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At least 37 records · Page 2
All sourcesNASA NTRSDOE OSTI
DOE OSTI2025

Ecosystem consequences of a nitrogen-fixing proto-organelle

Microscale symbioses can be critical to ecosystem functions, but the mechanisms of these interactions in nature are often cryptic. Here, we use a combination of stable isotope imaging and tracing to reveal carbon (C) and nitrogen (N) exchanges among three symbiotic primary producers that fuel a salmon-bearing river food web. Bulk isotope analysis, nanoSIMS (secondary ion mass spectrometry) isotope imaging, and density centrifugation for quantitative stable isotope probing enabled quantification of organism-specific C- and N-fixation rates from the subcellular scale to the ecosystem. After winters with riverbed-scouring floods, the macroalga Cladophora glomerata uses nutrients in spring runoff to grow streamers up to 10 m long. During summer flow recession, riverine N concentrations wane and Cladophora becomes densely epiphytized by three species of Epithemia, diatoms with N-fixing endosymbionts (proto-organelles) descended from a free-living Crocosphaera cyanobacterium. Over summertime epiphyte succession on Cladophora, N-fixation rates increased as Epithemia spp. became dominant, Cladophora C-fixation declined to near zero, and Epithemia C-fixation increased. Carbon transfer to caddisflies grazing on Cladophora with high densities of Epithemia was 10-fold higher than C transfer to caddisflies grazing Cladophora with low Epithemia loads. In response to demand for N, Epithemia allocates high levels of newly fixed C to its endosymbiont. Consequently, these endosymbionts have the highest rates of C and N accumulation of any taxon in this tripartite symbiosis during the biologically productive season and can produce one of the highest areal rates of N-fixation reported in any river ecosystem.

Marks, Jane C. [Northern Arizona Univ., Flagstaff,↗
DOE OSTI2024

Distinct cellular expression and subcellular localization of Kv2 voltage‐gated K + channel subtypes in dorsal root ganglion neurons conserved between mice and humans

Abstract The distinct organization of Kv2 voltage‐gated potassium channels on and near the cell body of brain neurons enables their regulation of action potentials and specialized membrane contact sites. Somatosensory neurons have a pseudounipolar morphology and transmit action potentials from peripheral nerve endings through axons that bifurcate to the spinal cord and the cell body within ganglia including the dorsal root ganglia (DRG). Kv2 channels regulate action potentials in somatosensory neurons, yet little is known about where Kv2 channels are located. Here, we define the cellular and subcellular localization of the Kv2 paralogs, Kv2.1 and Kv2.2, in DRG somatosensory neurons with a panel of antibodies, cell markers, and genetically modified mice. We find that relative to spinal cord neurons, DRG neurons have similar levels of detectable Kv2.1 and higher levels of Kv2.2. In older mice, detectable Kv2.2 remains similar, while detectable Kv2.1 decreases. Both Kv2 subtypes adopt clustered subcellular patterns that are distinct from central neurons. Most DRG neurons co‐express Kv2.1 and Kv2.2, although neuron subpopulations show preferential expression of Kv2.1 or Kv2.2. We find that Kv2 protein expression and subcellular localization are similar between mouse and human DRG neurons. We conclude that the organization of both Kv2 channels is consistent with physiological roles in the somata and stem axons of DRG neurons. The general prevalence of Kv2.2 in DRG as compared to central neurons and the enrichment of Kv2.2 relative to detectable Kv2.1 in older mice, proprioceptors, and axons suggest more widespread roles for Kv2.2 in DRG neurons.

Neurosciences & Neurology↗
DOE OSTI2026

Microfibril orientation and compositional heterogeneity in fiber and vessel cell walls of poplar xylem studied by AFM-IR and SFG spectroscopy

Understanding the structural organization of cellulose microfibrils (CMFs) within individual plant cell walls is essential for connecting cell wall architecture to its mechanical and physiological functions. However, due to the complex hierarchical structure and nanoscale heterogeneity of cell walls, it remains technically challenging to resolve detailed compositional and orientational information at subcellular levels of individual cell walls. This study investigates the internal 3D structure, chemical composition, and sublayer organization of fiber and vessel cell walls in the xylem tissue of a two-year-old field-grown hybrid poplar tree (Populus alba × P. glandulosa) using photothermal atomic force microscopy coupled with infrared spectroscopy (AFM-IR) and sum frequency generation (SFG) hyperspectral microscopy. AFM-IR provided nanoscale chemical imaging, revealing localized compositional heterogeneity, including variations between adjacent cell walls and transitional layers beyond the traditional S1, S2, and S3 sublayers. SFG microscopy revealed that CMFs in fiber walls are highly aligned along the stem axis, consistent with their role in mechanical support, while vessel cell walls exhibited slightly tilted CMFs, reflecting their function in hydraulic transport. Together, these results offer new insights into cell-type-specific CMF organization and compositional gradients in hybrid poplar xylem. These findings highlight the structural and chemical complexity of secondary cell walls in woody plants and demonstrate the value of AFM-IR and SFG spectroscopy in elucidating plant cell wall architecture.

60 APPLIED LIFE SCIENCES↗
DOE OSTI2025

Multi-Omics Reveals Temporal Scales of Carbon Metabolism in Synechococcus Elongatus PCC 7942 Under Light Disturbance

Central carbon metabolism in model cyanobacteria involves multiple pathways to adapt to energy-light limitations across diel cycles. However, the success in mechanistic modeling for phenotypic prediction of the protein regulators in the metabolic state depends on capturing the vast possibilities emerging from multiple regulatory pathways in complex biological processes. Here, we developed a physics-informed machine learning approach based on energy-landscape concepts to predict regulatory proteins responding to cyclic circadian and unforeseen light perturbations in cyanobacterial metabolic networks. Our approach provides interpretable de novo models for inferring gene expression dynamics from Synechococcus elongatus over diel cycles and using redox proteome analysis to distinguish immediate light-responsive elements from circadian-regulated processes in carbon metabolism pathways. We identified distinct temporal signatures with the analysis of the redox proteome: there was an immediate shift in cysteine redox states accompanied by a limited change in protein abundance under constant illumination and after 2 hours of darkness. This discovery indicates that the generation of reductants coordinates photoinduced electron transport with redox metabolic pathways in two discernable molecular mechanisms: fast redox-based protein modifications occur immediately after the light disturbance, followed by slow transcriptional regulations across networks. This temporal regulation reveals how metabolic networks integrate rapid light responses with programmed circadian rhythms to maintain cellular homeostasis under the light-energy limitations over the diel cycle.

Biomolecular & subcellular processes↗
DOE OSTI2024

Dramatic changes in mitochondrial subcellular location and morphology accompany activation of the CO 2 concentrating mechanism

Dynamic changes in intracellular ultrastructure can be critical for the ability of organisms to acclimate to environmental conditions. Microalgae, which are responsible for ~50% of global photosynthesis, compartmentalize their Ribulose 1,5 Bisphosphate Carboxylase/Oxygenase (Rubisco) into a specialized structure known as the pyrenoid when the cells experience limiting CO 2 conditions; this compartmentalization is a component of the CO 2 Concentrating Mechanism (CCM), which facilitates photosynthetic CO 2 fixation as environmental levels of inorganic carbon (Ci) decline. Changes in the spatial distribution of mitochondria in green algae have also been observed under CO 2 limitation, although a role for this reorganization in CCM function remains unclear. We used the green microalga Chlamydomonas reinhardtii to monitor changes in mitochondrial position and ultrastructure as cells transition between high CO 2 and Low/Very Low CO 2 (LC/VLC). Upon transferring cells to VLC, the mitochondria move from a central to a peripheral cell location and orient in parallel tubular arrays that extend along the cell’s apico-basal axis. We show that these ultrastructural changes correlate with CCM induction and are regulated by the CCM master regulator CIA5. The apico-basal orientation of the mitochondrial membranes, but not the movement of the mitochondrion to the cell periphery, is dependent on microtubules and the MIRO1 protein, with the latter involved in membrane–microtubule interactions. Furthermore, blocking mitochondrial respiration in VLC-acclimated cells reduces the affinity of the cells for Ci. Overall, our results suggest that mitochondrial repositioning functions in integrating cellular architecture and energetics with CCM activities and invite further exploration of how intracellular architecture can impact fitness under dynamic environmental conditions.

CO2 concentrating mechanism↗
DOE OSTI2022

Protein ubiquitination in plant peroxisomes

Protein ubiquitination regulates diverse cellular processes in eukaryotic organisms, from growth and development to stress response. Proteins subjected to ubiquitination can be found in virtually all subcellular locations and organelles, including peroxisomes, single-membrane and highly dynamic organelles ubiquitous in eukaryotes. Peroxisomes contain metabolic functions essential to plants and animals such as lipid catabolism, detoxification of reactive oxygen species (ROS), biosynthesis of vital hormones and cofactors, and photorespiration. Plant peroxisomes possess a complex proteome with functions varying among different tissue types and developmental stages, and during plant response to distinct environmental cues. However, how these diverse functions are regulated at the post-translational level is poorly understood, especially in plants. Here, in this review, we summarized current knowledge of the involvement of protein ubiquitination in peroxisome protein import, remodeling, pexophagy, and metabolism, focusing on plants, and referencing discoveries from other eukaryotic systems when relevant. Based on previous ubiquitinomics studies, we compiled a list of 56 ubiquitinated Arabidopsis peroxisomal proteins whose functions are associated with all the major plant peroxisomal metabolic pathways. This discovery suggests a broad impact of protein ubiquitination on plant peroxisome functions, therefore substantiating the need to investigate this significant regulatory mechanism in peroxisomes at more depths.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2023

Adaptive Optical Two-Photon Fluorescence Microscopy Probes Cellular Organization of Ocular Lenses In Vivo

The mammalian ocular lens is an avascular multicellular organ that grows continuously throughout life. Traditionally, its cellular organization is investigated using dissected lenses, which eliminates in vivo environmental and structural support. Therefore, in vivo optical imaging methods for studying lenses in their native context in live animals are urgently needed. Here, we demonstrated that two-photon fluorescence microscopy can visualize lens cells in vivo. To maintain subcellular resolution at depth, we used adaptive optics to correct aberrations owing to ocular and lens tissues, which led to substantial signal and resolution improvements. Imaging lens cells up to 980 µm deep, we observed novel cellular organizations including suture-associated voids, enlarged vacuoles, and large cavities, contrary to the conventional view of a highly ordered organization. We tracked these features longitudinally over weeks and observed the incorporation of new cells during growth. Taken together, noninvasive longitudinal in vivo imaging of lens morphology using adaptive optics two-photon fluorescence microscopy will allow us to observe the development or alterations of lens cellular organization in living animals directly.

60 APPLIED LIFE SCIENCES↗
DOE OSTI2021

Membrane Profiling by Free Flow Electrophoresis and SWATH-MS to Characterize Subcellular Compartment Proteomes in Mesembryanthemum crystallinum

The study of subcellular membrane structure and function facilitates investigations into how biological processes are divided within the cell. However, work in this area has been hampered by the limited techniques available to fractionate the different membranes. Free Flow Electrophoresis (FFE) allows for the fractionation of membranes based on their different surface charges, a property made up primarily of their varied lipid and protein compositions. In this study, high-resolution plant membrane fractionation by FFE, combined with mass spectrometry-based proteomics, allowed the simultaneous profiling of multiple cellular membranes from the leaf tissue of the plant Mesembryanthemum crystallinum. Comparisons of the fractionated membranes’ protein profile to that of known markers for specific cellular compartments sheds light on the functions of proteins, as well as provides new evidence for multiple subcellular localization of several proteins, including those involved in lipid metabolism.

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

MEMOTE for standardized genome-scale metabolic model testing

Reconstructing metabolic reaction networks enables the development of testable hypotheses of an organism’s metabolism under different conditions. State-of-the-art genome-scale metabolic models (GEMs) can include thousands of metabolites and reactions that are assigned to subcellular locations. Gene–protein–reaction (GPR) rules and annotations using database information can add meta-information to GEMs. GEMs with metadata can be built using standard reconstruction protocols, and guidelines have been put in place for tracking provenance and enabling interoperability, but a standardized means of quality control for GEMs is lacking. Here we report a community effort to develop a test suite named MEMOTE (for metabolic model tests) to assess GEM quality.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2023

A subcellular biochemical model for T6SS dynamics reveals winning competitive strategies

Abstract The type VI secretion system (T6SS) is a broadly distributed interbacterial weapon that can be used to eliminate competing bacterial populations. Although unarmed target populations are typically used to study T6SS function in vitro, bacteria most likely encounter other T6SS-armed competitors in nature. However, the connection between subcellular details of the T6SS and the outcomes of such mutually lethal battles is not well understood. Here, we incorporate biological data derived from natural competitors of Vibrio fischeri light organ symbionts to build a biochemical model for T6SS at the single-cell level, which we then integrate into an agent-based model (ABM). Using the ABM, we isolate and experiment with strain-specific physiological differences between competitors in ways not possible with biological samples to identify winning strategies for T6SS-armed populations. Through in vitro experiments, we discover that strain-specific differences exist in T6SS activation speed. ABM simulations corroborate that faster activation is dominant in determining survival during competition. Once competitors are fully activated, the energy required for T6SS creates a tipping point where increased weapon building and firing becomes too costly to be advantageous. Through ABM simulations, we identify the threshold where this transition occurs in the T6SS parameter space. We also find that competitive outcomes depend on the geometry of the battlefield: unarmed target cells survive at the edges of a range expansion where unlimited territory can be claimed. Alternatively, competitions within a confined space, much like the light organ crypts where natural V. fischeri compete, result in the rapid elimination of the unarmed population.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2022

Finding new friends and revisiting old ones – how plant lipid droplets connect with other subcellular structures

Summary The number of described contact sites between different subcellular compartments and structures in eukaryotic cells has increased dramatically in recent years and, as such, has substantially reinforced the well‐known premise that these kinds of connections are essential for overall cellular organization and the proper functioning of cellular metabolic and signaling pathways. Here, we discuss contact sites involving plant lipid droplets (LDs), including LD‐endoplasmic reticulum (ER) connections that mediate the biogenesis of new LDs at the ER, LD‐peroxisome connections, that facilitate the degradation of LD‐stored triacylglycerols (TAGs), and the more recently discovered LD‐plasma membrane connections, which involve at least three novel proteins, but have a yet unknown physiological function(s).

60 APPLIED LIFE SCIENCES↗
NASA NTRS1989

Thin film bioreactors in space

Studies from the Skylab, SL-3 and D-1 missions have demonstrated that biological organisms grown in microgravity have changes in basic cellular functions such as DNA, mRNA and protein synthesis, cytoskeleton synthesis, glucose utilization, and cellular differentiation. Since microgravity could affect prokaryotic and eukaryotic cells at a subcellular and molecular level, space offers an opportunity to learn more about basic biological systems with one inmportant variable removed. The thin film bioreactor will facilitate the handling of fluids in microgravity, under constant temperature and will allow multiple samples of cells to be grown with variable conditions. Studies on cell cultures grown in microgravity would make it possible to identify and quantify changes in basic biological function in microgravity which are needed to develop new applications of orbital research and future biotechnology.

Hughes-Fulford, M.↗
DOE OSTI2025

Lignin Removal in Subcellular Location of Poplar Cell Wall During Pretreatment Significantly Impacts Cellulose Digestibility

The γ-valerolactone (GVL) pretreatment is one of the leading solvent-based methods for producing high-quality lignin under mild conditions. However, the glucan conversion yield from GVL pretreated biomass remains unsatisfactory. To explore the discrepancies between the relatively low glucan conversion and high lignin extraction, we conducted GVL−HCl and NaOH pretreatments on poplar and investigated their effects on lignin content and location, as well as on enzymatic hydrolysis of poplar cell walls at the subcellular level. Under designated pretreatment conditions of GVL−HCl (90% GVL, 0.1 M HCl, 100 °C, 1 h) and NaOH (1 M, 121 °C, 2 h), the glucan conversion yields were 69.4% and 95.8%, with lignin removal rates of 67.8% and 47.7%, respectively. Four types of GFP-labeled carbohydrate binding modules were used to identify different forms of cellulose in the pretreated cell walls. The overall binding intensities to pretreated poplar were stronger for NaOH compared to GVL−HCl pretreatment. Stimulated Raman scattering microscopy imaging revealed that GVL−HCl preferentially extracted lignin from the compound middle lamella and cell corner areas, while NaOH effectively dissolved lignin in the secondary cell walls. Real-time imaging of cellulase degradation of pretreated cell walls further indicated that digestion started from both the cell lumen and the compound middle lamella areas for GVL, whereas it occurred uniformly across the secondary cell walls for NaOH. Our findings suggest that the location of lignin removal during pretreatment is crucial for enzymatic cellulose degradation, in addition to the total amount of lignin extraction.

37 INORGANIC, ORGANIC, PHYSICAL, AND ANALYTICAL CH↗
NASA NTRS1989

Physical phenomena and the microgravity response

The living biological cell is not a sack of Newtonian fluid containing systems of chemical reactions at equilibrium. It is a kinetically driven system, not a thermodynamically driven system. While the cell as a whole might be considered isothermal, at the scale of individual macromolecular events there is heat generated, and presumably sharp thermal gradients exist at the submicron level. Basic physical phenomena to be considered when exploring the cell's response to inertial acceleration include particle sedimentation, solutal convection, motility electrokinetics, cytoskeletal work, and hydrostatic pressure. Protein crystal growth experiments, for example, illustrate the profound effects of convection currents on macromolecular assembly. Reaction kinetics in the cell vary all the way from diffusion-limited to life-time limited. Transport processes vary from free diffusion, to facilitated and active transmembrane transport, to contractile-protein-driven motility, to crystalline immobilization. At least four physical states of matter exist in the cell: aqueous, non-aqueous, immiscible-aqueous, and solid. Levels of order vary from crystalline to free solution. The relative volumes of these states profoundly influence the cell's response to inertial acceleration. Such subcellular phenomena as stretch-receptor activation, microtubule re-assembly, synaptic junction formation, chemotactic receptor activation, and statolith sedimentation were studied recently with respect to both their basic mechanisms and their responsiveness to inertial acceleration. From such studies a widespread role of cytoskeletal organization is becoming apparent.

Todd, Paul↗
DOE OSTI2021

PhenoProfiling: Mapping phenotypic outcomes to molecular determinants of biochemical activity

The aggregate genomes of the trillions of microorganisms within soil, animal hosts, and aquatic systems encode for an extensive functional capacity for myriad biochemical activities. C, N, P, and S metabolism, synthesis of signaling molecules and vitamins, mineralization, and other activities are essential to microbe, community, and plant physiology, and more broadly to climate, water, animal, flora, and human health. The current understanding of the molecular basis for the function of microbial communities stems primarily from comparative metagenomic and metatranscriptomic studies. These same tools are employed to ascertain the impacts to the community resulting from perturbations, such as climate change, emerging pollutants, fires, and seawater infiltration for environmental communities, and dietary changes, xenobiotic exposure, and various disease states for the human gut microbiome. Such studies can identify the potential for a specific function, but they cannot determine that a particular cell is functionally active, nor can they determine the molecular architecture required for function. In short, genes and transcripts alone fail to reveal the complex subcellular arrangement of proteins and molecules that elicit a given phenotype in a microbial cell.

37 INORGANIC, ORGANIC, PHYSICAL, AND ANALYTICAL CH↗
NASA NTRS2017

Small Ion Channel Linking Molecular Simulations and Electrophysiology

Ion channels are pore-forming protein assemblies that mediate the transport of small ions across cell membranes. Otherwise, membrane bilayers would be almost impermeable to ions incapable to traverse the low dielectric constant, hydrophobic membrane core. Ion channels are ubiquitous to all life forms. In humans and other higher organisms they play the central role in conducting nerve impulses, cardiac functions, muscle contraction and apoptosis. On the other extreme of biological complexity, viral ion channels (viroporins) influence many stages of the virus infection cycle either through regulating virus replication, such as entry, assembly and release or modulating the electrochemical balance in the subcellular compartments of host cells. Ion channels were crucial components of protocells. Their emergence facilitated adaptation of nascent life to different environmental conditions. The earliest ion channels must have been much simpler than most of their modern ancestors. Viral channels are among only a few naturally occurring models to study the structure, function and evolution of primordial channels. Experimental studies of these properties are difficult and often unreliable. In principle, computational methods, and molecular dynamics (MD) simulations in particular, can aid in providing information about both the structure and the function of ion channels. However, MD suffers from its own problems, such as inability to access sufficiently long time scales or limited accuracy of force fields. It is, therefore, essential to determine the reliability of MD simulations. We propose to do so on the basis of two criteria. One is channel stability on time scales that extend for several microseconds or longer. The other is the ability to reproduce the measured ionic conductance as a function of applied voltage. If both the stability and the calculated ionic conductance are satisfactory it will greatly increase our confidence that the structure and the function of a channel are described sufficiently accurately. To our knowledge, long time scale stability (approx.10 micro-sec) and the correct electrophysiology have been shown so far for only one channel - the synthetic LS3 hexamer). In this presentation, this approach will be discussed in application to two viral channels - Vpu, encoded by the HIV-1 genome and p7 of hepatitis C.

Pohorille, Andrzej↗
NASA NTRS1985

Calcium and Calmodulin Localization in Gravitropically-responding Plant Organs

Antimonate staining procedures were used to detect calcium redistribution changes in corn roots. Results show that an asymmetric redistribution of Ca is induced by a gravitropic stimulus in roots as it is in shoots. Since this response occurs within 10 min, at least 5 min before any visible bending, it could play a role in the regulation of root gravitropism. Two different general approaches were used to localize calmodulin in plant tissue: radioimmunoassay of its content in tissue and in purified subcellular organelles, and immunocytochemical detection of it in roots and coleoptiles. Radioimmunoassay results indicate that calmodulin is present in large quantities in pllant cells and that it is specifically associated with mitochondria, etioplasts and nuclei. An assayed of an extract of soluble wall proteins revealed that over 1% of these proteins was calmodulin. Controls indicate that this calmodulin is not cytoplasmic in origin. A reaction product from anti-calmodulin was found mainly in the root cap cells, moderately in metazylem elements, in some cells in the stele surrounding metaxylem elements and in cortical cells.

Roux, S. J.↗