@article{mcarthur_cruz-teran_thatavarty_reeves_rao_2022, title={Experimental and Analytical Framework for "Mix-and-Read" Assays Based on Split Luciferase}, ISSN={["2470-1343"]}, DOI={10.1021/acsomega.2c02319}, abstractNote={The use of immunodetection assays including the widely used enzyme-linked immunosorbent assay (ELISA) in applications such as point-of-care detection is often limited by the need for protein immobilization and multiple binding and washing steps. Here, we describe an experimental and analytical framework for the development of simple and modular "mix-and-read" enzymatic complementation assays based on split luciferase that enable sensitive detection and quantification of analytes in solution. In this assay, two engineered protein binders targeting nonoverlapping epitopes on the target analyte were each fused to nonactive fragments of luciferase to create biosensor probes. Binding proteins to two model targets, lysozyme and Sso6904, were isolated from a combinatorial library of Sso7d mutants using yeast surface display. In the presence of the analyte, probes were brought into close proximity, reconstituting enzymatic activity of luciferase and enabling detection of low picomolar concentrations of the analyte by chemiluminescence. Subsequently, we constructed an equilibrium binding model that relates binding affinities of the binding proteins for the target, assay parameters such as the concentrations of probes used, and assay performance (limit of detection and concentration range over which the target can be quantified). Overall, our experimental and analytical framework provides the foundation for the development of split luciferase assays for detection and quantification of various targets.}, journal={ACS OMEGA}, author={McArthur, Nikki and Cruz-Teran, Carlos and Thatavarty, Apoorva and Reeves, Gregory T. and Rao, Balaji M.}, year={2022}, month={Jul} } @article{mcarthur_cruz-teran_thatavarty_reeves_rao_2022, title={Experimental and Analytical Framework for "Mix-and-Read" Assays Based on Split Luciferase}, volume={7}, ISSN={["2470-1343"]}, DOI={10.1021/acsomega.2c0231924551ACS}, number={28}, journal={ACS OMEGA}, author={McArthur, Nikki and Cruz-Teran, Carlos and Thatavarty, Apoorva and Reeves, Gregory T. and Rao, Balaji M.}, year={2022}, month={Jul}, pages={24551–24560} } @article{bowen_schloop_reeves_menegatti_rao_2020, title={Discovery of Membrane-Permeating Cyclic Peptides via mRNA Display}, volume={31}, ISSN={["1520-4812"]}, DOI={10.1021/acs.bioconjchem.0c00413}, abstractNote={Small synthetic peptides capable of crossing biological membranes represent valuable tools in cell biology and drug delivery. While several cell-penetrating peptides (CPPs) of natural or synthetic origin have been reported, no peptide is currently known to cross both cytoplasmic and outer embryonic membranes. Here, we describe a method to engineer membrane-permeating cyclic peptides (MPPs) with broad permeation activity by screening mRNA display libraries of cyclic peptides against embryos at different developmental stages. The proposed method was demonstrated by identifying peptides capable of permeating Drosophila melanogaster (fruit fly) embryos and mammalian cells. The selected peptide cyclo[Glut-MRKRHASRRE-K*] showed a strong permeation activity of embryos exposed to minimal permeabilization pretreatment, as well as human embryonic stem cells and a murine fibroblast cell line. Notably, in both embryos and mammalian cells, the cyclic peptide outperformed its linear counterpart and the control MPPs. Confocal microscopy and single cell flow cytometry analysis were utilized to assess the degree of permeation both qualitatively and quantitatively. These MPPs have potential application in studying and nondisruptively controlling intracellular or intraembryonic processes.}, number={10}, journal={BIOCONJUGATE CHEMISTRY}, author={Bowen, John and Schloop, Allison E. and Reeves, Gregory T. and Menegatti, Stefano and Rao, Balaji M.}, year={2020}, month={Oct}, pages={2325–2338} } @article{schloop_bandodkar_reeves_2020, title={Formation, interpretation, and regulation of the Drosophila Dorsal/NF-kappa B gradient}, volume={137}, ISBN={["978-0-12-812790-2"]}, ISSN={["0070-2153"]}, DOI={10.1016/bs.ctdb.2019.11.007}, abstractNote={The morphogen gradient of the transcription factor Dorsal in the early Drosophila embryo has become one of the most widely studied tissue patterning systems. Dorsal is a Drosophila homolog of mammalian NF-κB and patterns the dorsal-ventral axis of the blastoderm embryo into several tissue types by spatially regulating upwards of 100 zygotic genes. Recent studies using fluorescence microscopy and live imaging have quantified the Dorsal gradient and its target genes, which has paved the way for mechanistic modeling of the gradient. In this review, we describe the mechanisms behind the initiation of the Dorsal gradient and its regulation of target genes. The main focus of the review is a discussion of quantitative and computational studies of the Dl gradient system, including regulation of the Dl gradient. We conclude with a discussion of potential future directions.}, journal={GRADIENTS AND TISSUE PATTERNING}, author={Schloop, Allison E. and Bandodkar, Prasad U. and Reeves, Gregory T.}, year={2020}, pages={143–191} } @article{al asafen_bandodkar_carrell-noel_schloop_friedman_reeves_2020, title={Robustness of the Dorsal morphogen gradient with respect to morphogen dosage}, volume={16}, ISSN={["1553-7358"]}, DOI={10.1371/journal.pcbi.1007750}, abstractNote={In multicellular organisms, the timing and placement of gene expression in a developing tissue assigns the fate of each cell in the embryo in order for a uniform field of cells to differentiate into a reproducible pattern of organs and tissues. This positional information is often achieved through the action of spatial gradients of morphogens. Spatial patterns of gene expression are paradoxically robust to variations in morphogen dosage, given that, by definition, gene expression must be sensitive to morphogen concentration. In this work we investigate the robustness of the Dorsal/NF-κB signaling module with respect to perturbations to the dosage of maternally-expressed dorsal mRNA. The Dorsal morphogen gradient patterns the dorsal-ventral axis of the early Drosophila embryo, and we found that an empirical description of the Dorsal gradient is highly sensitive to maternal dorsal dosage. In contrast, we found experimentally that gene expression patterns are highly robust. Although the components of this signaling module have been characterized in detail, how their function is integrated to produce robust gene expression patterns to variations in the dorsal maternal dosage is still unclear. Therefore, we analyzed a mechanistic model of the Dorsal signaling module and found that Cactus, a cytoplasmic inhibitor for Dorsal, must be present in the nucleus for the system to be robust. Furthermore, active Toll, the receptor that dissociates Cactus from Dorsal, must be saturated. Finally, the vast majority of robust descriptions of the system require facilitated diffusion of Dorsal by Cactus. Each of these three recently-discovered mechanisms of the Dorsal module are critical for robustness. Our work highlights the need for quantitative understanding of biophysical mechanisms of morphogen gradients in order to understand emergent phenotypes, such as robustness. Author Summary The early stages of development of an embryo are crucial for laying the foundation of the body plan. The blueprint of this plan is encoded in long-range spatial protein gradients called morphogens. This positional information is then interpreted by nuclei that begin to differentiate by expressing different genes. In fruit fly embryos, the Dorsal morphogen forms a gradient along the dorsal-ventral axis, with a maximum at the ventral midline. This gradient, and the resulting gene expression patterns are extraordinarily robust to variations in developmental conditions, even during early stages of development. Since positional information is interpreted in terms of concentration of the morphogen, one would expect that doubling or halving dosage would result in disastrous consequences for the embryo. However, we observed that development remains robust. We quantified the effect of dosage by experimentally measuring the boundaries of 2 genes, - sna and sog, expressed along the DV axis and found that variation in the boundaries of these genes was minimal, across embryos with different dosages of Dl. We then used a mathematical model to discern components of the Dl system responsible for buffering the effects of dosage and found three specific mechanisms – deconvolution, Toll saturation and shuttling}, number={4}, journal={PLOS COMPUTATIONAL BIOLOGY}, author={Al Asafen, Hadel and Bandodkar, Prasad U. and Carrell-Noel, Sophia and Schloop, Allison E. and Friedman, Jeramey and Reeves, Gregory T.}, year={2020}, month={Apr} } @article{bandodkar_al asafen_reeves_2020, title={Spatiotemporal control of gene expression boundaries using a feedforward loop}, volume={249}, ISSN={["1097-0177"]}, DOI={10.1002/dvdy.150}, abstractNote={Abstract}, number={3}, journal={DEVELOPMENTAL DYNAMICS}, author={Bandodkar, Prasad U. and Al Asafen, Hadel and Reeves, Gregory T.}, year={2020}, month={Mar}, pages={369–382} } @article{reeves_bandodkar_al asafen_2020, title={Spatiotemporal control of gene expression boundaries using a feedforward loop}, volume={34}, ISSN={["1530-6860"]}, DOI={10.1096/fasebj.2020.34.s1.00347}, abstractNote={A feed forward loop (FFL) is commonly observed in several biological networks. The FFL network motif has been mostly been studied with respect to variation of the input signal in time, with only a few studies of FFL activity in a spatially distributed system such as morphogen‐mediated tissue patterning. However, most morphogen gradients also evolve in time. We studied the spatiotemporal behavior of a coherent FFL in two contexts: (1) a generic, oscillating morphogen gradient and (2) the dorsal‐ventral patterning of the early Drosophila embryo by a gradient of the NF‐κB homolog Dorsal with its early target Twist. In both models, we found features in the dynamics of the intermediate node – phase difference and noise filtering – that were largely independent of the parameterization of the models, and thus were functions of the structure of the FFL itself. In the Dorsal gradient model, we also found that the dynamics of Dorsal require maternal pioneering factor Zelda for proper target gene expression.}, journal={FASEB JOURNAL}, author={Reeves, Gregory and Bandodkar, Prasad and Al Asafen, Hadel}, year={2020}, month={Apr} } @article{jacobsen_yi_al asafen_jermusyk_beisel_reeves_2020, title={Tunable self-cleaving ribozymes for modulating gene expression in eukaryotic systems}, volume={15}, ISSN={["1932-6203"]}, DOI={10.1371/journal.pone.0232046}, abstractNote={Advancements in the field of synthetic biology have been possible due to the development of genetic tools that are able to regulate gene expression. However, the current toolbox of gene regulatory tools for eukaryotic systems have been outpaced by those developed for simple, single-celled systems. Here, we engineered a set of gene regulatory tools by combining self-cleaving ribozymes with various upstream competing sequences that were designed to disrupt ribozyme self-cleavage. As a proof-of-concept, we were able to modulate GFP expression in mammalian cells, and then showed the feasibility of these tools in Drosophila embryos. For each system, the fold-reduction of gene expression was influenced by the location of the self-cleaving ribozyme/upstream competing sequence (i.e. 5′ vs. 3′ untranslated region) and the competing sequence used. Together, this work provides a set of genetic tools that can be used to tune gene expression across various eukaryotic systems.}, number={4}, journal={PLOS ONE}, author={Jacobsen, Thomas and Yi, Gloria and Al Asafen, Hadel and Jermusyk, Ashley A. and Beisel, Chase L. and Reeves, Gregory T.}, year={2020}, month={Apr} } @article{reeves_2019, title={The engineering principles of combining a transcriptional incoherent feedforward loop with negative feedback}, volume={13}, ISSN={["1754-1611"]}, DOI={10.1186/s13036-019-0190-3}, abstractNote={Regulation of gene expression is of paramount importance in all living systems. In the past two decades, it has been discovered that certain motifs, such as the feedforward motif, are overrepresented in gene regulatory circuits. Feedforward loops are also ubiquitous in process control engineering, and are nearly always structured so that one branch has the opposite effect of the other, which is a structure known as an "incoherent" feedforward loop in biology. In engineered systems, feedforward control loops are subject to several engineering constraints, including that (1) they are finely-tuned so that the system returns to the original steady state after a disturbance occurs (perfect adaptation), (2) they are typically only implemented in the combination with negative feedback, and (3) they can greatly improve the stability and dynamical characteristics of the conjoined negative feedback loop. On the other hand, in biology, incoherent feedforward loops can serve many purposes, one of which may be perfect adaptation. It is an open question as to whether those that achieve perfect adaptation are subject to the above engineering principles.We analyzed an incoherent feedforward gene regulatory motif from the standpoint of the above engineering principles. In particular, we showed that an incoherent feedforward loop Type 1 (I1-FFL), from within a gene regulatory circuit, can be finely-tuned for perfect adaptation after a stimulus, and that the robustness of this behavior is increased by the presence of moderate negative feedback. In addition, we analyzed the advantages of adding a feedforward loop to a system that already operated under negative feedback, and found that the dynamical properties of the combined feedforward/feedback system were superior.Our analysis shows that many of the engineering principles used in engineering design of feedforward control are also applicable to feedforward loops in biological systems. We speculate that principles found in other domains of engineering may also be applicable to analogous structures in biology.}, journal={JOURNAL OF BIOLOGICAL ENGINEERING}, author={Reeves, Gregory T.}, year={2019}, month={Jul} } @article{trisnadi_altinok_stathopoulos_reeves_2013, title={Image analysis and empirical modeling of gene and protein expression}, volume={62}, ISSN={1046-2023}, url={http://dx.doi.org/10.1016/j.ymeth.2012.09.016}, DOI={10.1016/j.ymeth.2012.09.016}, abstractNote={Protein gradients and gene expression patterns are major determinants in the differentiation and fate map of the developing embryo. Here we discuss computational methods to quantitatively measure the positions of gene expression domains and the gradients of protein expression along the dorsal-ventral axis in the Drosophila embryo. Our methodology involves three layers of data. The first layer, or the primary data, consists of z-stack confocal images of embryos processed by in situ hybridization and/or antibody stainings. The secondary data are relationships between location, usually an x-axis coordinate, and fluorescent intensity of gene or protein detection. Tertiary data comprise the optimal parameters that arise from fits of the secondary data to empirical models. The tertiary data are useful to distill large datasets of imaged embryos down to a tractable number of conceptually useful parameters. This analysis allows us to detect subtle phenotypes and is adaptable to any set of genes or proteins with a canonical pattern. For example, we show how insights into the Dorsal transcription factor protein gradient and its target gene ventral-neuroblasts defective (vnd) were obtained using such quantitative approaches.}, number={1}, journal={Methods}, publisher={Elsevier BV}, author={Trisnadi, Nathanie and Altinok, Alphan and Stathopoulos, Angelike and Reeves, Gregory T.}, year={2013}, month={Jul}, pages={68–78} } @article{garcia_nahmad_reeves_stathopoulos_2013, title={Size-dependent regulation of dorsal–ventral patterning in the early Drosophila embryo}, volume={381}, ISSN={0012-1606}, url={http://dx.doi.org/10.1016/J.YDBIO.2013.06.020}, DOI={10.1016/J.YDBIO.2013.06.020}, abstractNote={How natural variation in embryo size affects patterning of the Drosophila embryo dorsal-ventral (DV) axis is not known. Here we examined quantitatively the relationship between nuclear distribution of the Dorsal transcription factor, boundary positions for several target genes, and DV axis length. Data were obtained from embryos of a wild-type background as well as from mutant lines inbred to size select embryos of smaller or larger sizes. Our data show that the width of the nuclear Dorsal gradient correlates with DV axis length. In turn, for some genes expressed along the DV axis, the boundary positions correlate closely with nuclear Dorsal levels and with DV axis length; while the expression pattern of others is relatively constant and independent of the width of the Dorsal gradient. In particular, the patterns of snail (sna) and ventral nervous system defective (vnd) correlate with nuclear Dorsal levels and exhibit scaling to DV length; while the pattern of intermediate neuroblasts defective (ind) remains relatively constant with respect to changes in Dorsal and DV length. However, in mutants that exhibit an abnormal expansion of the Dorsal gradient which fails to scale to DV length, only sna follows the Dorsal distribution and exhibits overexpansion; in contrast, vnd and ind do not overexpand suggesting some additional mechanism acts to refine the dorsal boundaries of these two genes. Thus, our results argue against the idea that the Dorsal gradient works as a global system of relative coordinates along the DV axis and suggest that individual targets respond to changes in embryo size in a gene-specific manner.}, number={1}, journal={Developmental Biology}, publisher={Elsevier BV}, author={Garcia, Mayra and Nahmad, Marcos and Reeves, Gregory T. and Stathopoulos, Angelike}, year={2013}, month={Sep}, pages={286–299} } @article{reeves_trisnadi_truong_nahmad_katz_stathopoulos_2012, title={Dorsal-Ventral Gene Expression in the Drosophila Embryo Reflects the Dynamics and Precision of the Dorsal Nuclear Gradient}, volume={22}, ISSN={1534-5807}, url={http://dx.doi.org/10.1016/j.devcel.2011.12.007}, DOI={10.1016/j.devcel.2011.12.007}, abstractNote={

Summary

Patterning of the dorsal-ventral axis in the early Drosophila embryo depends on the nuclear distribution of the Dorsal transcription factor. Using live two-photon light-sheet microscopy, we quantified the nuclear Dorsal gradient in space and time and found that its amplitude and basal levels display oscillations throughout early embryonic development. These dynamics raise questions regarding how cells can reproducibly establish patterns of gene expression from a rapidly varying signal. We therefore quantified domains of Dorsal target genes, discovering their expression patterns are also dynamic. Computational modeling of this system reveals a correlation between Dorsal gradient dynamics and changes in target gene expression and suggests that these dynamics, together with time averaging of noise, results in the formation of graded gene expression borders in regions where the gradient is nearly flat. We propose that mRNA levels remain plastic during transient signaling events, allowing tissues to refine patterns in the face of genetic or environmental variation.}, number={3}, journal={Developmental Cell}, publisher={Elsevier BV}, author={Reeves, Gregory T. and Trisnadi, Nathanie and Truong, Thai V. and Nahmad, Marcos and Katz, Sophie and Stathopoulos, Angelike}, year={2012}, month={Mar}, pages={544–557} } @article{nahmad_reeves_stathopoulos_2011, title={Size-dependent regulation of dorsal-ventral patterning in the early Drosophila embryo}, volume={356}, ISSN={0012-1606}, url={http://dx.doi.org/10.1016/j.ydbio.2011.05.061}, DOI={10.1016/j.ydbio.2011.05.061}, number={1}, journal={Developmental Biology}, publisher={Elsevier BV}, author={Nahmad, Marcos and Reeves, Gregory and Stathopoulos, Angelike}, year={2011}, month={Aug}, pages={119} } @article{liberman_reeves_stathopoulos_2009, title={Quantitative imaging of the Dorsal nuclear gradient reveals limitations to threshold-dependent patterning in Drosophila}, volume={106}, ISSN={0027-8424 1091-6490}, url={http://dx.doi.org/10.1073/pnas.0906227106}, DOI={10.1073/pnas.0906227106}, abstractNote={ The NF-κB-related transcription factor, Dorsal, forms a nuclear concentration gradient in the early Drosophila embryo, patterning the dorsal-ventral (DV) axis to specify mesoderm, neurogenic ectoderm, and dorsal ectoderm cell fates. The concentration of nuclear Dorsal is thought to determine these patterning events; however, the levels of nuclear Dorsal have not been quantified previously. Furthermore, existing models of Dorsal-dependent germ layer specification and patterning consider steady-state levels of Dorsal relative to target gene expression patterns, yet both Dorsal gradient formation and gene expression are dynamic. We devised a quantitative imaging method to measure the Dorsal nuclear gradient while simultaneously examining Dorsal target gene expression along the DV axis. Unlike observations from other insects such as Tribolium , we find the Dorsal gradient maintains a constant bell-shaped distribution during embryogenesis. We also find that some classical Dorsal target genes are located outside the region of graded Dorsal nuclear localization, raising the question of whether these genes are direct Dorsal targets. Additionally, we show that Dorsal levels change in time during embryogenesis such that a steady state is not reached. These results suggest that the multiple gene expression outputs observed along the DV axis do not simply reflect a steady-state Dorsal nuclear gradient. Instead, we propose that the Dorsal gradient supplies positional information throughout nuclear cycles 10-14, providing additional evidence for the idea that compensatory combinatorial interactions between Dorsal and other factors effect differential gene expression along the DV axis. }, number={52}, journal={Proceedings of the National Academy of Sciences}, publisher={Proceedings of the National Academy of Sciences}, author={Liberman, Louisa M. and Reeves, Gregory T. and Stathopoulos, Angelike}, year={2009}, month={Dec}, pages={22317–22322} } @article{goentoro_reeves_kowal_martinelli_schüpbach_shvartsman_2006, title={Quantifying the Gurken Morphogen Gradient in Drosophila Oogenesis}, volume={11}, ISSN={1534-5807}, url={http://dx.doi.org/10.1016/j.devcel.2006.07.004}, DOI={10.1016/j.devcel.2006.07.004}, abstractNote={

Summary

Quantitative information about the distribution of morphogens is crucial for understanding their effects on cell-fate determination, yet it is difficult to obtain through direct measurements. We have developed a parameter estimation approach for quantifying the spatial distribution of Gurken, a TGFα-like EGFR ligand that acts as a morphogen in Drosophila oogenesis. Modeling of Gurken/EGFR system shows that the shape of the Gurken gradient is controlled by a single dimensionless parameter, the Thiele modulus, which reflects the relative importance of ligand diffusion and degradation. By combining the model with genetic alterations of EGFR levels, we have estimated the value of the Thiele modulus in the wild-type egg chamber. This provides a direct characterization of the shape of the Gurken gradient and demonstrates how parameter estimation techniques can be used to quantify morphogen gradients in development.}, number={2}, journal={Developmental Cell}, publisher={Elsevier BV}, author={Goentoro, Lea A. and Reeves, Gregory T. and Kowal, Craig P. and Martinelli, Luigi and Schüpbach, Trudi and Shvartsman, Stanislav Y.}, year={2006}, month={Aug}, pages={263–272} } @article{reeves_muratov_schüpbach_shvartsman_2006, title={Quantitative Models of Developmental Pattern Formation}, volume={11}, ISSN={1534-5807}, url={http://dx.doi.org/10.1016/j.devcel.2006.08.006}, DOI={10.1016/j.devcel.2006.08.006}, abstractNote={Pattern formation in developing organisms can be regulated at a variety of levels, from gene sequence to anatomy. At this level of complexity, mechanistic models of development become essential for integrating data, guiding future experiments, and predicting the effects of genetic and physical perturbations. However, the formulation and analysis of quantitative models of development are limited by high levels of uncertainty in experimental measurements, a large number of both known and unknown system components, and the multiscale nature of development. At the same time, an expanding arsenal of experimental tools can constrain models and directly test their predictions, making the modeling efforts not only necessary, but feasible. Using a number of problems in fruit fly development, we discuss how models can be used to test the feasibility of proposed patterning mechanisms and characterize their systems-level properties.}, number={3}, journal={Developmental Cell}, publisher={Elsevier BV}, author={Reeves, Gregory T. and Muratov, Cyrill B. and Schüpbach, Trudi and Shvartsman, Stanislav Y.}, year={2006}, month={Sep}, pages={289–300} } @article{reeves_kalifa_klein_lemmon_shvartsman_2005, title={Computational analysis of EGFR inhibition by Argos}, volume={284}, ISSN={0012-1606}, url={http://dx.doi.org/10.1016/j.ydbio.2005.05.013}, DOI={10.1016/j.ydbio.2005.05.013}, abstractNote={Argos, a secreted inhibitor of the Drosophila epidermal growth factor receptor, and the only known secreted receptor tyrosine kinase inhibitor, acts by sequestering the EGFR ligand Spitz. We use computational modeling to show that this biochemically-determined mechanism of Argos action can explain available genetic data for EGFR/Spitz/Argos interactions in vivo. We find that efficient Spitz sequestration by Argos is key for explaining the existing data and for providing a robust feedback loop that modulates the Spitz gradient in embryonic ventral ectoderm patterning. Computational analysis of the EGFR/Spitz/Argos module in the ventral ectoderm shows that Argos need not be long-ranged to account for genetic data, and can actually have very short range. In our models, Argos with long or short length scale functions to limit the range and action of secreted Spitz. Thus, the spatial range of Argos does not have to be tightly regulated or may act at different ranges in distinct developmental contexts.}, number={2}, journal={Developmental Biology}, publisher={Elsevier BV}, author={Reeves, Gregory T. and Kalifa, Rachel and Klein, Daryl E. and Lemmon, Mark A. and Shvartsman, Stanislav Y.}, year={2005}, month={Aug}, pages={523–535} } @article{klein_nappi_reeves_shvartsman_lemmon_2004, title={Argos inhibits epidermal growth factor receptor signalling by ligand sequestration}, volume={430}, ISSN={0028-0836 1476-4687}, url={http://dx.doi.org/10.1038/nature02840}, DOI={10.1038/nature02840}, abstractNote={The epidermal growth factor receptor (EGFR) has critical functions in development and in many human cancers. During development, the spatial extent of EGFR signalling is regulated by feedback loops comprising both well-understood activators and less well-characterized inhibitors. In Drosophila melanogaster the secreted protein Argos functions as the only known extracellular inhibitor of EGFR, with clearly identified roles in multiple stages of development. Argos is only expressed when the Drosophila EGFR (DER) is activated at high levels, and downregulates further DER signalling. Although there is ample genetic evidence that Argos inhibits DER activation, the biochemical mechanism has not been established. Here we show that Argos inhibits DER signalling without interacting directly with the receptor, but instead by sequestering the DER-activating ligand Spitz. Argos binds tightly to the EGF motif of Spitz and forms a 1:1 (Spitz:Argos) complex that does not bind DER in vitro or at the cell surface. Our results provide an insight into the mechanism of Argos function, and suggest new strategies for EGFR inhibitor design.}, number={7003}, journal={Nature}, publisher={Springer Science and Business Media LLC}, author={Klein, Daryl E. and Nappi, Valerie M. and Reeves, Gregory T. and Shvartsman, Stanislav Y. and Lemmon, Mark A.}, year={2004}, month={Aug}, pages={1040–1044} } @article{reeves_narang_pilyugin_2004, title={Growth of mixed cultures on mixtures of substitutable substrates: the operating diagram for a structured model}, volume={226}, ISSN={0022-5193}, url={http://dx.doi.org/10.1016/S0022-5193(03)00280-7}, DOI={10.1016/S0022-5193(03)00280-7}, abstractNote={The growth of mixed microbial cultures on mixtures of substrates is a problem of fundamental biological interest. In the last two decades, several unstructured models of mixed-substrate growth have been studied. It is well known, however, that the growth patterns in mixed-substrate environments are dictated by the enzymes that catalyse the transport of substrates into the cell. We have shown previously that a model taking due account of transport enzymes captures and explains all the observed patterns of growth of a single species on two substitutable substrates (J. Theor. Biol. 190 (1998) 241). Here, we extend the model to study the steady states of growth of two species on two substitutable substrates. The model is analysed to determine the conditions for existence and stability of the various steady states. Simulations are performed to determine the flow rates and feed concentrations at which both species coexist. We show that if the interaction between the two species is purely competitive, then at any given flow rate, coexistence is possible only if the ratio of the two feed concentrations lies within a certain interval; excessive supply of either one of the two substrates leads to annihilation of one of the species. This result simplifies the construction of the operating diagram for purely competing species. This is because the two-dimensional surface that bounds the flow rates and feed concentrations at which both species coexist has a particularly simple geometry: It is completely determined by only two coordinates, the flow rate and the ratio of the two feed concentrations. We also study commensalistic interactions between the two species by assuming that one of the species excretes a product that can support the growth of the other species. We show that such interactions enhance the coexistence region.}, number={2}, journal={Journal of Theoretical Biology}, publisher={Elsevier BV}, author={Reeves, Gregory T. and Narang, Atul and Pilyugin, Sergei S.}, year={2004}, month={Jan}, pages={143–157} } @article{pilyugin_reeves_narang_2004, title={Predicting stability of mixed microbial cultures from single species experiments: 1. Phenomenological model}, volume={192}, ISSN={0025-5564}, url={http://dx.doi.org/10.1016/j.mbs.2004.10.003}, DOI={10.1016/j.mbs.2004.10.003}, abstractNote={The growth of mixed microbial cultures on mixtures of substrates is a fundamental problem of both theoretical and practical interest. On the one hand, the literature is abundant with experimental studies of mixed-substrate phenomena [T. Egli, The ecological and physiological significance of the growth of heterotrophic microorganisms with mixtures of substrates, Adv. Microbiol. Ecol. 14 (1995) 305-386]. On the other hand, a number of mathematical models of mixed-substrate growth have been analyzed in the last three decades. These models typically assume specific kinetic expressions for substrate uptake and biomass growth rates and their predictions are formulated in terms of parameters of the model. In this work, we formulate and analyze a general mathematical model of mixed microbial growth on mixtures of substitutable substrates. Using this model, we study the effect of mutual inhibition of substrate uptake rates on the stability of the equilibria of the model. Specifically, we address the following question: How much of the dynamics exhibited by two competing species can be inferred from single species data? We provide geometric criteria for stability of various types of equilibria corresponding to non-competitive exclusion, competitive exclusion, and coexistence of two competing species in terms of growth isoclines and consumption curves. A growth isocline is a curve in the plane of substrate concentrations corresponding to the zero net growth of a given species. In [G.T. Reeves, A. Narang, S.S. Pilyugin, Growth of mixed cultures on mixtures of substitutable substrates: The operating diagram for a structured model, J. Theor. Biol. 226 (2004) 143-157], we introduced consumption curves as sets of all possible combinations of substrate concentrations corresponding to balanced growth of a single microbial species. Both types of curves can be obtained in single species experiments.}, number={2}, journal={Mathematical Biosciences}, publisher={Elsevier BV}, author={Pilyugin, Sergei S. and Reeves, Gregory T. and Narang, Atul}, year={2004}, month={Dec}, pages={85–109} } @article{pilyugin_reeves_narang_2004, title={Predicting stability of mixed microbial cultures from single species experiments: 2. Physiological model}, volume={192}, ISSN={0025-5564}, url={http://dx.doi.org/10.1016/j.mbs.2004.10.002}, DOI={10.1016/j.mbs.2004.10.002}, abstractNote={In this paper, we study the equilibria of a physiological model describing the continuous culture in which two microbial populations compete for two substitutable resources. This work is an extension of the stability analysis of the phenomenological model of mixed microbial growth [M.M. Ballyk, G.S.K. Wolkowicz, Exploitative competition in the chemostat for two perfectly substitutable resources, Math. Biosci. 118 (1993) 127–180; S.S. Pilyugin, G.T. Reeves, A. Narang, Predicting stability of mixed microbial cultures from single species experiments: 2. Phenomenological model]. Here, we investigate the influence of the peripheral enzymes that catabolize the substrate uptake on the stability of the mixed culture. We show that, under steady state conditions, an increase in the concentration of one substrate inhibits the uptake of the other substrate(s). We present the criteria for existence, uniqueness, and stability of various types of equilibria. We formulate these criteria in terms of growth isoclines and consumption curves for each of the competing species. Since both types of curves can be obtained from a single species experiment, our approach provides a direct connection between theory and experiment and allows one to infer the dynamics of mixed cultures from the dynamics of single species cultures. By expressing the stability criteria in terms of intracellular properties, the model establishes a link between ecology and molecular biology.}, number={2}, journal={Mathematical Biosciences}, publisher={Elsevier BV}, author={Pilyugin, Sergei S. and Reeves, Gregory T. and Narang, Atul}, year={2004}, month={Dec}, pages={111–136} }