Biology subjects

O'Leary, T. S.

Publications and source records attributed to O'Leary, T. S..

7 recordsLinked to original sources

bioRxiv2026

Heat stress drives opposing redox shifts in temperate versus tropical Drosophila melanogaster embryos

Redox balance is central to aerobic metabolism, yet acute heat stress can destabilize this balance by increasing metabolic rates and shifting the balance of critical electron carriers such as NADH. In early Drosophila melanogaster embryos, maintaining redox balance is particularly critical as embryos undergo a developmental redox shift and rely on oxidative phosphorylation to power nuclear divisions. Here, we assayed six isofemale D. melanogaster lines from temperate (Vermont, USA; France; Japan) and tropical (St. Kitts; Ghana; India) climates to assess metabolic responses to heat in heat-sensitive versus heat-tolerant embryos. We used untargeted LC-MS to measure 33 metabolites and the major redox couples (NADH/NAD+, NADPH/NADP+, and GSH/GSSG) at 25{degrees}C and after a 32{degrees}C heat shock. In all embryos, heat shock induced shared shifts in metabolic profiles, with increases in nucleotide monophosphates (e.g., AMP, CMP, and GMP) and amino acids (e.g., alanine, glutamic acid, serine). In contrast, redox metabolites diverged by region: heat-sensitive temperate embryos shifted toward a more oxidized state (46.6% decrease in NADH/NAD+ ratio and 4-fold increase in oxidized glutathione), while heat-tolerant tropical embryos maintained glutathione balance and increased the NADH/NAD+ ratio by 52.9%, indicating a more reduced state. These patterns are consistent with higher NADH oxidation and greater oxidative stress (inferred from oxidized glutathione) in the temperate embryos, versus better maintenance of redox balance in tropical embryos. Together, our results suggest that maintaining redox balance is a key determinant of acute heat tolerance, and healthy development overall, during early embryogenesis.

evolutionary biology↗

bioRxiv2026

Modes of natural selection on maternal and zygotic gene expression in Drosophila melanogaster embryos

Early embryonic development involves the coordination of gene expression from two distinct genomes, as mothers load eggs with gene products prior to the beginning of zygotic transcription. Because the maternal transcriptome is controlled by the mothers regulatory genotype rather than that of the embryo, these two sequential developmental programs may experience distinct evolutionary constraints and selection pressures despite both existing within the embryo. To infer modes of selection on gene expression at maternal and zygotic developmental stages, we used single-embryo RNA-sequencing and conducted variance tests of selection on transcriptomes of parents and F2 segregants of tropical and temperate Drosophila melanogaster embryos. We found that approximately 10% of maternal transcripts and 5% of zygotic transcripts showed signals of selection. Genome-wide, directional selection was more common than stabilizing selection. However, among core early developmental genes and transcription factors, maternal transcription showed patterns of both stabilizing and directional selection, whereas zygotic transcription was predominantly under stabilizing selection. Many heat shock genes showed patterns of directional selection between tropical and temperate embryos, consistent with local adaptation. Additionally, directional selection in piRNA-pathway genes suggests a role for germline defense during embryogenesis. Overall, while our data support the canonical view that core developmental networks are constrained by stabilizing selection, the genome-wide prevalence of directional selection highlights a substantial and previously underappreciated contribution of diversifying selection to the evolution of early development. Article SummaryThe evolution of embryonic gene expression remains poorly characterized on microevolutionary timescales. We conducted genetic crosses between tropical and temperate Drosophila melanogaster, combining single-embryo RNA sequencing with variance tests of selection, to infer patterns of adaptive evolution in gene expression. We found pervasive signals of selection. While core developmental genes were largely under stabilizing selection, directional selection was the predominant mode of selection genome-wide. These findings challenge the paradigm that early embryogenesis is evolutionarily inflexible, suggesting that diversifying selection and local adaptation play key roles in the evolution of development on short evolutionary timescales.

evolutionary biology↗

bioRxiv2025

Single-nuclei multiome ATAC and RNA sequencing reveals the molecular basis of thermal plasticity in Drosophila melanogaster embryos

Embryogenesis is remarkably robust to temperature variability, yet the homeostatic mechanisms that offset thermal effects during early development remain poorly understood. We measured how acclimation shifts the upper thermal limit and profiled chromatin accessibility and gene expression in D. melanogaster embryos using single-nucleus multiome sequencing. Acclimation preserved shared primordial cell types yet rapidly shifted heat tolerance. Cool-acclimated embryos showed a homeostatic response characterized by increased accessibility at binding motifs for the transcriptional activator Zelda, along with enhanced activity of gene regulatory networks in a subset of primordial cell types including the gut, muscle, and nervous system. Cool-acclimated embryos also had higher expression of ribosomal and oxidative phosphorylation genes, reflecting a coordinated increase in translation and energy production to buffer slower biochemical kinetics in the cold. Our results indicate that maintaining developmental robustness across temperature requires homeostatic gene expression responses that may carry metabolic costs constraining upper thermal limits.

developmental biology↗

bioRxiv2024

Heat tolerance, oxidative stress response tuning, and robust gene activation in early-stage Drosophila melanogaster embryos

In organisms with complex life cycles, life stages that are most susceptible to environmental stress may determine species persistence in the face of climate change. Early embryos of Drosophila melanogaster are particularly sensitive to acute heat stress, yet tropical embryos have higher heat tolerance than temperate embryos, suggesting adaptive variation in embryonic heat tolerance. We compared transcriptomic responses to heat stress among tropical and temperate embryos to elucidate the gene regulatory basis of divergence in embryonic heat tolerance. The transcriptomes of tropical and temperate embryos were differentiated by the expression of relatively few genes, including genes involved in oxidative stress. But most of the transcriptomic response to heat stress was shared among all embryos. Further, embryos shifted the expression of thousands of genes and showed robust gene activation, demonstrating that, contrary to previous reports, early embryos are not transcriptionally silent. The involvement of oxidative stress genes in embryonic heat tolerance corroborates recent reports on the critical role of redox homeostasis in coordinating developmental transitions. By characterizing adaptive variation in the transcriptomic basis of embryonic heat tolerance, this study is a novel contribution to the literature on developmental physiology and genetics, which often lacks ecological and evolutionary context.

genetics↗

bioRxiv2021

Biomechanics and Myofibrillar Alignment Enhance Contractile Development and Reproducibility in Stem Cell Derived Cardiac Muscle

Human pluripotent stem cell derived cardiomyocytes (hPSC-CMs) allow novel investigations of human cardiac disease, but disorganized mechanics and immaturity of hPSC-CMs on two-dimensional (2D) surfaces have been hurdles for efficient and reproducible study of these cells. Here, we developed a platform of micron-scale 2D cardiac tissues (M2DCTs) to precisely control biomechanics in arrays of thousands of purified, independently contracting cardiac muscle strips in 2D. By defining geometry and workload in M2DCTs in this reductionist platform that does not incorporate other cell types, we show that myofibrillar alignment and auxotonic contractions at physiologic workload critically drive maturation of cardiac contractile function, calcium handling, and electrophysiology. Additionally, the organized biomechanics in this system facilitates rapid and automated extraction of contractile kinetic parameters from brightfield microscopy images, increasing the reproducibility and throughput of pharmacologic testing. Finally, we show that M2DCTs enable precise and efficient dissection of contractile kinetics in cardiomyopathy disease models.

cell biology↗

bioRxiv2020

CryoEM Structure of Drosophila Flight Muscle Thick Filaments at 7A Resolution

Striated muscle thick filaments are composed of myosin II and several non-myosin proteins. Myosin IIs long -helical coiled-coil tail forms the dense protein backbone of filaments while its N-terminal globular head containing the catalytic and actin binding activities extends outward from the backbone. Here we report the structure of thick filaments of the flight muscle of the fruit fly Drosophila melanogaster at 7 [A] resolution. Its myosin tails are arranged in curved molecular crystalline layers identical to flight muscles of the giant waterbug Lethocerus indicus. Four non-myosin densities are observed, three of which correspond to ones found in Lethocerus; one new density, possibly stretchin-Mlck, is found on the backbone outer surface. Surprisingly, the myosin heads are disordered rather than ordered along the filament backbone. Our results show striking myosin tail similarity within flight muscle filaments of two insect orders separated by several hundred million years of evolution. Significance StatementMyosin thick filaments are one of striated muscles key structures, but also one of its least understood. A key question is how the myosin a-helical coiled-coil tail is arranged in the backbone. At 7[A] resolution, sufficient to resolve individual a-helices, the myosin tail arrangement in thick filaments from the flight muscle of the fruit fly Drosophila melanogaster is strikingly similar to the myosin tail arrangement in flight muscles of the giant waterbug Lethocerus indicus. Nearly every other thick filament feature is different. Drosophila and Lethocerus evolved separately >245 million years ago suggesting myosin tail packing into curved molecular crystalline layers forms a highly conserved thick filament building block and different properties are obtained by alterations in non-myosin proteins.

biophysics↗

bioRxiv2019

SKELETAL MyBP-C ISOFORMS TUNE THE MOLECULAR CONTRACTILITY OF DIVERGENT SKELETAL MUSCLE SYSTEMS

Skeletal muscle myosin-binding protein C (MyBP-C) is a myosin thick filament-associated protein; localized through its C terminus to distinct regions (C-zones) of the sarcomere. MyBP-C modulates muscle contractility, presumably through its N terminus extending from the thick filament and interacting with either the myosin head region and/or the actin thin filament. Two isoforms of MyBP-C (fast- and slow-type) are expressed in a muscle-type specific manner. Are the expression, localization, and Ca2+-dependent modulatory capacities of these isoforms different in fast-twitch extensor digitorum longus (EDL) and slow-twitch soleus (SOL) muscles derived from Sprague-Dawley rats? By mass spectrometry, four MyBP-C isoforms (one fast-type MyBP-C and three N-terminally spliced slow-type MyBP-C) were expressed in EDL but only the three slow-type MyBP-C isoforms in SOL. Using EDL and SOL native thick filaments in which the MyBP-C stoichiometry and localization are preserved, native thin filament sliding over these thick filaments showed that only in the C-zone, MyBP-C Ca2+-sensitizes the thin filament and slows thin filament velocity. These modulatory properties depended on MyBP-Cs N-terminus, as N-terminal proteolysis attenuated MyBP-Cs functional capacities. To determine each MyBP-C isoforms contribution to thin filament Ca2+-sensitization and slowing in the C-zone, we used a combination of in vitro motility assays using expressed recombinant N-terminal fragments and in silico mechanistic modeling. Our results suggest that each skeletal MyBP-C isoforms N terminus is functionally distinct and has modulatory capacities that depend on the muscle-type in which they are expressed, providing the potential for molecular tuning of skeletal muscle performance through differential MyBP-C expression.\n\nSIGNIFICANCEMyosin-binding protein C (MyBP-C) is a critical component of the skeletal muscle sarcomere, muscles smallest contractile unit. MyBP-Cs importance is evident by genetic mutations leading to human myopathies, such as distal arthrogryposis (i.e. club foot). However, the molecular basis of MyBP-Cs functional impact on skeletal muscle contractility is far from certain. Complicating matters further is the expression of fast- and slow-type MyBP-C isoforms that depend on whether the muscle is fast- or slow-twitch. Using multi-scale proteomic, biophysical and mathematical modeling approaches, we define the expression, localization, and modulatory capacities of these distinct skeletal MyBP-C isoforms in rat skeletal muscles. Each MyBP-C isoform appears to modulate muscle contractility differentially; providing the capacity to fine-tune muscles mechanical performance as physiological demands arise.

physiology↗