Biology subjects

Miller, E. A.

Publications and source records attributed to Miller, E. A..

At least 19 recordsLinked to original sources

bioRxiv2026

Bilayer thickness and transmembrane domain dynamics drive directional ER-Golgi traffic via Erv14/cornichon

Directionality of protein traffic during secretion is driven by sorting receptors that bind cargo in one compartment and release them in another. Erv14/cornichon family receptors mediate delivery from the endoplasmic reticulum (ER) to the Golgi. Erv14 traffics its client, Mid2, via a conserved interface formed by two transmembrane helices (TMHs). Molecular dynamics simulations of Erv14-Mid2 suggest that Erv14 TMHs undergo dynamic movement in response to both client binding and lipid bilayer thickness. Interaction between charged residues that flank the TMHs of Mid2 and Erv14 couple client binding to stabilization of the Erv14 export signal and thus engagement with the COPII coat. We term this mechanism Helix Engagement Induced Signal-mediated Traffic (HEIST) and suggest it as a general mechanism where changes in membrane biophysical properties drive directional transport.

cell biology↗

bioRxiv2026

Sequential restriction of SNARE-mediated fusion by the COPII inner coat and SNARE chaperones

SNARE-mediated fusion requires assembly of four SNARE domains (R, Qa, Qb, and Qc) distributed across two membranes, with an SM (Sec1/Munc-18 family) chaperone catalyzing this assembly. We investigated topological requirements for the four SNAREs that mediate ER-Golgi fusion using in vitro assays. In the presence of the cognate SM Sly1, we find that a single topology drives efficient fusion: the R SNARE on one membrane versus Qa/Qb/Qc SNAREs on the other. These results prompted us to look upstream, at COPII-SNARE interactions. The assembled COPII coat is known to block fusion. We discovered that of five COPII core subunits, the Sar1 GTPase and Sec23/Sec24 subunits were necessary and sufficient to prevent fusion for prolonged periods, and that Sar1 was dispensable for inhibition over short periods. When Sec24- interactions with vesicle SNAREs were disrupted, inhibition was relieved, indicating that SNARE sequestration by COPII prevents fusion. These observations help to explain how appropriate fusion events are facilitated while inappropriate events are deterred.

cell biology↗

bioRxiv2025

The microcephaly-associated protein YIPF5 differentially regulates ER-export

YIPF5 is a small ER-membrane protein implicated in ER-Golgi transport. Mutations in YIPF5 cause MEDS2 (microcephaly with simplified gyral pattern, epilepsy, and neonatal diabetes syndrome), a fatal disorder manifesting in early childhood. We demonstrate that YIPF5 is involved in ER export of a subset of proteins, including cargoes of the ER-export receptor SURF4, with which it directly interacts. In YIPF5 knockout cells, we observe a shift in the cell surface and secretome composition, marked by reduced neuronal adhesion molecules and increased secretion of ER chaperones influencing cell migration. YIPF5 depletion enhances cell migration in a wound-healing assay and alters SURF4 localization, causing elongated ERGIC53- and Rab1-positive tubules from COPII-labeled ER exit sites. Kinetic analysis suggests that YIPF5 negatively regulates SURF4-mediated ER export. In utero knockdown of Yipf5 in embryonic mouse brains induces premature neuronal migration and abnormal neuronal morphology. These findings suggest that YIPF5 and SURF4 coordinate ER export, and disruption may underlie cortical development defects leading to microcephaly.

cell biology↗

bioRxiv2024

Discovery of a novel bandavirus using metagenomic sequencing in a retrospective analysis of an unresolved 2020 mortality event involving black vultures in the northeastern United States.

Investigations of wildlife diseases and mortality events can sometimes lead to inconclusive results due to limitations in diagnostics combined with an ever-increasing number of emerging viruses. The use of tools such as unbiased metagenomic next generation sequencing (mNGS) can facilitate the identification of causative agents where conventional investigation methods fail. We performed a retrospective mNGS analysis on RNA isolated from postmortem samples collected during a black vulture (Coragyps atratus, family: Cathartidae) mortality event that occurred in eastern Pennsylvania and western New Jersey in 2020. We describe the discovery and identification of a novel species of bandavirus (Phenuiviridae family) in case specimens from this die-off, as well as some of the associated pathological findings. The Bandavirus genus comprises tickborne viral species that have been reported across five continents and implicated in outbreaks in a variety of mammalian hosts, including humans, and in avian species making them important potential sources of zoonotic spillover events. Genomic and phylogenetic analysis of the bandavirus detected in this study indicate its closest relative corresponds to Hunter Island virus, a bandavirus previously implicated in albatross mortality events off the coast of Tasmania, Australia. Follow-up PCR testing of samples from additional vultures from the same cohort confirmed that this new bandavirus is the likely cause of death.

genomics↗

bioRxiv2024

Identification of Cellular Signatures Associated with Chinese Hamster Ovary (CHO) Cell Adaptation for Secretion of Antibodies

The secretory capacity of Chinese hamster ovary (CHO) cells remains a fundamental bottleneck in the manufacturing of protein-based therapeutics. Unconventional biological drugs with complex structures and processing requirements are particularly problematic. Although engineered vector DNA elements can achieve rapid and high-level therapeutic protein production, a high metabolic and protein folding burden is imposed on the host cell. Cellular adaptations to these conditions include differential gene expression profiles that can in turn influence the productivity and quality control of recombinant proteins. In this study, we used quantitative transcriptomics and proteomics analyses to investigate how biological pathways change with antibody titre. Gene and protein expression profiles of CHO pools and clones producing a panel of different monoclonal and bispecific antibodies were analysed during fed-batch production. Antibody-expressing CHO pools were heterogeneous, resulting in few discernible genetic signatures. Clonal lines derived from these pools, selected for high and low production, yielded a small number of differentially expressed proteins that correlated with productivity and were shared across biotherapeutics. However, the dominant feature associated with higher protein production was transgene copy number and resulting mRNA expression level. Moreover, variability between clones suggested that the process of cellular adaptation is variable with diverse cellular changes associated with individual adaptation events.

bioengineering↗

bioRxiv2024

NLRP3 and AIM2 inflammasomes exacerbate the pathogenic Th17 cell response to eggs of the helminth Schistosoma mansoni

Infection with the helminth Schistosoma mansoni can cause exacerbated morbidity and mortality via a pathogenic host CD4 T cell-mediated immune response directed against parasite egg antigens, with T helper (Th) 17 cells playing a major role in the development of severe granulomatous hepatic immunopathology. The role of inflammasomes in intensifying disease has been reported; however, neither the types of caspases and inflammasomes involved, nor their impact on the Th17 response are known. Here we show that enhanced egg-induced IL-1{beta} secretion and pyroptotic cell death required both caspase-1 and caspase-8 as well as NLRP3 and AIM2 inflammasome activation. Schistosome genomic DNA activated AIM2, whereas reactive oxygen species, potassium efflux and cathepsin B, were the major activators of NLRP3. NLRP3 and AIM2 deficiency led to a significant reduction in pathogenic Th17 responses, suggesting their crucial and non-redundant role in promoting inflammation. Additionally, we show that NLRP3- and AIM2-induced IL-1{beta} suppressed IL-4 and protective Type I IFN (IFN-I) production, which further enhanced inflammation. IFN-I signaling also curbed inflammasome-mediated IL-1{beta} production suggesting that these two antagonistic pathways shape the severity of disease. Lastly, Gasdermin D (Gsdmd) deficiency resulted in a marked decrease in egg-induced granulomatous inflammation. Our findings establish NLRP3/AIM2-Gsdmd axis as a central inducer of pathogenic Th17 responses which is counteracted by IFN-I pathway in schistosomiasis. SummarySchistosomiasis is a major tropical parasitic disease caused by trematode worms of the genus Schistosoma. Morbidity and mortality in infection with the species Schistosoma mansoni are due to a pathogenic CD4 T cell-mediated immune response directed against parasite eggs, resulting in granulomatous inflammation. In severe cases of schistosomiasis, there is liver fibrosis, hepatosplenomegaly, portal hypertension, gastro-intestinal hemorrhage and death. Here we describe the role of two proteins, the NLRP3 and AIM2 inflammasomes, in intensifying disease. We found that upstream proteins which activate these inflammasomes are caspase-1 and caspase 8; these in turn lead to the activation of another protein, Gasdermin D (Gsdmd), which facilitates the release of the proinflammatory cytokine IL-1{beta}. Importantly, we observed that mice deficient in Gsdmd exhibit diminished pathology. Finally, we discovered that the protective Type I Interferon (IFN-I) pathway counteracts the caspase/inflammasome/Gsdmd axis thereby controlling egg mediated inflammation. These results give us a deeper understanding of the functional features of the crosstalk between inflammasome and IFN-I pathway, which may lead to the identification of novel targets for therapeutic intervention.

immunology↗

bioRxiv2023

Metagenomics for pathogen detection during a wildlife mortality event in songbirds

Mass mortality events in wildlife can be indications of an emerging infectious disease. During the spring and summer of 2021, hundreds of dead passerines were reported across the eastern US. Birds exhibited a range of clinical signs including swollen conjunctiva, ocular discharge, ataxia, and nystagmus. As part of the diagnostic investigation, high-throughput metagenomic next-generation sequencing was performed across three molecular laboratories on samples from affected birds. Many potentially pathogenic microbes were detected, with bacteria comprising the largest proportion; however, no singular agent was consistently identified, with many of the detected microbes also found in unaffected (control) birds, and thus considered to be subclinical infections. Congruent results across laboratories have helped drive further investigation into alternative causes including environmental contaminants and nutritional deficiencies. This work highlights the utility of metagenomic approaches in investigations of emerging diseases and provides a framework for future wildlife mortality events. Article Summary LineThe causative agent of a mass mortality event in passerines remains inconclusive after metagenomic high-throughput sequencing with results prompting further investigation into non-pathogenic causes.

genomics↗

bioRxiv2022

Tumor-specific CD4 T cells instruct monocyte differentiation in pancreatic ductal adenocarcinoma

Pancreatic ductal adenocarcinoma (PDA) is a lethal malignancy resistant to immunotherapy. The pancreatic tumor microenvironment is shaped and maintained by myeloid cells that outnumber tumor cells. Here, using monocyte fate-mapping PDA mouse models and human tumor tissues, we identify monocytes give rise to most heterogeneous macrophage subpopulations in PDA. We show that monocyte differentiation is governed by the local presence of CD4, but not CD8, T cells. We demonstrate that tumor specific CD4 T cells induce monocyte differentiation into antitumor MHCIIhi proinflammatory macrophages dependent on non-redundant IFN{gamma} and CD40 signaling pathways that suppress tumor growth. Pancreatic tissue-resident macrophages exhibit an immunosuppressive pro-tumor state that is refractory to the modulatory effects of antitumor CD4 T cells. Intratumoral monocytes adopt a pro-tumor fate indistinguishable from tissue-resident macrophages following CD4 T cell depletion. Thus, tumor-specific CD4 T cell governance of monocyte fate promotes immune-mediated control of solid tumors. Highlights{blacksquare} Circulating monocytes are progenitors to most heterogeneous macrophage subsets in PDA {blacksquare}Monocyte-derived macrophage acquisition of an MHCIIhi phenotype is dependent on tumor-specific CD4 T cells {blacksquare}In the absence of CD4 T cells, monocyte-derived macrophages acquire tissue resident macrophage traits and tumors rapidly progress {blacksquare}IFN{gamma} and CD40 signaling are nonredundant and critical determinants of intratumoral monocyte fate

immunology↗

bioRxiv2022

Habitat use differences mediate anthropogenic threat exposure in white sturgeon

Understanding intraspecific variation in habitat use, particularly of long-lived fishes across multiple life history stages, is core to improved conservation management. Here, we present results from a synthesis of acoustic telemetry data for large juvenile and adult white sturgeon (Acipenser transmontanus) from 2010 to 2017 in the San Francisco Estuary and Sacramento River ecosystems. We focused primarily on uncovering spatial patterns of inferred habitat occupancy across life stages, and on linking habitat use to population threats. We found substantial differences in habitat use across individuals and over time that was related to fish age class. However, differences in habitat use were not explained by fish sex or water year flow conditions. We estimated an index of angling exposure, which showed that fish entering reproductive maturity, which historically were of harvestable size, were detected less often than other sizes in areas with high angler pressure, suggesting possible behavioral avoidance of areas of high angler pressure. Additionally, we used historical data to evaluate potential exposure of white sturgeon to a severe red tide event in late summer 2022. We found that >50% of reproductive-age fish may have been residing in areas affected by the red tide. Future monitoring and management of white sturgeon might benefit from examining multiple phases of white sturgeon life history. For example, additional tracking studies could improve understanding of juvenile habitat use, adult survival rates, patterns of anadromy, and cross-basin habitat utilization.

ecology↗

bioRxiv2022

A molecular mechanism for membrane chaperoning by a late embryogenesis abundant protein

Environmental stress can result in substantial damage to proteins, membranes, and genetic material, impacting organismal survival1-3. Stress tolerance can be conferred by intrinsically disordered proteins (IDPs)4 that lack stable tertiary structure. IDPs from the large family of late embryogenesis abundant (LEA) proteins confer a fitness advantage when heterologously expressed5,6. Such protection suggests a general molecular function leading to stress tolerance, although the mechanisms remain unclear. Here, we report that a tardigrade LEA protein that confers stress tolerance in yeast acts as a molecular chaperone for the mitochondrial membrane. This protein, named HeLEA1, localizes to the mitochondrial matrix, and harbors conserved LEA sequence motifs that undergo dynamic disorder-to-helical transition upon binding to negatively charged membranes. Yeast expressing HeLEA1 show increased mitochondrial membrane fluidity, increased membrane potential, and enhanced tolerance to hyperosmotic stress under non-fermentative growth without significantly altering mitochondrial lipid composition or triggering a generic stress response. We demonstrate that membrane binding ameliorates excess surface tension, possibly by stabilizing lipid packing defects. Evolutionary analysis suggests that HeLEA1 homologs localize to different membrane-bound organelles and share similar sequence and biophysical features. We suggest that membrane chaperoning by LEA proteins represents a general biophysical solution that can operate across the domains of life.

biophysics↗

bioRxiv2022

Supramolecular architecture of the ER-mitochondria encounter structure in its native environment

The endoplasmic reticulum and mitochondria are main hubs of eukaryotic membrane biogenesis which rely on lipid exchange via membrane contact sites, but the underpinning mechanisms remain poorly understood. In yeast, tethering and lipid transfer between the two organelles is mediated by the ER-mitochondria encounter structure ERMES, a four-subunit complex of unclear stoichiometry and architecture. We determined the molecular organization of ERMES within cells using integrative structural biology, combining quantitative live-imaging, cryo-correlative microscopy, subtomogram averaging and molecular modeling. ERMES assembles into approximately 25 discrete bridge-like complexes distributed irregularly across a contact site. Each bridge consists of three lipid-binding SMP domains arranged in zig-zag fashion. Our molecular model of ERMES reveals an unconventional restrained pathway for lipids. These findings resolve a supramolecular architecture controlling interorganelle lipid fluxes.

cell biology↗

bioRxiv2022

Ultrastructure of COPII vesicle formation characterised by correlative light and electron microscopy.

Traffic of proteins out of the endoplasmic reticulum (ER) is driven by the COPII coat, a layered protein scaffold that mediates the capture of cargo proteins and the remodelling of the ER membrane into spherical vesicular carriers. Although the components of this machinery have been genetically defined, and the mechanisms of coat assembly extensively explored in vitro, understanding the physical mechanisms of membrane remodelling in cells remains a challenge. Here we use correlative light and electron microscopy (CLEM) to visualize the nanoscale ultrastructure of membrane remodelling at ER exit sites (ERES) in yeast cells. Using various COPII mutants, we have determined the broad contribution that each layer of the coat makes in membrane remodelling. Our data suggest that inner coat components define the radius of curvature whereas outer coat components facilitate membrane fission. The organization of the coat in conjunction with membrane biophysical properties determine the ultrastructure of vesicles and thus the efficiency of protein transport.

cell biology↗

bioRxiv2022

Selective inhibition of protein secretion by abrogating receptor-coat interactions during ER export.

Protein secretion is an essential cellular process that permits cell growth, movement and communication. Traffic of proteins within the eukaryotic secretory pathway is mediated by transport intermediates that bud from one compartment, populated with appropriate cargo proteins, and fuse with a downstream compartment to deliver their contents. Here, we explore the possibility that protein secretion can be selectively inhibited by perturbing protein-protein interactions that drive capture into transport vesicles. Human PCSK9 is a major determinant of cholesterol metabolism, whose secretion is mediated by a specific cargo adaptor of the ER export machinery, SEC24A. We map a series of protein-protein interactions between PCSK9, its ER export receptor, SURF4, and SEC24A, that mediate secretion of PCSK9. We show that the interaction between SURF4 and SEC24A can be inhibited by 4-PBA, a small molecule that occludes a cargo-binding domain of SEC24. This inhibition reduces secretion of PCSK9 and additional SURF4 clients that we identify by mass spectrometry, leaving other secreted cargoes unaffected. We propose that selective small molecule inhibition of cargo recognition by SEC24 is a potential therapeutic intervention for atherosclerosis and other diseases that are modulated by secreted proteins.

cell biology↗

bioRxiv2021

Structures of core eukaryotic protein complexes

Protein-protein interactions play critical roles in biology, but despite decades of effort, the structures of many eukaryotic protein complexes are unknown, and there are likely many interactions that have not yet been identified. Here, we take advantage of recent advances in proteome-wide amino acid coevolution analysis and deep-learning-based structure modeling to systematically identify and build accurate models of core eukaryotic protein complexes, as represented within the Saccharomyces cerevisiae proteome. We use a combination of RoseTTAFold and AlphaFold to screen through paired multiple sequence alignments for 8.3 million pairs of S. cerevisiae proteins and build models for strongly predicted protein assemblies with two to five components. Comparison to existing interaction and structural data suggests that these predictions are likely to be quite accurate. We provide structure models spanning almost all key processes in Eukaryotic cells for 104 protein assemblies which have not been previously identified, and 608 which have not been structurally characterized. One-sentence summaryWe take advantage of recent advances in proteome-wide amino acid coevolution analysis and deep-learning-based structure modeling to systematically identify and build accurate models of core eukaryotic protein complexes.

biochemistry↗

bioRxiv2020

Structure and mechanism of TRAPPIII-mediated Rab1 activation

The GTPase Rab1 is a master regulator of both the early secretory pathway and autophagy. Rab1 activation is controlled by its GEF (guanine nucleotide exchange factor), the multi-subunit TRAPPIII complex. The Trs85 regulatory subunit is critical for robust activation of Rab1 but its mechanistic role within the complex has remained unclear. Here we report the cryo-EM structure of the intact yeast TRAPPIII complex bound to its substrate Rab1/Ypt1. The orientation of the Rab1/Ypt1 hypervariable domain when bound to the complex leads to a model for how TRAPPIII associates with and activates Rab1/Ypt1 at the membrane surface. We identify a conserved amphipathic -helix motif within Trs85 and demonstrate that this helix is required for stable membrane binding and Rab1/Ypt1 activation by TRAPPIII. Taken together, our results provide a comprehensive analysis of the structure and function of the yeast TRAPPIII complex and reveal that the key function of Trs85 is to serve as a membrane anchor, via its amphipathic helix, for the entire TRAPPIII complex.

cell biology↗

bioRxiv2020

Structure of the complete, membrane-assembled COPII coat reveals a complex interaction network.

The COPII coat mediates Endoplasmic Reticulum (ER) to Golgi trafficking for thousands of proteins. Five essential coat proteins assemble at the ER into a characteristic two-layer architecture, which recruits cargo proteins whilst sculpting membrane carriers with diverse morphologies. How coat architecture drives membrane curvature whilst ensuring morphological plasticity is largely unknown, yet is central to understanding mechanisms of carrier formation. Here, we use an established reconstitution system to visualise the complete, membrane-assembled COPII coat with unprecedented detail by cryo-electron tomography and subtomogram averaging. We discover a network of interactions within and between coat layers, including multiple interfaces that were previously unknown. We reveal the physiological importance of these interactions using genetic and biochemical approaches. A newly resolved Sec31 C-terminal domain provides order to the coat and is essential to drive membrane curvature in cells. Moreover, a novel outer coat assembly mode provides a basis for coat adaptability to varying membrane curvatures. Furthermore, a newly resolved region of Sec23, which we term the L-loop, imparts coat stability and in part dictates membrane shape. Our results suggest these interactions collectively contribute to coat organisation and membrane curvature, providing a structural framework to understand regulatory mechanisms of COPII trafficking and secretion.

cell biology↗

bioRxiv2020

The architecture of EMC reveals a path for membrane protein insertion

Approximately 25% of eukaryotic genes code for integral membrane proteins that are assembled at the endoplasmic reticulum. An abundant and widely conserved multi-protein complex termed EMC has been implicated in membrane protein biogenesis, but its mechanism of action is poorly understood. Here, we define the composition and architecture of human EMC using biochemical assays, crystallography of individual subunits, site-specific photocrosslinking, and cryo-EM reconstruction. Our results show that EMCs cytosolic domain contains a large, moderately hydrophobic vestibule that binds a substrates transmembrane domain (TMD). The cytosolic vestibule leads into a lumenally-sealed, lipid-exposed intramembrane groove large enough to accommodate a single substrate TMD. A gap between the cytosolic vestibule and intramembrane groove provides a path for substrate egress from EMC. These findings suggest how EMC facilitates energy-independent membrane insertion of TMDs, explain why only short lumenal domains are translocated by EMC, and constrain models of EMCs proposed chaperone function.

biochemistry↗

bioRxiv2020

A multivalent fuzzy interface drives reversible COPII coat assembly.

Protein secretion is initiated at the endoplasmic reticulum by the COPII coat, which self-assembles to form vesicles. Here, we examine the mechanisms by which the outer scaffolding layer of the coat drives local assembly of a structure rigid enough to enforce membrane curvature, yet able to readily disassemble at the Golgi. An intrinsically disordered region in the outer coat protein, Sec31, drives binding with an inner coat layer via multiple distinct interfaces. Interactions are individually dispensable but combinatorially reinforce each other, suggesting coat oligomerization is driven by the cumulative effects of multivalent interactions. Such a multimodal assembly platform could be readily reversed at the Golgi via perturbation of each individual interface. These design principles provide an explanation for how cells build a powerful yet transient scaffold to direct vesicle traffic.

cell biology↗