Transposable elements, which are known to influence stress response across organisms, were not found to be enriched nearby differentially expressed genes. Finally, we identified several tRNA-derived small RNA fragments that differentially targeted genes in response to desiccation stress. ConclusionsOur results showed that by integrating transcriptomics with physiological trait analysis we can pinpoint the genetic basis of the differences in tolerance to desiccation stress found in natural D. melanogaster populations. Moreover, we showed that, beyond starvation and aging, tRNA-derived small RNA fragments (tRFs) appear to be relevant post-transcriptional gene regulators in response to desiccation stress.

evolutionary biology↗

bioRxiv2022

A distinct subset of oligodendrocyte lineage cells interact with the developing dorsal root entry during its genesis

Oligodendrocytes are the myelinating cell of the CNS and are critical for the functionality of the nervous system. In the packed CNS, we know distinct profiles of oligodendrocytes are present. Here, we used intravital imaging in zebrafish to identify a distinct oligodendrocyte lineage cell (OLC) that resides on the dorsal root ganglia sensory neurons in the spinal cord. Our profiling of OLC cellular dynamics revealed a distinct cell cluster that interacts with peripheral sensory neurons at the dorsal root entry zone (DREZ). With pharmacological, physical and genetic manipulations, we show that the entry of dorsal root ganglia pioneer axons across the DREZ is important to produce sensory located oligodendrocyte lineage cells. These oligodendrocyte lineage cells on peripherally derived sensory neurons display distinct processes that are stable and do not express mbp. Upon their removal, sensory behavior related to the DRG neurons is abolished. Together, these data support the hypothesis that peripheral neurons at the DREZ can also impact oligodendrocyte development.

neuroscience↗

bioRxiv2021

Population-scale long-read sequencing uncovers transposable elements contributing to gene expression variation and associated with adaptive signatures in Drosophila melanogaster

High quality reference genomes are crucial to understanding genome function, structure and evolution. The availability of reference genomes has allowed us to start inferring the role of genetic variation in biology, disease, and biodiversity conservation. However, analyses across organisms demonstrate that a single reference genome is not enough to capture the global genetic diversity present in populations. In this work, we generated 32 high-quality reference genomes for the well-known model species D. melanogaster and focused on the identification and analysis of transposable element variation as they are the most common type of structural variant. We showed that integrating the genetic variation across natural populations from five climatic regions increases the number of detected insertions by 58%. Moreover, 26% to 57% of the insertions identified using long-reads were missed by short-reads methods. We also identified hundreds of transposable elements associated with gene expression variation and new TE variants likely to contribute to adaptive evolution in this species. Our results highlight the importance of incorporating the genetic variation present in natural populations to genomic studies, which is essential if we are to understand how genomes function and evolve.

evolutionary biology↗

bioRxiv2021

Natural variation in copper tolerance in Drosophila melanogaster is shaped by transcriptional and physiological changes in the gut

Increases in industrialization and anthropogenic activity have resulted in an increase of pollutants released into the environment. Of these pollutants, heavy metals such as copper are particularly concerning due to their bio-accumulative nature. Due to its highly heterogeneous distribution and its dual nature as an essential micronutrient and toxic element, the genetic basis of copper tolerance is likely shaped by a complex interplay of genetic and environmental factors. In this study, we utilized the natural variation present in multiple populations of Drosophila melanogaster collected across Europe to screen for variation in copper tolerance. We found that latitude and the degree of urbanization at the collection sites, rather than any other combination of environmental factors, were linked to copper tolerance. While previously identified copper-related genes were not differentially expressed in tolerant vs. sensitive strains, genes involved in metabolism, reproduction, and protease induction contributed to the differential stress response. Additionally, the greatest transcriptomic and physiological responses to copper toxicity were seen in the midgut; where we found that preservation of gut acidity is strongly linked to greater tolerance. Finally, we identified transposable element insertions likely to play a role in copper stress response. Overall, by combining genome-wide approaches with environmental association analysis, and functional analysis of candidate genes, our study provides a unique perspective on the genetic and environmental factors that shape copper tolerance in natural D. melanogaster populations, and identifies new genes, transposable elements and physiological traits involved in this complex phenotype.

evolutionary biology↗

bioRxiv2021

The role of ventromedial prefrontal cortex in reward valuation and future thinking during intertemporal choice

Intertemporal choices require trade-offs between short-term and long-term outcomes. Ventromedial prefrontal cortex (vmPFC) damage causes steep discounting of future rewards (delay discounting; DD) and impoverished episodic future thinking (EFT). The role of vmPFC in reward valuation, EFT, and their interaction during intertemporal choice is still unclear. Here, twelve patients with lesions to vmPFC and forty-one healthy controls chose between smaller-immediate and larger-delayed hypothetical monetary rewards while we manipulated reward magnitude and the availability of EFT cues. In the EFT condition, participants imagined personal events to occur at the delays associated with the larger-delayed rewards. We found that DD was steeper in vmPFC patients compared to controls, and not modulated by reward magnitude. However, EFT cues downregulated DD in vmPFC patients as well as controls. These findings indicate that vmPFC integrity is critical for the valuation of (future) rewards, but not to instill EFT in intertemporal choice.

neuroscience↗

bioRxiv2020

Increasing Chlorophyll a Amid Stable Nutrient Concentrations in Rhode Island Lakes and Reservoirs

Addressing anthropogenic impacts on aquatic ecosystems is a focus of lake management. Controlling phosphorus and nitrogen can mitigate these impacts, but determining management effectiveness requires long-term datasets. Recent analysis of the LAke multi-scaled GeOSpatial and temporal database for the Northeast (LAGOSNE) United States found stable water quality in the northeastern and midwestern United States, however, sub-regional trends may be obscured. We analyze a sub-regional (i.e., 3000 km2) trend with the University of Rhode Islands Watershed Watch Volunteer Monitoring Program (URIWW) dataset. URIWW has collected water quality data on Rhode Island lakes and reservoirs for over 25 years. The LAGOSNE and URIWW datasets allow for comparison of water quality trends at regional and sub-regional extents, respectively. We assess regional (LAGOSNE) and state (URIWW) trends with yearly mean anomalies calculated on a per-station basis. Sub-regionally, temperature and chlorophyll a increased from 1993 to 2016. Total nitrogen shows a weak increase driven by low years in the early 1990s. Total phosphorus and the nitrogen:phosphorus ratio (N:P) were stable. At the regional scale, the LAGOSNE dataset shows similar trends to prior studies of the LAGOSNE with chlorophyll a, total nitrogen, total phosphorus, and N:P all stable over time. In short, algal biomass, as measured by chlorophyll a in Rhode Island lakes and reservoirs is increasing, despite stability in total nitrogen, total phosphorus, and the nitrogen to phosphorus ratio. This analysis suggests an association between lake temperature and primary production. Additionally, we demonstrate both the value of long-term monitoring programs, like URIWW, for identifying trends in environmental condition, and the utility of site-specific anomalies for analyzing for long-term water quality trends.

ecology↗

bioRxiv2019

In vitro reconstitution of branched microtubule nucleation

Eukaryotic cell division requires the mitotic spindle, a microtubule (MT)-based structure which accurately aligns and segregates duplicated chromosomes. The dynamics of spindle formation are determined primarily by correctly localising the MT nucleator,{gamma} -Tubulin Ring Complex ({gamma}-TuRC)1-4, within the cell. A conserved MT-associated protein complex, Augmin, recruits{gamma} -TuRC to pre-existing spindle MTs, amplifying their number, in an essential cellular phenomenon termed \"branched\" MT nucleation5-9. Here, we purify endogenous, GFP-tagged Augmin and{gamma} -TuRC from Drosophila embryos to near homogeneity using a novel one-step affinity technique. We demonstrate that, in vitro, while Augmin alone does not affect Tubulin polymerisation dynamics, it stimulates{gamma} -TuRC-dependent MT nucleation in a cell cycle-dependent manner. We also assemble and visualise the MT-Augmin-{gamma}-TuRC-MT junction using light microscopy. Our work therefore conclusively reconstitutes branched MT nucleation. It also provides a powerful synthetic approach with which to investigate the emergence of cellular phenomena, such as mitotic spindle formation, from component parts.

cell biology↗

bioRxiv2018

Cis and trans-acting variants contribute to survivorship in a naive Drosophila melanogaster population exposed to ryanoid insecticides

Insecticide resistance is a paradigm of microevolution and insecticides are responsible for the strongest cases of recent selection in the genome of Drosophila melanogaster. Here we use a naive population and a novel insecticide class to examine the ab initio genetic architecture of a potential selective response. Genome wide association studies of chlorantraniliprole susceptibility reveal variation in a gene of major effect, Stretchin Myosin light chain kinase (Strn-Mlck), which we validate with linkage mapping and transgenic manipulation of gene expression. We propose that allelic variation in Strn-Mlck alters sensitivity to the calcium depletion attributable to chlorantraniliproles mode of action. Genome-wide association studies also reveal a network of genes involved in neuromuscular biology. In contrast, phenotype to transcriptome associations identify differences in constitutive levels of multiple transcripts regulated by cnc, the homologue of mammalian Nrf2. This suggests that genetic variation acts in trans to regulate multiple metabolic enzymes in this pathway. The most outstanding association is with the transcription level of Cyp12d1 which is also affected in cis by copy number variation. Transgenic overexpression of Cyp12d1 reduces susceptibility to both chlorantraniliprole and the closely related insecticide cyantraniliprole. This systems genetics study reveals multiple allelic variants segregating at intermediate frequency in a population that is completely naive to this new insecticide chemistry and it adumbrates a selective response among natural populations to these chemicals.\n\nSignificanceAround the world insecticides are being deregistered and banned, as their environmental costs are deemed too great or their efficacy against pest insects is reduced through the evolution of insecticide resistance. With the introduction of replacement insecticides comes the responsibility to assess the way new insecticides perturb various levels of biological systems; from insect physiology to ecosystems. We used a systems genetics approach to identify genetic variants affecting survivorship of Drosophila melanogaster exposed to chlorantraniliprole. The study population was completely naive to this insecticide chemistry and yet we find associations with variants in neuromuscular genes and co-regulated detoxification genes. We predict that these variants will increase in populations of this sentinel species as these insecticides are applied in the environment.

genetics↗

bioRxiv2018

Structural variants and selective sweep foci contribute to insecticide resistance in the Drosophila melanogaster genetic reference panel

Patterns of nucleotide polymorphism within populations of Drosophila melanogaster suggest that insecticides have been the selective agents driving the strongest recent bouts of positive selection. However, there is a need to explicitly link selective sweep loci to the particular insecticide phenotypes that could plausibly account for the drastic selective responses that are observed in these non-target insects. Here, we screen the Drosophila Genetic Reference Panel with two common insecticides; malathion (an organophosphate) and permethrin (a pyrethroid). Genome wide association studies map survival-on-malathion to two of the largest sweeps in the D. melanogaster genome; Ace and Cyp6g1. Malathion survivorship also correlates with lines which have high levels of Cyp12d1 and Jheh1 and Jheh2 transcript abundance. Permethrin phenotypes map to the largest cluster of P450 genes in the Drosophila genome, however in contrast to a selective sweep driven by insecticide use, the derived state seems to be associated with susceptibility. These results underscore previous findings that highlight the importance of structural variation to insecticide phenotypes: Cyp6g1 exhibits copy number variation and transposable element insertions, Cyp12d1 is tandemly duplicated, the Jheh loci are associated with a Bari1 transposable element insertion, and a Cyp6a17 deletion is associated with susceptibility.

genetics↗