Biology subjects

Miller, D. M.

Publications and source records attributed to Miller, D. M..

At least 19 recordsLinked to original sources

bioRxiv2026

Losing the split ends: unraveling the mechanisms of fiber branching during blood clot formation

The coagulation cascade generates a three-dimensional fibrin gel that seals wounds and prevents the loss of blood. Extensive research has defined the structural and mechanical characteristics of fibrin gels and the various biochemical, physiological, and pathological factors that alter those properties. However, there has been no direct visualization of fibrin polymerization with the spatial and temporal resolution to characterize the fibrin branching process. Thus, a mechanistic explanation for the origins of fibrin structures remains unresolved, with several hypothesized models for how fibrin branching occurs. Using SimView light-sheet microscopy, we captured videos of fibrin fibers forming junctions and gelling. Results: We observed four distinct pathways for fibrin fibers to interact and bind, all predicated on the observation that tumbling and diffusing fibers collide and stick together. Branching occurred due to the binding of mature fibers, not through the splitting of fibers or through the lateral association of small fibrin oligomers (protofibrils), as had been previously hypothesized. Diffusion simulations of rod-like fibers confirmed the branching mechanisms, the relative frequency of the various binding pathways, and the distribution of fiber angles after binding. Scanning Electron Microscopy (SEM) imaging also corroborated the relative frequency of various types of fibrin junctions. Moreover, SEM enabled the visualization of junctions at different timepoints, demonstrating that additional fibrin strands adhere to the initial binding site, strengthening the interaction. These results establish a mechanistic framework for fibrin branching and gel formation and define an innovative experimental approach for future interrogations of fibrin polymerization.

biophysics↗

bioRxiv2026

The Adhesion GPCR Flamingo-Like 1 (FMIL-1) Directs Synapse Formation in a Nociceptive Circuit

The species-specific anatomy of nervous systems suggests that circuit architecture is largely encoded by genetic blueprints. In C. elegans, PVD nociceptive neurons synapse with PVC and AVA interneurons to drive escape responses to noxious stimuli. We used fluorescent markers for PVD synapses with PVC and AVA in a candidate screen to detect connectivity genes. This approach revealed that the LIM homeodomain transcription factor MEC-3 and its target, FMIL-1 (Flamingo-like), function in PVD to direct connectivity with PVC and AVA. FMIL-1 is an adhesion class G Protein-coupled receptor (aGPCR), a protein family with members also implicated in mammalian synapse formation. We show that FMIL-1 acts early in PVD and is also sufficient to induce ectopic synapses in another circuit, thus suggesting that FMIL-1 promotes synaptogenesis. Our work establishes a new experimental circuit in C. elegans for investigating neuronal connectivity and provides evidence that FMIL-1/aGPCRs regulate synapse formation.

neuroscience↗

bioRxiv2026

ContrasTED: contrastive domain embeddings for scalable remote homology classification

Protein structure prediction has expanded structural databases to hundreds of millions of domains. Classifying these domains into homologous superfamilies reveals evolutionary and functional relationships that can persist despite low sequence similarity. As the size of structural databases continues to grow, homology classification requires methods that combine scalability with accuracy. Here we present ContrasTED, which uses CATH-supervised center-contrastive learning to project structure-aware embeddings into a domain-level metric space for nearest-centroid superfamily assignment. On a sequence-filtered S20 benchmark (n = 1,028), superfamily assignment accuracy reached 92.9% (1-NN) and 91.4% (nearest centroid), exceeding sequence search, profile HMMs, Foldseek, and a classifier trained on embeddings. The learned latent space separates superfamilies while retaining structural information below 20% sequence identity, with the largest gains among sparsely represented superfamilies. ContrasTED produces 4.67 million new candidate assignments across 3,796 superfamilies in The Encyclopedia of Domains (TED), extending annotation coverage beyond previous structure-based methods.

bioinformatics↗

bioRxiv2025

ProFam: Open-Source Protein Family Language Modelling for Fitness Prediction and Design

Protein language models have become essential tools for engineering novel functional proteins. The emerging paradigm of family-based language models makes use of homologous sequences to steer protein design and enhance zero-shot fitness prediction, by imbuing models with an ability to explicitly reason over evolutionary context. To provide an open foundation for this modelling approach, we introduce ProFam-1, a 251M-parameter autoregressive protein family language model (pfLM) trained with next-token prediction on millions of protein families represented as concatenated, unaligned sets of sequences. ProFam-1 is competitive with state-of-the-art models on the ProteinGym zero-shot fitness prediction benchmark, achieving Spearman correlations of 0.47 for substitutions and 0.53 for indels. For homology-guided generation, ProFam-1 generates diverse sequences with predicted structural similarity, while preserving residue conservation and covariance patterns. All of ProFams training and inference pipelines, together with our curated, large-scale training dataset ProFam Atlas, are released fully open source, lowering the barrier to future method development.

bioinformatics↗

bioRxiv2025

A gene expression atlas of a juvenile nervous system

Although the fundamental architecture of metazoan nervous systems is typically established in the embryo, substantial numbers of neurons are added during post-natal development while existing neurons expand in size, refine connectivity, and undergo additional differentiation. To reveal the underlying molecular determinants of postembryonic neurogenesis and maturation, we have produced gene expression profiles of all neuron types and their progenitors in the first larval stage (L1) of C. elegans. Comparisons of the L1 profile to the embryo and to the later L4 larval stage identified thousands of differentially expressed genes across individual neurons throughout the nervous system. Key neuropeptide signaling networks, for example, are remodeled during larval development. Gene regulatory network analysis revealed potential transcription factors driving the temporal changes in gene expression across the nervous system, including a broad role for the heterochronic transcription factor lin-14. We utilized available connectomic data of juvenile animals in combination with our neuron-specific atlas to identify potential molecular determinants of membrane contact and synaptic connectivity. These expression data are available through a user-friendly interface at CeNGEN.org for independent investigations of the maturation, connectivity and function of a developing nervous system.

neuroscience↗

bioRxiv2025

The forkhead transcription factor FKH-7/FOXP acts in chemosensory neurons to regulate developmental decision-making

Autism is a complex neurodevelopmental disorder with many associated genetic factors, including the forkhead transcription factor FOXP1. Although FOXP1s neuronal role is well-studied, the specific molecular consequences of different FOXP1 pathogenic variants in physiologically-relevant contexts are unknown. Here we ascribe the first function to Caenorhabditis elegans FKH-7/FOXP, which acts in two chemosensory neuron classes to promote the larval decision to enter the alternative, developmentally-arrested dauer life stage. We demonstrate that human FOXP1 can functionally substitute for C. elegans FKH-7 in these neurons and that engineering analogous FOXP1 hypomorphic missense mutations in the endogenous fkh-7 locus also impairs developmental decision-making. In a fkh-7/FOXP1 missense variant, single-cell transcriptomics identifies downregulated expression of autism-associated kcnl-2/KCNN2 calcium-activated potassium channel in a serotonergic sensory neuron. Our findings establish a novel framework linking two evolutionarily-conserved autism-associated genes for deeper characterization of variant-specific molecular pathology at single neuron resolution in the context of a developmental decision-making paradigm.

neuroscience↗

bioRxiv2025

Integrating bulk and single cell RNA-seq refines transcriptomic profiles of individual C. elegans neurons

Neuron-specific morphology and function are fundamentally tied to differences in gene expression across the nervous system. We previously generated a single cell RNA-seq (scRNA-Seq) dataset for every anatomical neuron class in the C. elegans hermaphrodite. Here we present a complementary set of bulk RNA-seq samples for 52 of the 118 canonical neuron classes in C. elegans. We show that the bulk RNA-seq dataset captures both lowly expressed and noncoding RNAs that are not detected in the scRNA-Seq profile, but also includes false positives due to contamination by other cell types. We present an analytical strategy that integrates the two datasets, preserving both the specificity of scRNA-Seq data and the sensitivity of bulk RNA-Seq. We show that this integrated dataset enhances the sensitivity and accuracy of transcript detection and differential gene analysis. In addition, we show that the bulk RNA-Seq data set detects differentially expressed non-coding RNAs across neuron types, including multiple families of non-polyadenylated transcripts. We propose that our approach provides a new strategy for interrogating gene expression by bridging the gap between bulk and single cell methodologies for transcriptomic studies. We suggest that these datasets advance the goal of delineating the mechanisms that define morphology and connectivity in the nervous system.

genomics↗

bioRxiv2024

Multi-Tissue Metabolomics Reveal mtDNA- and Diet-Specific Metabolite Profiles in a Mouse Model of Cardiometabolic Disease

RationaleExcess consumption of sugar- and fat-rich foods has heightened the prevalence of cardiometabolic disease, which remains a driver of cardiovascular disease- and type II diabetes-related mortality globally. Skeletal muscle insulin resistance is an early feature of cardiometabolic disease and is a precursor to diabetes. Insulin resistance risk varies with self-reported race, whereby, African-Americans have a greater risk of diabetes development relative to their White counterparts. Self-reported race is strongly associated with mitochondrial DNA (mtDNA) haplogroups, and previous reports have noted marked differences in bioenergetic and metabolic parameters in cells belonging to distinct mtDNA haplogroups, but the mechanism of these associations remains unknown. Additionally, distinguishing nuclear DNA (nDNA) and mtDNA contributions to cardiometabolic disease remains challenging in humans. The Mitochondrial-Nuclear eXchange (MNX) mouse model enables in vivo preclinical investigation of the role of mtDNA in cardiometabolic disease development, and has been implemented in studies of insulin resistance, fatty liver disease, and obesity in previous reports. MethodsSix-week-old male C57nDNA:C57mtDNA and C3HnDNA:C3HmtDNA wild-type mice, and C57nDNA:C3HmtDNA and C3HnDNA:C57mtDNA MNX mice, were fed sucrose-matched high-fat (45% kcal fat) or control diet (10% kcal fat) until 12 weeks of age (n = 5/group). Mice were weighed weekly and total body fat was collected at euthanasia. Gastrocnemius skeletal muscle and plasma metabolomes were characterized using untargeted dual-chromatography mass spectrometry; both hydrophilic interaction liquid chromatography (HILIC) and C18 columns were used, in positive- and negative-ion modes, respectively. ResultsComparative analyses between nDNA-matched wild-type and MNX strains demonstrated significantly increased body fat percentage in mice possessing C57mtDNA regardless of nDNA background. High-fat diet in mice possessing C57mtDNA was associated with differential abundance of phosphatidylcholines, lysophosphatidylcholines, phosphatidylethanolamines, and glucose. Conversely, high-fat diet in mice possessing C3HmtDNA was associated with differential abundance of phosphatidylcholines, cardiolipins, and alanine. Glycerophospholipid metabolism and beta-alanine signaling pathways were enriched in skeletal muscle and plasma, indicating mtDNA-directed priming of mitochondria towards oxidative stress and increased fatty acid oxidation in C57nDNA:C57mtDNA wild-type and C3HnDNA:C57mtDNA MNX mice, relative to their nDNA-matched counterparts. In mtDNA-matched mice, C57mtDNA was associated with metabolite co-expression related to the pentose phosphate pathway and sugar-related metabolism; C3HmtDNA was associated with branched chain amino acid metabolite co-expression. ConclusionsThese results reveal novel nDNA-mtDNA interactions that drive significant changes in metabolite levels. Alterations to key metabolites involved in mitochondrial bioenergetic dysfunction and electron transport chain activity are implicated in elevated beta-oxidation during high-fat diet feeding; abnormally elevated rates of beta-oxidation may be a key driver of insulin resistance. The results reported here support the hypothesis that mtDNA influences cardiometabolic disease-susceptibility by modulating mitochondrial function and metabolic pathways.

molecular biology↗

bioRxiv2024

Alternative splicing across the C. elegans nervous system

Alternative splicing is a key mechanism that shapes neuronal transcriptomes, helping to define neuronal identity and modulate function. Here, we present an atlas of alternative splicing across the nervous system of Caenorhabditis elegans. Our analysis identifies novel alternative splicing in key neuronal genes such as unc-40/DCC and sax-3/ROBO. Globally, we delineate patterns of differential alternative splicing in almost 2,000 genes, and estimate that a quarter of neuronal genes undergo differential splicing. We introduce a web interface for examination of splicing patterns across neuron types. We explore the relationship between neuron type and splicing patterns, and between splicing patterns and differential gene expression. We identify RNA features that correlate with differential alternative splicing, and describe the enrichment of microexons. Finally, we compute a splicing regulatory network that can be used to generate hypotheses on the regulation and targets of alternative splicing in neurons.

neuroscience↗

bioRxiv2023

A molecular atlas of adult C. elegans motor neurons reveals ancient diversity delineated by conserved transcription factor codes

Motor neurons (MNs) constitute an ancient cell type targeted by multiple adult-onset diseases. It is therefore important to define the molecular makeup of adult MNs in animal models and extract organizing principles. Here, we generated a comprehensive molecular atlas of adult Caenorhabditis elegans MNs and a searchable database (http://celegans.spinalcordatlas.org). Single-cell RNA-sequencing of 13,200 cells revealed that ventral nerve cord MNs cluster into 29 molecularly distinct subclasses. All subclasses are delineated by unique expression codes of either neuropeptide or transcription factor gene families. Strikingly, we found that combinatorial codes of homeodomain transcription factor genes define adult MN diversity both in C. elegans and mice. Further, molecularly defined MN subclasses in C. elegans display distinct patterns of connectivity. Hence, our study couples the connectivity map of the C. elegans motor circuit with a molecular atlas of its constituent MNs, and uncovers organizing principles and conserved molecular codes of adult MN diversity.

developmental biology↗

bioRxiv2022

The Epithelial Na+ Channel UNC-8 promotes an endocytic mechanism that recycles presynaptic components from old to new boutons in remodeling neurons

SummaryPresynaptic terminals are actively relocated during development to refine circuit function, but the underlying cell biological mechanisms are largely unknown. In C. elegans, the presynaptic boutons of GABAergic DD neurons are moved to new locations during early larval development. We show that developmentally regulated expression of a presynaptic Epithelial Na+ Channel (ENaC), UNC-8, promotes a Ca2+-dependent mechanism, resembling Activity-Dependent Bulk Endocytosis (ADBE), that dismantles presynaptic material for reassembly at nascent DD synapses. ADBE normally functions in highly active neurons to accelerate local recycling of synaptic vesicles. We show that DD presynaptic remodeling depends on canonical features of ADBE including elevated intracellular Ca2+, the phosphatase Calcineurin and its targets, dynamin and the F-BAR protein syndapin, and Arp2/3-driven actin polymerization. Thus, our findings suggest that a native mechanism (ADBE) for maintaining neurotransmitter release at local synapses has been repurposed, in this case, to dismantle presynaptic terminals for reassembly at new locations. HighlightsO_LIDeveloping GABAergic neurons dismantle presynaptic terminals for reassembly at new locations. C_LIO_LIThe DEG/ENaC protein, UNC-8, promotes presynaptic disassembly and recycling C_LIO_LICa2+-dependent endocytosis drives presynaptic disassembly and recycling to new boutons C_LI

neuroscience↗

bioRxiv2022

Integrating bulk and single cell RNA-seq refines transcriptomic profiles of specific C. elegans neurons.

genetics↗

bioRxiv2021

cAMP controls a trafficking mechanism that directs the neuron specificity and subcellular placement of electrical synapses

Electrical synapses are established between specific neurons and within distinct subcellular compartments, but the mechanisms that direct gap junction assembly in the nervous system are largely unknown. Here we show that a transcriptional program tunes cAMP signaling to direct the neuron-specific assembly and placement of electrical synapses in the C. elegans motor circuit. For these studies, we use live cell imaging to visualize electrical synapses in vivo and a novel optogenetic assay to confirm that they are functional. In VA motor neurons, the UNC-4 transcription factor blocks expression of cAMP antagonists that promote gap junction miswiring. In unc-4 mutants, VA electrical synapses are established with an alternative synaptic partner and are repositioned from the VA axon to soma. We show that cAMP counters these effects by driving gap junction trafficking into the VA axon for electrical synapse assembly. Thus, our experiments in an intact nervous system establish that cAMP regulates gap junction trafficking for the biogenesis of electrical synapses.

neuroscience↗

bioRxiv2021

Selective stimulation of the ferret abdominal vagus nerve with multi-contact nerve cuff electrodes

Dysfunction and diseases of the gastrointestinal (GI) tract are a major driver of medical care. The vagus nerve innervates and controls multiple organs of the GI tract and vagus nerve stimulation (VNS) could provide a means for affecting GI function and treating disease. However, the vagus nerve also innervates many other organs throughout the body, and off-target effects of VNS could cause major side effects such as changes in blood pressure. In this study, we aimed to achieve selective stimulation of populations of vagal afferents using a multi-contact cuff electrode wrapped around the abdominal trunks of the vagus nerve. Four-contact nerve cuff electrodes were implanted around the dorsal (N=3) or ventral (N=3) abdominal vagus nerve in six ferrets, and the response to stimulation was measured via a 32-channel microelectrode array (MEA) inserted into the nodose ganglion. Selectivity was characterized by the ability to evoke responses in MEA channels through one bipolar pair of cuff contacts but not through the other bipolar pair. We demonstrated that is was possible to selectively activate subpopulations of vagal afferents using abdominal VNS. Additionally, we quantified the conduction velocity of evoked responses to determine what types of nerve fibers (i.e. A{delta} vs. C) responded to stimulation. We also quantified the spatial organization of evoked responses in the nodose MEA to determine if there is somatotopic organization of the neurons in that ganglion. Finally, we demonstrated in a separate set of three ferrets that stimulation of the abdominal vagus via a four-contact cuff could selectively alter gastric myoelectric activity, suggesting that abdominal VNS can potentially be used to control GI function.

bioengineering↗

bioRxiv2020

Molecular topography of an entire nervous system

Nervous systems are constructed from a deep repertoire of neuron types but the underlying gene expression programs that specify individual neuron identities are poorly understood. To address this deficit, we have produced an expression profile of all 302 neurons of the C. elegans nervous system that matches the single cell resolution of its anatomy and wiring diagram. Our results suggest that individual neuron classes can be solely identified by combinatorial expression of specific gene families. For example, each neuron class expresses unique codes of [~]23 neuropeptide-encoding genes and [~]36 neuropeptide receptors thus pointing to an expansive "wireless" signaling network. To demonstrate the utility of this uniquely comprehensive gene expression catalog, we used computational approaches to (1) identify cis-regulatory elements for neuron-specific gene expression across the nervous system and (2) reveal adhesion proteins with potential roles in synaptic specificity and process placement. These data are available at cengen.org and can be interrogated at the web application CengenApp. We expect that this neuron-specific directory of gene expression will spur investigations of underlying mechanisms that define anatomy, connectivity and function throughout the C. elegans nervous system.

neuroscience↗

bioRxiv2020

Responses of Neurons in the Medullary Lateral Tegmental Field and Nucleus Tractus Solitarius to Vestibular Stimuli in Conscious Felines

Considerable evidence shows that the vestibular system contributes to adjusting sympathetic nervous system activity to maintain adequate blood pressure during movement and changes in posture. However, only a few prior experiments entailed recordings in conscious animals from brainstem neurons presumed to convey baroreceptor and vestibular inputs to neurons in the rostral ventrolateral medulla (RVLM) that provide inputs to sympathetic preganglionic neurons in the spinal cord. In this study, recordings were made in conscious felines from neurons in the medullary lateral tegmental field (LTF) and nucleus tractus solitarius (NTS) identified as regulating sympathetic nervous system activity by exhibiting changes in firing rate related to the cardiac cycle, or cardiac-related activity (CRA). Approximately 38% of LTF and NTS neurons responded to static 40{degrees} head up tilts with a change in firing rate of ~50%. However, few of these neurons responded to 10{degrees} sinusoidal rotations in the pitch plane, in contrast to prior findings in decerebrate animals that the firing rates of both NTS and LTF neurons are modulated by small-amplitude body rotations. Thus, as previously demonstrated for RVLM neurons, in conscious animals NTS and LTF neurons only respond to large rotations that lead to changes in sympathetic nervous system activity. The similar responses to head-up rotations of LTF and NTS neurons with those documented for RVLM neurons suggest that LTF and NTS neurons are components of the vestibulo-sympathetic reflex pathway. However, a difference between NTS/LTF neurons and RVLM was variability in CRA over time. This variability was significantly greater for RVLM neurons, raising the hypothesis that the responsiveness of these neurons to baroreceptor input is adjusted based on the animals vigilance and alertness.

neuroscience↗

bioRxiv2020

Transcriptional control of parallel-acting pathways that remove discrete presynaptic proteins in remodeling neurons

Synapses are actively dismantled to mediate circuit refinement, but the developmental pathways that regulate synaptic disassembly are largely unknown. We have previously shown that the epithelial sodium channel UNC-8 triggers an activity-dependent mechanism that drives the removal of presynaptic proteins liprin-/SYD-2, Synaptobrevin/SNB-1, RAB-3 and Endophilin/UNC-57 in remodeling GABAergic neurons in C. elegans (Miller-Fleming et al., 2016). Here, we report that the transcription factor Iroquois/IRX-1 regulates UNC-8 expression as well as an additional pathway, independent of UNC-8, that functions in parallel to dismantle functional presynaptic terminals. We show that the additional IRX-1-regulated pathway is selectively required for the removal of the presynaptic proteins, Munc13/UNC-13 and ELKS, which normally mediate synaptic vesicle fusion and neurotransmitter release. Our findings are notable because they highlight the key role of transcriptional regulation in synapse elimination and reveal parallel-acting pathways that orchestrate synaptic disassembly by removing specific active zone proteins.

neuroscience↗

bioRxiv2019

Expression profiling of the mature C. elegans nervous system by single-cell RNA-Sequencing

A single neuron and its synapses define the fundamental structural motif of the brain but the underlying gene expression programs that specify individual neuron types are poorly understood. To address this question in a model organism, we have produced a gene expression profile of >90% of the individual neuron classes in the C. elegans nervous system, an ensemble of neurons for which both the anatomy and connectivity are uniquely defined at single cell resolution. We generated single cell transcriptomes for 52,412 neurons that resolve as clusters corresponding to 109 of the canonical 118 neuron classes in the mature hermaphrodite nervous system. Detailed analysis revealed molecular signatures that further subdivide identified classes into specific neuronal subtypes. Notably, neuropeptide-related genes are often differentially expressed between subtypes of the given neuron class which points to distinct functional characteristics. All of these data are publicly available at our website (http://www.cengen.org) and can be interrogated at the web application SCeNGEA (https://cengen.shinyapps.io/SCeNGEA). We expect that this gene expression catalog will spur the goal of delineating the underlying mechanisms that define the developmental lineage, detailed anatomy, synaptic connectivity and function of each type of C. elegans neuron.

neuroscience↗