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All sourcesNASA NTRSDOE OSTI
DOE OSTI2022

A Subunit of ESCRT-III, MoIst1, Is Involved in Fungal Development, Pathogenicity, and Autophagy in Magnaporthe oryzae

The culprit of rice blast, Magnaporthe oryzae , is a filamentous fungus that seriously affects the yield and quality of rice worldwide. MoIst1, a subunit of ESCRT-III, is involved in identified ubiquitinated proteins and transports them into the intraluminal vesicles of multivesicular bodies (MVBs) for degradation in lysosomes. Here, we identify and characterize MoIst1 in M. oryzae . Disruption of MoIst1 leads to a significant decrease in sporulation and formation of appressoria, defects in response to oxidative stress, cell wall stress, hyperosmotic stress, and reduced pathogenicity. Deletion of MoIst1 also caused the decreased Pmk1 phosphorylation levels, appressorium formation, the delayed translocation and degradation of lipid droplets and glycogen, resulting in a decreased appressorium turgor. In addition, deletion of MoIst1 leads to an abnormal autophagy. In summary, our results indicate that MoIst1 is involved in sporulation, appressorium development, plant penetration, pathogenicity, and autophagy in M. oryzae .

Sun, Lixiao↗
DOE OSTI2023

Two related families of metal transferases, ZNG1 and ZNG2, are involved in acclimation to poor Zn nutrition in Arabidopsis

Metal homeostasis has evolved to tightly modulate the availability of metals within the cell, avoiding cytotoxic interactions due to excess and protein inactivity due to deficiency. Even in the presence of homeostatic processes, however, low bioavailability of these essential metal nutrients in soils can negatively impact crop health and yield. While research has largely focused on how plants assimilate metals, acclimation to metal-limited environments requires a suite of strategies that are not necessarily involved in metal transport across membranes. The identification of these mechanisms provides a new opportunity to improve metal-use efficiency and develop plant foodstuffs with increased concentrations of bioavailable metal nutrients. Here, we investigate the function of two distinct subfamilies of the nucleotide-dependent metallochaperones (NMCs), named ZNG1 and ZNG2, that are found in plants, using Arabidopsis thaliana as a reference organism. AtZNG1 (AT1G26520) is an ortholog of human and fungal ZNG1, and like its previously characterized eukaryotic relatives, localizes to the cytosol and physically interacts with methionine aminopeptidase type I (AtMAP1A). Analysis of At ZNG1 , At MAP1A , At MAP2A , and At MAP2B transgenic mutants are consistent with the role of Arabidopsis ZNG1 as a Zn transferase for AtMAP1A, as previously described in yeast and zebrafish. Structural modeling reveals a flexible cysteine-rich loop that we hypothesize enables direct transfer of Zn from AtZNG1 to AtMAP1A during GTP hydrolysis. Based on proteomics and transcriptomics, loss of this ancient and conserved mechanism has pleiotropic consequences impacting the expression of hundreds of genes, including those involved in photosynthesis and vesicle transport. Members of the plant-specific family of NMCs, ZNG2A1 (AT1G80480) and ZNG2A2 (AT1G15730), are also required during Zn deficiency, but their target protein(s) remain to be discovered. In conclusion, RNA-seq analyses reveal wide-ranging impacts across the cell when the genes encoding these plastid-localized NMCs are disrupted.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2020

Duration of Contagion of Foot-And-Mouth Disease Virus in Infected Live Pigs and Carcasses

Data-driven modeling of incursions of high-consequence, transboundary pathogens of animals is a critical component of veterinary preparedness. However, simplifying assumptions and excessive use of proxy measures to compensate for gaps in available data may compromise modeled outcomes. The current investigation was prospectively designed to address two major gaps in current knowledge of foot-and-mouth disease virus (FMDV) pathogenesis in pigs: the end (duration) of the infectious period and the viability of FMDV in decaying carcasses. By serial exposure of sentinel groups of pigs to the same group of donor pigs infected by FMDV A24 Cruzeiro, it was demonstrated that infected pigs transmitted disease at 10 days post infection (dpi), but not at 15 dpi. Assuming a latent period of 1 day, this would result in a conservative estimate of an infectious duration of 9 days, which is considerably longer than suggested by a previous report from an experiment performed in cattle. Airborne contagion was diminished within two days of removal of infected pigs from isolation rooms. FMDV in muscle was inactivated within 7 days in carcasses stored at 4°C. By contrast, FMDV infectivity in vesicle epithelium harvested from intact carcasses stored under similar conditions remained remarkably high until the study termination at 11 weeks post mortem. The output from this study consists of experimentally determined data on contagion associated with FMDV-infected pigs. This information may be utilized to update parameterization of models used for foot-and-mouth disease outbreak simulations involving areas of substantial pig production.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2022

Characterization of Senecavirus A Isolates Collected From the Environment of U.S. Sow Slaughter Plants

Vesicular disease caused by Senecavirus A (SVA) is clinically indistinguishable from foot-and-mouth disease (FMD) and other vesicular diseases of swine. When a vesicle is observed in FMD-free countries, a costly and time-consuming foreign animal disease investigation (FADI) is performed to rule out FMD. Recently, there has been an increase in the number of FADIs and SVA positive samples at slaughter plants in the U.S. The objectives of this investigation were to: (1) describe the environmental burden of SVA in sow slaughter plants; (2) determine whether there was a correlation between PCR diagnostics, virus isolation (VI), and swine bioassay results; and (3) phylogenetically characterize the genetic diversity of contemporary SVA isolates. Environmental swabs were collected from three sow slaughter plants (Plants 1-3) and one market-weight slaughter plant (Plant 4) between June to December 2020. Of the 426 samples taken from Plants 1-3, 304 samples were PCR positive and 107 were VI positive. There was no detection of SVA by PCR or VI at Plant 4. SVA positive samples were most frequently found in the summer (78.3% June-September, vs. 59.4% October-December), with a peak at 85% in August. Eighteen PCR positive environmental samples with a range of C t values were selected for a swine bioassay: a single sample infected piglets ( n = 2). A random subset of the PCR positive samples was sequenced; and phylogenetic analysis demonstrated co-circulation and divergence of two genetically distinct groups of SVA. These data demonstrate that SVA was frequently found in the environment of sow slaughter plants, but environmental persistence and diagnostic detection was not indicative of whether a sampled was infectious to swine. Consequently, a more detailed understanding of the epidemiology of SVA and its environmental persistence in the marketing chain is necessary to reduce the number of FADIs and aide in the development of control measures to reduce the spread of SVA.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2020

Proteomic Analysis of Exosomes for Discovery of Protein Biomarkers for Prostate and Bladder Cancer

Extracellular vesicles (EVs) are released by nearly all cell types as part of normal cell physiology, transporting biological cargo, including nucleic acids and proteins, across the cell membrane. In pathological states such as cancer, EV-derived cargo may mirror the altered state of the cell of origin. Exosomes are the smaller, 50–150 nanometer-sized EVs released from fusion of multivesicular endosomes with the plasma membrane. Exosomes play important roles in cell-cell communication and participate in multiple cancer processes, including invasion and metastasis. Therefore, proteomic analysis of exosomes is a promising approach to discover potential cancer biomarkers, even though it is still at an early stage. Herein, we critically review the advances in exosome isolation methods and their compatibility with mass spectrometry (MS)-based proteomic analysis, as well as studies of exosomes in pathogenesis and progression of prostate and bladder cancer, two common urologic cancers whose incidence rates continue to rise annually. As urological tumors, both urine and blood samples are feasible for noninvasive or minimally invasive analysis. A better understanding of the biological cargo and functions of exosomes via high-throughput proteomics will help provide new insights into complex alterations in cancer and provide potential therapeutic targets and personalized treatment for patients.

60 APPLIED LIFE SCIENCES↗
DOE OSTI2022

The Magnetosome Protein, Mms6 from Magnetospirillum magneticum Strain AMB-1, Is a Lipid-Activated Ferric Reductase

Magnetosomes of magnetotactic bacteria consist of magnetic nanocrystals with defined morphologies enclosed in vesicles originated from cytoplasmic membrane invaginations. Although many proteins are involved in creating magnetosomes, a single magnetosome protein, Mms6 from Magnetospirillum magneticum strain AMB-1, can direct the crystallization of magnetite nanoparticles in vitro. The in vivo role of Mms6 in magnetosome formation is debated, and the observation that Mms6 binds Fe3+ more tightly than Fe2+ raises the question of how, in a magnetosome environment dominated by Fe3+, Mms6 promotes the crystallization of magnetite, which contains both Fe3+ and Fe2+. Here we show that Mms6 is a ferric reductase that reduces Fe3+ to Fe2+ using NADH and FAD as electron donor and cofactor, respectively. Reductase activity is elevated when Mms6 is integrated into either liposomes or bicelles. Analysis of Mms6 mutants suggests that the C-terminal domain binds iron and the N-terminal domain contains the catalytic site. Although Mms6 forms multimers that involve C-terminal and N-terminal domain interactions, a fusion protein with ubiquitin remains a monomer and displays reductase activity, which suggests that the catalytic site is fully in the monomer. However, the quaternary structure of Mms6 appears to alter the iron binding characteristics of the C-terminal domain. These results are consistent with a hypothesis that Mms6, a membrane protein, promotes the formation of magnetite in vivo by a mechanism that involves reducing iron.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2026

Protocol for Engineered Compositional Asymmetry Within Nanodiscs

Membrane proteins remain the most challenging targets for structural characterization, yet their elucidation provides valuable insights into protein function, disease mechanisms, and drug specificity. Structural biology platforms have advanced rapidly in recent years, notably through the development and implementation of nanodiscs—discoidal lipid–protein complexes that encapsulate and solubilize membrane proteins within a controlled, native-like environment. While nanodiscs have become powerful tools for studying membrane proteins, faithfully reconstituting the compositional asymmetry intrinsic to nearly all biological membranes has not yet been achieved. Proper membrane leaflet lipid distribution is critical for accurate protein folding, stability, and insertion. Here, we share a protocol for reconstituting tailored compositional asymmetry within nanodiscs through membrane extraction from giant unilamellar vesicles (GUVs) treated with a leaflet-specific methyl-β-cyclodextrin (mβCD) lipid exchange. Nanodisc asymmetry is verified through a geometric approach: biotin-DPPE-preloaded mβCD engages in lipid exchange with the outer leaflet of POPC GUVs solubilized by the lipid-free membrane scaffold protein (MSP) Δ49ApoA-I to form nanodisc structures. Once isolated, nanodiscs are introduced to the biotin-binding bacterial protein streptavidin. High-speed atomic force microscopy imaging depicts nanodisc–dimer complexes, indicating that biotin-DPPE was successfully reconstituted into a single leaflet of the nanodiscs. This finding outlines the first step toward engineering tailored nanodisc asymmetry and mimicking the native environment of integral proteins—a potentially powerful tool for accurately reconstituting and structurally analyzing integral membrane proteins whose functions are modulated by lipid asymmetry.

Biological and medical sciences↗
DOE OSTI2024

The Exometabolome of Xylella fastidiosa in Contact with Paraburkholderia phytofirmans Supernatant Reveals Changes in Nicotinamide, Amino Acids, Biotin, and Plant Hormones

Microbial competition within plant tissues affects invading pathogens’ fitness. Metabolomics is a great tool for studying their biochemical interactions by identifying accumulated metabolites. Xylella fastidiosa, a Gram-negative bacterium causing Pierce’s disease (PD) in grapevines, secretes various virulence factors including cell wall-degrading enzymes, adhesion proteins, and quorum-sensing molecules. These factors, along with outer membrane vesicles, contribute to its pathogenicity. Previous studies demonstrated that co-inoculating X. fastidiosa with the Paraburkholderia phytofirmans strain PsJN suppressed PD symptoms. Here, we further investigated the interaction between the phytopathogen and the endophyte by analyzing the exometabolome of wild-type X. fastidiosa and a diffusible signaling factor (DSF) mutant lacking quorum sensing, cultivated with 20% P. phytofirmans spent media. Liquid chromatography–mass spectrometry (LC-MS) and the Method for Metabolite Annotation and Gene Integration (MAGI) were used to detect and map metabolites to genomes, revealing a total of 121 metabolites, of which 25 were further investigated. These metabolites potentially relate to host adaptation, virulence, and pathogenicity. Notably, this study presents the first comprehensive profile of X. fastidiosa in the presence of a P. phytofirmans spent media. The results highlight that P. phytofirmans and the absence of functional quorum sensing affect the ratios of glutamine to glutamate (Gln:Glu) in X. fastidiosa. Additionally, two compounds with plant metabolism and growth properties, 2-aminoisobutyric acid and gibberellic acid, were downregulated when X. fastidiosa interacted with P. phytofirmans. These findings suggest that P. phytofirmans-mediated disease suppression involves modulation of the exometabolome of X. fastidiosa, impacting plant immunity.

60 APPLIED LIFE SCIENCES↗
DOE OSTI2023

Genetic Characterization of the Acidic and Neutral Glycosphingolipid Biosynthetic Pathways in Neurospora crassa

Fungal glycosphingolipids (GSLs) are important membrane components which play a key role in vesicle trafficking. To assess the importance of GSLs in the fungal life cycle, we performed a mutant phenotypic study of the acidic and neutral GSL biosynthetic pathways in Neurospora crassa. GSL biosynthesis begins with two reactions leading up to the formation of dihydrosphingosine. The first of these reactions is catalyzed by serine palmitoyltransferase and generates 3-keto dihydrosphinganine. In N. crassa, this reaction is catalyzed by GSL-1 and GSL-2 and is required for viability. The second reaction is carried out by GSL-3, a 3-keto dihydrosphinoganine reductase to generate dihydrosphingosine, which is used for the synthesis of neutral and acidic GSLs. We found that deletion mutations in the acidic GSL pathway leading up to the formation of mannosylinositol-phosphoceramide are lethal, indicating that acidic GSLs are essential for viability in N. crassa. Once mannosylinositol-phosphoceramide is made, it is further modified by GSL-5, an inositol-phosphoceramide-B C26 hydroxylase, which adds a hydroxyl group to the amide-linked fatty acid. GSL-5 is not required for viability but gives a clear mutant phenotype affecting all stages of the life cycle. Our results show that the synthesis of mannosylinositol-phosphoceramide is required for viability and that the modification of the amide-linked fatty acid is important for acidic GSL functionality. We also examined the neutral GSL biosynthetic pathway and identified the presence of glucosylceramide. The deletion of neutral GSL biosynthetic genes affected hyphal morphology, vegetative growth rate, conidiation, and female development. Our results indicate that the synthesis of neutral GSLs is essential for normal growth and development of N. crassa.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2024

Simultaneous Two- and Three-Photon Deep Imaging of Autofluorescence in Bacterial Communities

The intrinsic fluorescence of bacterial samples has a proven potential for label-free bacterial characterization, monitoring bacterial metabolic functions, and as a mechanism for tracking the transport of relevant components through vesicles. The reduced scattering and axial confinement of the excitation offered by multiphoton imaging can be used to overcome some of the limitations of single-photon excitation (e.g., scattering and out-of-plane photobleaching) to the imaging of bacterial communities. In this work, we demonstrate in vivo multi-photon microscopy imaging of Streptomyces bacterial communities, based on the excitation of blue endogenous fluorophores, using an ultrafast Yb-fiber laser amplifier. Its parameters, such as the pulse energy, duration, wavelength, and repetition rate, enable in vivo multicolor imaging with a single source through the simultaneous two- and three-photon excitation of different fluorophores. Three-photon excitation at 1040 nm allows fluorophores with blue and green emission spectra to be addressed (and their corresponding ultraviolet and blue single-photon excitation wavelengths, respectively), and two-photon excitation at the same wavelength allows fluorophores with yellow, orange, or red emission spectra to be addressed (and their corresponding green, yellow, and orange single-photon excitation wavelengths). We demonstrate that three-photon excitation allows imaging over a depth range of more than 6 effective attenuation lengths to take place, corresponding to an 800 micrometer depth of imaging, in samples with a high density of fluorescent structures.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2021

Partial connectomes of labeled dopaminergic circuits reveal non-synaptic communication and axonal remodeling after exposure to cocaine

Dopaminergic (DA) neurons exert profound influences on behavior including addiction. However, how DA axons communicate with target neurons and how those communications change with drug exposure remains poorly understood. We leverage cell type-specific labeling with large volume serial electron microscopy to detail DA connections in the nucleus accumbens (NAc) of the mouse (Mus musculus) before and after exposure to cocaine. We find that individual DA axons contain different varicosity types based on their vesicle contents. Spatially ordering along individual axons further suggests that varicosity types are non-randomly organized. DA axon varicosities rarely make specific synapses (<2%, 6/410), but instead are more likely to form spinule-like structures (15%, 61/410) with neighboring neurons. Days after a brief exposure to cocaine, DA axons were extensively branched relative to controls, formed blind-ended ‘bulbs’ filled with mitochondria, and were surrounded by elaborated glia. Finally, mitochondrial lengths increased by ~2.2 times relative to control only in DA axons and NAc spiny dendrites after cocaine exposure. We conclude that DA axonal transmission is unlikely to be mediated via classical synapses in the NAc and that the major locus of anatomical plasticity of DA circuits after exposure to cocaine are large-scale axonal re-arrangements with correlated changes in mitochondria.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2024

Structural mechanisms for VMAT2 inhibition by tetrabenazine

The vesicular monoamine transporter 2 (VMAT2) is a proton-dependent antiporter responsible for loading monoamine neurotransmitters into synaptic vesicles. Dysregulation of VMAT2 can lead to several neuropsychiatric disorders including Parkinson’s disease and schizophrenia. Furthermore, drugs such as amphetamine and MDMA are known to act on VMAT2, exemplifying its role in the mechanisms of actions for drugs of abuse. Despite VMAT2’s importance, there remains a critical lack of mechanistic understanding, largely driven by a lack of structural information. Here, we report a 3.1 Å resolution cryo-electron microscopy (cryo-EM) structure of VMAT2 complexed with tetrabenazine (TBZ), a non-competitive inhibitor used in the treatment of Huntington’s chorea. We find TBZ interacts with residues in a central binding site, locking VMAT2 in an occluded conformation and providing a mechanistic basis for non-competitive inhibition. We further identify residues critical for cytosolic and lumenal gating, including a cluster of hydrophobic residues which are involved in a lumenal gating strategy. Our structure also highlights three distinct polar networks that may determine VMAT2 conformational dynamics and play a role in proton transduction. The structure elucidates mechanisms of VMAT2 inhibition and transport, providing insights into VMAT2 architecture, function, and the design of small-molecule therapeutics.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2020

Cell-based composite materials with programmed structures and functions

The present invention is directed to the use of silicic acid to transform biological materials, including cellular architecture into inorganic materials to provide biocomposites (nanomaterials) with stabilized structure and function. In the present invention, there has been discovered a means to stabilize the structure and function of biological materials, including cells, biomolecules, peptides, proteins (especially including enzymes), lipids, lipid vesicles, polysaccharides, cytoskeletal filaments, tissue and organs with silicic acid such that these materials may be used as biocomposites. In many instances, these materials retain their original biological activity and may be used in harsh conditions which would otherwise destroy the integrity of the biological material. In certain instances, these biomaterials may be storage stable for long periods of time and reconstituted after storage to return the biological material back to its original form. In addition, by exposing an entire cell to form CSCs, the CSCs may function to provide a unique system to study enzymes or a cascade of enzymes which are otherwise unavailable.

37 INORGANIC, ORGANIC, PHYSICAL, AND ANALYTICAL CH↗
DOE OSTI2020

Hierarchical Membrane Dynamics in Phase-Separated Model Membranes

Lipid membranes, the primary matrix of cell membranes, exhibit a hierarchy of dynamics from molecular motions to collective undulations, which control spatiotemporal membrane phenomena and regulate various cellular functions. Thus, understanding how various dynamic modes influence membrane behavior is crucial to uncovering the design rules of cell membranes and implementing them in future technologies. Previous membrane dynamics studies using neutron spin-echo spectroscopy (NSE) have shed light on the dependence of collective membrane fluctuations on viscoelastic membrane properties, demonstrating that thickness fluctuation modes (100ns) are dictated by membrane viscosity, which directly governs fast (ps) molecular diffusive motions. Here, we focus on phase-separating lipid membranes, composed of DMPC:DSPC mixtures, to understand the effect of domain formation/growth on hierarchical matrix dynamics using selective lipid deuteration and different neutron spectroscopy techniques. NSE measurements on DMPC:DSPC-d83 (70:30 mol%) show that bending rigidity of the DMPC-rich matrix graduallyincreases with decreasing temperature - as the fully-fluid membrane approaches the upper phase transition (i.e. with the formation of transient DSPC clusters) - and increases further in the phase coexistence region with the growth of DSPC domains. Comparatively, NSE measurements on DMPC-d54:DSPC-d83 vesicles show pronounced matrix thickness fluctuations in the fully-fluid phase and drastic suppression of this fluctuation mode in the gel-fluid coexistence phase, even when the DMPC-rich matrix is still in its fluid phase. This suppression in fluctuations suggests an increase in the matrix viscosity with the growth of gel DSPC domains, which is further investigated by selectively measuring lipid diffusion in the DMPC-rich matrix using neutron backscattering spectroscopy. Comparisons of the experimental results with coarse-grained MD simulationsillustrate the underlying coupling mechanisms. Overall, these observations indicate a clear mechanical and viscoelastic coupling betweenlipid domains and their host matrix, suggesting that domain formation influences membrane properties beyond local effects.

Carrillo, Jan Michael↗
DOE OSTI2023

MEMPHIS (Mesoscle Multiphysics Phase Field Simulator v.0

SAND2024-01427O Mesoscale Multiphysics Phase Field Simulator (MEMPHIS) is used for solving interfacial problems and can be applied to many evolutionary problems. Some examples are solidification dynamics, viscous fingering, fracture mechanics, hydrogen embrittlement, vesicle dynamics, electro-mechanical degradation of batteries, aging of microstructures, and segregation. MEMPHIS is written in Fortran 90 in a modular fashion, where model and numerical solvers are decoupled from one another. Sandia National Laboratories is a multimission laboratory managed and operated by National Technology & Engineering Solutions of Sandia, LLC, a wholly owned subsidiary of Honeywell International Inc., for the U.S. Department of Energy’s National Nuclear Security Administration under contract DE-NA0003525.

Dingreville, Remi↗
DOE OSTI2023

Relaxing Wrinkles in Jammed Interfacial Assemblies

Dynamic covalent bonding has emerged as a mean by which stresses in a network can be relaxed. Here, the strength of the bonding of ligands to nanoparticles at the interface between two immiscible liquids affect the same results in jammed assemblies of nanoparticle surfactants. Beyond a critical degree of overcrowding induced by the compression of jammed interfacial assemblies, the bonding of ligands to nanoparticles (NPs) can be broken, resulting in a desorption of the NPs from the interface. This reduces the areal density of nanoparticle surfactants at the interface, allowing the assemblies to relax, not to a fluid state but rather another jammed state. The relaxation of the wrinkles caused by the compression reflects the tendency of these assemblies to eliminate areas of high curvature, favoring a more planar geometry. Importantly, this enabled the generation of giant vesicular and multivesicular structures from these assemblies.

37 INORGANIC, ORGANIC, PHYSICAL, AND ANALYTICAL CH↗