Biology subjects

Roberts, K. E.

Publications and source records attributed to Roberts, K. E..

9 recordsLinked to original sources

bioRxiv2024

A negative feedback loop between small GTPase Rap1 and mammalian tumor suppressor homolog KrsB regulates cell-substrate adhesion in Dictyostelium

Cell adhesion to the substrate influences a variety of cell behaviors and its proper regulation is essential for migration, although details of the molecular pathways regulating cell adhesion during migration are lacking. Rap1 is a small GTPase that regulates adhesion in mammalian cells, as well as in Dictyostelium discoideum social amoeba, which is an established model for studying directed cell migration. In Dictyostelium, Rap1 controls adhesion via its effects on adhesion mediator talin and Ser/Thr kinase Phg2, which inhibits myosin II function. Kinase responsive to stress B (KrsB), a homolog of mammalian tumor suppressor MST1/2 and Drosophila Hippo, also regulates cell adhesion and migration, although the molecular mechanism of KrsB action is not understood. Since KrsB has been shown to interact with active Rap1 by mass spectroscopy, we investigated the genetic interaction between Rap1 and KrsB. Cells lacking KrsB have increased adhesion to the substrate, which leads to reduced movement. Expression of constitutively active Rap1 G12V increased cell spreading and adhesion even in the absence of KrsB, suggesting that Rap1 does not require KrsB to mediate cell adhesion. In contrast, dominant negative Rap1 S17N completely reversed the over-adhesive phenotype of KrsB-null cells and impaired KrsB phosphorylation, suggesting that KrsB activation and function in adhesion requires Rap1. Even though Rap1 did not require KrsB for its function in adhesion, KrsB negatively regulates Rap1 function as seen by increased cortical localization of Rap1 in KrsB-null cells. Consistent with this, chemoattractant-induced activation of downstream effectors of Rap1, TalB and Phg2, was increased in the absence of KrsB. Taken together, these findings suggest that Rap1 leads to activation of KrsB, which inhibits Rap1 and its downstream targets, shutting off adhesion. The existence of a negative feedback loop between Rap1 and KrsB may contribute to the dynamic regulation of cell adhesion that is necessary for rapid amoeboid-type migration.

cell biology↗

bioRxiv2022

Investigating the outcomes of virus coinfection within and across host species

Interactions between coinfecting pathogens have the potential to alter the course of infection and can act as a source of phenotypic variation in susceptibility between hosts. This phenotypic variation may influence the evolution of host-pathogen interactions within host species and interfere with patterns in the outcomes of infection across host species. Here, we examine experimental coinfections of two Cripaviruses - Cricket Paralysis Virus (CrPV), and Drosophila C Virus (DCV) -across a panel of 25 Drosophila melanogaster inbred lines and 47 Drosophilidae host species. We find that interactions between these viruses alter viral loads across D. melanogaster genotypes, with a ~3 fold increase in the viral load of DCV and a ~2.5 fold decrease in CrPV in coinfection compared to single infection, but we find little evidence of a host genetic basis for these effects. Across host species, we find no evidence of systematic changes in susceptibility during coinfection, with no interaction between DCV and CrPV detected in the majority of host species. These results suggest that phenotypic variation in coinfection interactions within host species can occur independently of natural host genetic variation in susceptibility, and that patterns of susceptibility across host species to single infections can be robust to the added complexity of coinfection.

evolutionary biology↗

bioRxiv2022

Sex and tissue differences in virus susceptibility across species

Species vary in their susceptibility to pathogens, and this can alter the ability of a pathogen to infect a novel host. However, many factors can generate heterogeneity in infection outcomes, obscuring our ability to understand pathogen emergence. Such heterogeneities can alter the consistency of responses across individuals and host species. For example, sexual dimorphism in susceptibility means males are often intrinsically more susceptible than females (although this can vary by host and pathogen). Further, we know little about whether the tissues infected by a pathogen in one host are the same in another species, and how this relates to the harm a pathogen does to its host. Here, we first take a comparative approach to examine sex differences in susceptibility across 31 species of Drosophilidae infected with Drosophila C Virus (DCV). We found a strong positive inter-specific correlation in viral load between males and females, with a close to 1:1 relationship, suggesting that susceptibility to DCV across species is not sex specific. Next, we made comparisons of the tissue tropism of DCV across seven species of fly. We found differences in viral load between the tissues of the seven host species, but no evidence of tissues showing different patterns of susceptibility in different host species. We conclude that, in this system, patterns of viral infectivity across host species are robust between males and females, and susceptibility in a given host is general across tissue types.

evolutionary biology↗

bioRxiv2022

The evolution of host resistance to a virus is determined by resources, historical contingency, and time scale

Hosts can often evolve resistance to parasites (and other stressors), but such resistance is generally thought to be constrained by trade-offs with other traits. These trade-offs determine the hosts optimal resistance strategy and whether resistance cycles, diversifies, and/or is maintained in the absence of parasite. However, trade-offs are often inconsistently measured across experiments and can depend on environmental conditions. Here, we extend a selection experiment evolving resistance to viral infection under variable resource quality in the Plodia interpunctella model system to explore the evolutionary conditions leading to an incongruent earlier measurement of costless resistance. We find that environmental resource quality, historical contingency, and the time scale of selection all affect trade-offs in our long-term selection experiment. Specifically, populations selected for resistance with the dual stressor of low resource quality are slowed, but not prevented, from evolving resistance. Second, variation in starting populations or early sampled adaptations led to contingency towards context-dependent resistance. Finally, some costs to resistance observed at early time points were compensated over longer evolutionary time scales. Our work therefore informs perspectives for the predictability of adaptation and how variation in specific evolutionary conditions can alter the evolutionary trajectories of a population towards costly or costless resistance strategies.

evolutionary biology↗

bioRxiv2021

Virus relatedness predicts susceptibility in novel host species

Virus host shifts are a major source of outbreaks and emerging infectious diseases, and predicting the outcome of novel host and virus interactions remains a key challenge for virus research. The evolutionary relationships between host species can explain variation in transmission rates, virulence, and virus community composition between hosts, but the potential for different viruses to interact with host species effects has yet to be established. Here, we measure correlations in viral load of four Cripavirus isolates across experimental infections of 45 Drosophilidae host species. We find positive correlations between every pair of viruses tested, suggesting that broadly susceptible host clades could act as reservoirs and donors for certain types of viruses. Additionally, we find evidence of genotype-by-genotype interactions between viruses and host species, highlighting the importance of both host and virus traits in determining the outcome of virus host shifts. More closely related viruses tended to be more strongly correlated, providing tentative evidence that virus evolutionary relatedness may be a useful proxy for determining the likelihood of novel virus emergence, which warrants further research. Impact SummaryMany new infectious diseases are caused by viruses jumping into novel host species. Estimating the probability that jumps will occur, what the characteristics of new viruses will be, and how they are likely to evolve after jumping to new host species are major challenges. To solve these challenges, we require a detailed understanding of the interactions between different viruses and hosts, or metrics that can capture some of the variation in these interactions. Previous studies have shown that the evolutionary relationships between host species can be used to predict traits of infections in different hosts, including transmission rates and the damage caused by infection. However, the potential for different viruses to influence the patterns of these host species effects has yet to be determined. Here, we use four viruses of insects in experimental infections across 45 different fruit fly host species to begin to answer this question. We find similarities in the patterns of replication and persistence between all four viruses, suggesting susceptible groups of related hosts could act as reservoirs and donors for certain types of virus. However, we also find evidence that different virus genotypes interact in different ways with some host species. Viruses that were more closely related tended to behave in similar ways, and so we suggest that virus evolutionary relatedness may prove to be a useful metric for predicting the traits of novel infections and should be explored further in future studies.

evolutionary biology↗

bioRxiv2020

Diet has no effect on susceptibility to an RNA virus across host species

The likelihood of a successful host shift of a parasite to a novel host species can be influenced by environmental factors that can act on both the host and parasite. Changes in nutritional resource availability have been shown to alter pathogen susceptibility and the outcome of infection in a range of systems. Here we examined how dietary protein to carbohydrate altered susceptibility in a large cross infection experiment. We infected 27 species of Drosophilidae with an RNA virus on three food types of differing protein to carbohydrate ratios. We then measured how viral load and mortality across species was affected by changes in diet. We found that changes in the protein:carbohydrate in the diet did not alter the outcomes of infection, with strong positive inter-species correlations in both viral load and mortality across diets, suggesting no species by diet interaction. Mortality and viral load were strongly positively correlated, and this association was consistent across diets. This suggests changes in diet may give consistent outcomes across host species, and may not be universally important in determining host susceptibility to pathogens. Twitter summaryNo role of host diet in susceptibility to a novel viral pathogen across host species Impact StatementA successful host shift of a parasite from one susceptible species to a novel host can be influenced by many ecological factors. Changes in host diet can alter the immune response and outcomes of host-parasite interactions and could potentially alter the outcome of a virus host shift. To investigate, we infected 27 species of Drosophilidae with an RNA virus (DCV) across three food types with differing protein to carbohydrate ratios. We then looked at pathogen loads and survival of infected hosts compared to uninfected controls. Changes in the ratio of protein to carbohydrate did not alter susceptibility to DCV across host species.

evolutionary biology↗

bioRxiv2019

A new lineage of segmented RNA viruses infecting animals

Metagenomic sequencing has revolutionised our knowledge of virus diversity, with new virus sequences being reported faster than ever before. However, virus discovery from metagenomic sequencing usually depends on detectable homology: without a sufficiently close relative, so-called dark virus sequences remain unrecognisable. An alternative approach is to use virus-identification methods that do not depend on detecting homology, such as virus recognition by host antiviral immunity. For example, virus-derived small RNAs have previously been used to propose dark virus sequences associated with the Drosophilidae (Diptera). Here we combine published Drosophila data with a comprehensive search of transcriptomic sequences and selected meta-transcriptomic datasets to identify a completely new lineage of segmented positive-sense single-stranded RNA viruses that we provisionally refer to as the Quenyaviruses. Each of the five segments contains a single open reading frame, with most encoding proteins showing no detectable similarity to characterised viruses, and one sharing a small number of residues with the RNA-dependent RNA polymerases of single- and double-stranded RNA viruses. Using these sequences, we identify close relatives in approximately 20 arthropods, including insects, crustaceans, spiders and a myriapod. Using a more conserved sequence from the putative polymerase, we further identify relatives in meta-transcriptomic datasets from gut, gill, and lung tissues of vertebrates, reflecting infections of vertebrates or of their associated parasites. Our data illustrate the utility of small RNAs to detect viruses with limited sequence conservation, and provide robust evidence for a new deeply divergent and phylogenetically distinct RNA virus lineage.

evolutionary biology↗

bioRxiv2019

MNC-RED a Chemically Defined Method to Produce Enucleated Red Blood Cells from Adult Peripheral Blood Mononuclear Cells

Many methods have been developed to produce red blood cells in vitro but translational applications have been hampered by the high cost of production. We have developed R6, a chemically-defined, albumin-free, low-transferrin culture medium, and MNC-RED, a protocol to differentiate peripheral blood mononuclear cells into enucleated erythroid cells that does not require any albumin or any animal components. Erythropoiesis requires large amounts of iron for hemoglobin synthesis. In all existing protocols, these large iron needs are met by increasing the concentration of holo-transferrin. This is necessary because transferrin recycling does not take place in existing erythroid culture conditions. In the R6 medium, iron is provided to the differentiating erythroblasts by small amounts of recombinant transferrin supplemented with FeIII-EDTA, an iron chelator that allows transferrin recycling to take place in cell culture. As a result of the absence of albumin and the use of low amounts of transferrin, the production of cultured red blood cells using the MNC-RED protocol is much less expensive than with existing protocols. The MNC-RED protocol should therefore help make the many translational applications of cultured RBCs economically more feasible.\n\nHighlightsWe have developed R6, a chemically-defined, albumin-free low-transferrin culture medium, and MNC-RED, a protocol to differentiate peripheral blood mononuclear cells into enucleated erythroid E\n\nR6 is suitable for red blood cell culture despite the low transferrin amounts because of the presence of FeIII-EDTA, an iron chelator that allows transferrin recycling to take place in cell culture.\n\nThe MNC-RED protocol should help make the many translational applications of cultured RBCs more economically feasible.

bioengineering↗

bioRxiv2019

PSC-RED, an Albumin-Free Robust Erythroid Differentiation Method to Produce Enucleated Red Blood Cells from Human Pluripotent Stem Cells

Cultured red blood cells (cRBCs) have many potential applications in transfusion medicine and drug delivery. We report that we have developed chemically defined, albumin-free Robust Erythroid Differentiation (RED) methods to produce enucleated cRBCs from human induced pluripotent stem cells (iPSCs). Human iPSC-derived cRBCs produced with either the short or long variation of the RED protocol respectively express embryonic/fetal or a mixture of fetal and adult hemoglobins. The long version of the protocol produces up to 50% of enucleated cells at an unprecedented yield. RED is scalable and relies on inexpensive components and therefore dramatically increases the feasibility and economic viability of all translational applications of cRBCs.\n\nHighlightsO_LIPSC-RED: A chemically-defined, albumin-free Robust Erythroid Differentiation (RED) methods to produce cRBCs from human induced pluripotent stem cells.\nC_LIO_LIPSC-RED produces up to 50% enucleated cells at an unprecedented yield.\nC_LIO_LIPSC-RED is scalable and relies on inexpensive components and therefore increases the feasibility and economic viability of translational applications of cRBCs.\nC_LI

bioengineering↗