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Search indexed NASA NTRS and DOE OSTI research on propulsion, heat transfer, battery materials and energy systems. Follow report and document links to the original sources.

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At least 55 records · Page 3
All sourcesNASA NTRSDOE OSTI
DOE OSTI2020

Distinct Preflowering Drought Tolerance Strategies of Sorghum bicolor Genotype RTx430 Revealed by Subcellular Protein Profiling

Drought is the largest stress affecting agricultural crops, resulting in substantial reductions in yield. Plant adaptation to water stress is a complex trait involving changes in hormone signaling, physiology, and morphology. Sorghum (Sorghum bicolor (L.) Moench) is a C4 cereal grass; it is an agricultural staple, and it is particularly drought-tolerant. To better understand drought adaptation strategies, we compared the cytosolic- and organelle-enriched protein profiles of leaves from two Sorghum bicolor genotypes, RTx430 and BTx642, with differing preflowering drought tolerances after 8 weeks of growth under water limitation in the field. In agreement with previous findings, we observed significant drought-induced changes in the abundance of multiple heat shock proteins and dehydrins in both genotypes. Interestingly, our data suggest a larger genotype-specific drought response in protein profiles of organelles, while cytosolic responses are largely similar between genotypes. Organelle-enriched proteins whose abundance significantly changed exclusively in the preflowering drought-tolerant genotype RTx430 upon drought stress suggest multiple mechanisms of drought tolerance. These include an RTx430-specific change in proteins associated with ABA metabolism and signal transduction, Rubisco activation, reactive oxygen species scavenging, flowering time regulation, and epicuticular wax production. We discuss the current understanding of these processes in relation to drought tolerance and their potential implications.

59 BASIC BIOLOGICAL SCIENCES↗
NASA NTRS1981

Symbiosis in cell evolution: Life and its environment on the early earth

The book treats cell evolution from the viewpoint of the serial endosymbiosis theory of the origin of organelles. Following a brief outline of the symbiotic theory, which holds that eukaryotes evolved by the association of free-living bacteria with a host prokaryote, the diversity of life is considered, and five kingdoms of organisms are distinguished: the prokaryotic Monera and the eukaryotic Protoctista, Animalia, Fungi and Plantae. Symbiotic and traditional direct filiation theories of cell evolution are compared. Recent observations of cell structure and biochemistry are reviewed in relation to early cell evolution, with attention given to the geological context for the origin of eukaryotic cells, the origin of major bacterial anaerobic pathways, the relationship between aerobic metabolism and atmospheric oxygen, criteria for distinguishing symbiotic organelles from those that originated by differentiation, and the major classes of eukaryotic organelles: mitochondria, cilia, microtubules, the mitotic and meiotic apparatuses, and pastids. Cell evolution during the Phanerozoic is also discussed with emphasis on the effects of life on the biosphere

Margulis, L.↗
NASA NTRS1997

Root cytoskeleton: its role in perception of and response to gravity

We have critically evaluated the possible functions of the plant cytoskeleton in root gravisensing and graviresponse and discussed the evidence that microtubules (MTs) and actin microfilaments (MFs) do not control differential cell growth during bending of roots. On the other hand, MF and MT networks are envisaged to participate in gravisensing because of the mechanical properties of the cytoskeletal structures that interconnect plant cell organelles with the plasma membrane. In restrained gravisensing, forces are suggested to be transmitted to membranes because large-scale gravity-dependent repositioning of organelles is effectively prevented due to the cytoskeleton-mediated anchorage of their envelopes at the plasma membrane. From the cytoskeletal point of view, we can also envisage an unrestrained gravity sensing when cytoskeletal tethers are not strong enough to preserve the tight control over distribution of organelles and the latter, if heavy enough, are allowed to sediment towards the physical bottom of cells. This situation obviously occurs in root cap statocytes because these uniquely organized cells are depleted of prominent actin MF bundles, endoplasmic MT arrays, and ER elements in their internal cytoplasm. Nevertheless, indirect evidence clearly indicates that sedimented root cap statoliths are enmeshed within fine but dynamic MF networks and that their behaviour is obviously under, at least partial, cytoskeletal control. The actomyosin-enriched domain among and around amyloplasts is proposed to increase the perception of gravity due to the grouping effect of sedimenting statoliths. Cytoskeletal links between myosin-rich statoliths, and cell peripheries well equipped with dense cortical MTs, membrane-associated cytoskeleton, as well as with ER elements, would allow efficient restrained gravisensing only at the statocyte cell cortex. As a consequence of cytoskeletal depletion in the internal statocyte cytoplasm and bulk sedimentation of large amyloplasts, restrained gravisensing is spatially restricted to the bottom of the statocyte irrespective of whether roots are vertical or horizontal. This spatial aspect allows for efficient gravisensing via amplification of gravity-induced impacts on the cellular architecture, a phenomenon which is unique to root cap statocytes.

NASA Discipline Plant Biology↗
NASA NTRS2000

Release of kinesin from vesicles by hsc70 and regulation of fast axonal transport

The nature of kinesin interactions with membrane-bound organelles and mechanisms for regulation of kinesin-based motility have both been surprisingly difficult to define. Most kinesin is recovered in supernatants with standard protocols for purification of motor proteins, but kinesin recovered on membrane-bound organelles is tightly bound. Partitioning of kinesin between vesicle and cytosolic fractions is highly sensitive to buffer composition. Addition of either N-ethylmaleimide or EDTA to homogenization buffers significantly increased the fraction of kinesin bound to organelles. Given that an antibody against kinesin light chain tandem repeats also releases kinesin from vesicles, these observations indicated that specific cytoplasmic factors may regulate kinesin release from membranes. Kinesin light tandem repeats contain DnaJ-like motifs, so the effects of hsp70 chaperones were evaluated. Hsc70 released kinesin from vesicles in an MgATP-dependent and N-ethylmaleimide-sensitive manner. Recombinant kinesin light chains inhibited kinesin release by hsc70 and stimulated the hsc70 ATPase. Hsc70 actions may provide a mechanism to regulate kinesin function by releasing kinesin from cargo in specific subcellular domains, thereby effecting delivery of axonally transported materials.

Non-NASA Center↗
NASA NTRS1999

Model system for plant cell biology: GFP imaging in living onion epidermal cells

The ability to visualize organelle localization and dynamics is very useful in studying cellular physiological events. Until recently, this has been accomplished using a variety of staining methods. However, staining can give inaccurate information due to nonspecific staining, diffusion of the stain or through toxic effects. The ability to target green fluorescent protein (GFP) to various organelles allows for specific labeling of organelles in vivo. The disadvantages of GFP thus far have been the time and money involved in developing stable transformants or maintaining cell cultures for transient expression. In this paper, we present a rapid transient expression system using onion epidermal peels. We have localized GFP to various cellular compartments (including the cell wall) to illustrate the utility of this method and to visualize dynamics of these compartments. The onion epidermis has large, living, transparent cells in a monolayer, making them ideal for visualizing GFP. This method is easy and inexpensive, and it allows for testing of new GFP fusion proteins in a living tissue to determine deleterious effects and the ability to express before stable transformants are attempted.

NASA Discipline Plant Biology↗
DOE OSTI2020

SPLIT: Stable Protein Coacervation Using a Light Induced Transition

Protein coacervates serve as hubs to concentrate and sequester proteins and nucleotides and thus function as membraneless organelles to manipulate cell physiology. We have engineered a coacervating protein to create tunable, synthetic membraneless organelles that assemble in response to a single pulse of light. Coacervation is driven by the intrinsically disordered RGG domain from the protein LAF-1, and opto-responsiveness is coded by the protein PhoCl, which cleaves in response to 405 nm light. We developed a fusion protein containing a solubilizing maltose-binding protein domain, PhoCl, and two copies of the RGG domain. Several seconds of illumination at 405 nm is sufficient to cleave PhoCl, removing the solubilization domain and enabling RGG-driven coacervation within minutes in cellularsized water-in-oil emulsions. An optimized version of this system displayed light-induced coacervation in Saccharomyces cerevisiae. Finally, the methods described here provide novel strategies for inducing protein phase separation using light.

32 ENERGY CONSERVATION, CONSUMPTION, AND UTILIZATI↗
DOE OSTI2022

Defining upstream enhancing and inhibiting sequence patterns for plant peroxisome targeting signal type 1 using large–scale in silico and in vivo analyses

Peroxisomes are universal eukaryotic organelles essential to plants and animals. Most peroxisomal matrix proteins carry peroxisome targeting signal type 1 (PTS1), a C-terminal tripeptide. Studies from various kingdoms have revealed influences from sequence upstream of the tripeptide on peroxisome targeting, supporting the view that positive charges in the upstream region are the major enhancing elements. However, a systematic approach to better define the upstream elements influencing PTS1 targeting capability is needed. Here, we used protein sequences from 177 plant genomes to perform large-scale and in-depth analysis of the PTS1 domain, which includes the PTS1 tripeptide and upstream sequence elements. We identified and verified 12 low-frequency PTS1 tripeptides and revealed upstream enhancing and inhibiting sequence patterns for peroxisome targeting, which were subsequently validated in vivo. Follow-up analysis revealed that nonpolar and acidic residues have relatively strong enhancing and inhibiting effects, respectively, on peroxisome targeting. However, in contrast to the previous understanding, positive charges alone do not show the anticipated enhancing effect and that both the position and property of the residues within these patterns are important for peroxisome targeting. We further demonstrated that the three residues immediately upstream of the tripeptide are the core influencers, with a ‘basic-nonpolar-basic’ pattern serving as a strong and universal enhancing pattern for peroxisome targeting. These findings have significantly advanced our knowledge of the PTS1 domain in plants and likely other eukaryotic species as well. The principles and strategies employed in the present study may also be applied to deciphering auxiliary targeting signals for other organelles.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2020

Solution structure for an Encephalitozoon cuniculi adrenodoxin-like protein in the oxidized state

Encephalitozoon cuniculi is a unicellular, obligate intracellular eukaryotic parasite in the Microsporidia family and one of the agents responsible for microsporidosis infections in humans. Like most Microsporidia, the genome of E. cuniculi is markedly reduced and the organism contains mitochondria-like organelles called mitosomes instead of mitochondria. Here we report the solution NMR structure for a protein physically associated with mitosome-like organelles in E. cuniculi, the 128-residue, adrenodoxin-like protein Ec-Adx (UniProt ID Q8SV19) in the [2Fe-2S] ferredoxin superfamily. Oxidized Ec-Adx contains a mixed four-strand β-sheet, β2-β1-β4-β3 (↓↑↑↓), loosely encircled by three α-helices and two 310-helices. This fold is similar to the structure observed in other adrenodoxin and adrenodoxin-like proteins except for the absence of a fifth anti-parallel β-strand next to β3 and the position of α3. Cross peaks are missing or cannot be unambiguously assigned for 20 amide resonances in the 1 H- 15 N HSQC spectrum of Ec-Adx. These missing residues are clustered primarily in two regions, G48-V61 and L94-L98, containing the four cysteine residues predicted to ligate the paramagnetic [2Fe-2S] cluster. Missing amide resonances in 1 H- 15 N HSQC spectra are detrimental to NMR-based solution structure calculations because 1 H- 1 H NOE restraints are absent (glass half-empty) and this may account for the absent β-strand (β5) and the position of α3 in oxidized Ec-Adx. On the other hand, the missing amide resonances unambiguously identify the presence, and immediate environment, of the paramagnetic [2Fe-2S] cluster in oxidized Ec-Adx (glass half-full).

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2025

Steric effects of central dogma processes on the compaction and segregation of bacterial nucleoids

The bacterial cytoplasm is characterized by a distinctive membrane-less organelle, the nucleoid, which harbors the chromosomal DNA. Here, we investigate the steric effects of dynamic processes associated with transcription and translation on the structure of this organelle using coarse-grained molecular dynamics simulations that incorporate out-of-equilibrium reactions. Our model captures the scale of the entire cell and incorporates a reaction-diffusion system for ribosomes and polyribosomes, coupling their nonequilibrium kinetics to DNA excluded volume interactions. Our findings demonstrate that out-of-equilibrium reactions increase the size of the nucleoid and the number of ribosomes in this subcellular region. In addition, we show that nucleoid size is proportional to transcriptional activity. Our model reproduces the time-dependent change in nucleoid size observed in rifampicin treatment experiments, where the pool of polyribosomes is depleted. Furthermore, we find that these active processes are essential for complete sister chromosome separation and correct nucleoid positioning within the cell. Overall, our study reveals the effects of the central dogma processes on the internal organization and localization of bacterial nucleoids.

Chang, Mu-Hung [Univ. of Tennessee, Knoxville, TN ↗
DOE OSTI2020

Self-assembling Shell Proteins PduA and PduJ have Essential and Redundant Roles in Bacterial Microcompartment Assembly

Protein self-assembly is a common and essential biological phenomenon, and bacterial microcompartments present a promising model system to study this process. Bacterial microcompartments are large, protein-based organelles which natively carry out processes important for carbon fixation in cyanobacteria and the survival of enteric bacteria. These structures are increasingly popular with biological engineers due to their potential utility as nanobioreactors or drug delivery vehicles. However, the limited understanding of the assembly mechanism of these bacterial microcompartments hinders efforts to repurpose them for non-native functions. Here, we comprehensively investigate proteins involved in the assembly of the 1,2-propanediol utilization bacterial microcompartment from Salmonella enterica serovar Typhimurium LT2, one of the most widely studied microcompartment systems. We first demonstrate that two shell proteins, PduA and PduJ, have a high propensity for self-assembly upon overexpression, and we provide a novel method for self-assembly quantification. Using genomic knock-outs and knock-ins, we systematically show that these two proteins play an essential and redundant role in bacterial microcompartment assembly that cannot be compensated by other shell proteins. At least one of the two proteins PduA and PduJ must be present for the bacterial microcompartment shell to assemble. We also demonstrate that assembly-deficient variants of these proteins are unable to rescue microcompartment formation, highlighting the importance of this assembly property. Our work provides insight into the assembly mechanism of these bacterial organelles and will aid downstream engineering efforts.

1,2-propanediol utilization MCP↗
DOE OSTI2022

Putting the brakes on chloroplast stress signaling

As sessile organisms, plants must be able to sense their surroundings and adjust. One way plants do this is by using their energy-producing organelles (chloroplasts and mitochondria). During environmental stress, these organelles experience metabolic changes that induce signals for acclimation. While many metabolites have been proposed as signaling factors, reactive oxygen species (ROS) are known to play prominent roles. In the chloroplast, the ROS singlet oxygen ( 1 O 2 ) is naturally produced during impaired photosynthesis and can lead to retrograde signaling to the nucleus (to control the expression of hundreds of genes), chloroplast degradation, and cell death. The mechanisms controlling these pathways have mostly remained obscure. Recently, Dogra et al. (2022) reported a new role for EXECUTER2 (EX2) in these chloroplast 1 O 2 signaling pathways, demonstrating that EX2 acts as a buffer to prevent premature activation of 1 O 2 signaling. Furthermore, these exciting findings reveal an unexpected complexity to chloroplast stress signaling, and identify a decoy mechanism to prevent early activation of cell death.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2024

Comparative Pore Structure and Dynamics for Bacterial Microcompartment Shell Protein Assemblies in Sheets or Shells

Bacterial microcompartments (BMCs) are protein-bound organelles found in some bacteria that encapsulate enzymes for enhanced catalytic activity. These compartments spatially sequester enzymes within semipermeable shell proteins, analogous to many membrane-bound organelles. The shell proteins assemble into multimeric tiles; hexamers, trimers, and pentamers, and these tiles self-assemble into larger assemblies with icosahedral symmetry. While icosahedral shells are the predominant form in vivo , the tiles can also form nanoscale cylinders or sheets. The individual multimeric tiles feature central pores that are key to regulating transport across the protein shell. Our primary interest is to quantify pore shape changes in response to alternative component morphologies at the nanoscale. We used molecular modeling tools to develop atomically detailed models for both planar sheets of tiles and curved structures representative of the complete shells found in vivo . Subsequently, these models were animated using classical molecular dynamics simulations. From the resulting trajectories, we analyzed the overall structural stability, water accessibility to individual residues, water residence time, and pore geometry for the hexameric and trimeric protein tiles from the Haliangium ochraceu m model BMC shell. These exhaustive analyses suggest no substantial variation in pore structure or solvent accessibility between the flat and curved shell geometries. We additionally compare our analysis to hydroxyl radical footprinting data to serve as a check against our simulation results, highlighting specific residues where water molecules are bound for a long time. Although with little variation in morphology or water interaction, we propose that the planar and capsular morphology can be used interchangeably when studying permeability through BMC pores.

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

Integrative Quantitative-Phase and Airy Light-Sheet Imaging

Light-sheet microscopy enables considerable speed and phototoxicity gains, while quantitative-phase imaging confers label-free organelle recognition and metabolic information that are inaccessible by conventional methods. We report the fusion of these two modalities onto a standard inverted microscope that retains compatibility with microfluidics. We describe the utilization of an accelerating Airy-beam light-sheet yielding identical imaging areas with interferometry, and an application in unmasking the effects of cellular noise on metabolic compartmentalization.

Biological sciences, Biological techniques, Micros↗
DOE OSTI2020

Three-dimensional imaging of mitochondrial cristae complexity using cryo-soft X-ray tomography

Mitochondria are dynamic organelles that change morphology to adapt to cellular energetic demands under both physiological and stress conditions. Cardiomyopathies and neuronal disorders are associated with structure-related dysfunction in mitochondria, but three-dimensional characterizations of the organelles are still lacking. In this study, we combined high-resolution imaging and 3D electron density information provided by cryo-soft X-ray tomography to characterize mitochondria cristae morphology isolated from murine. Using the linear attenuation coefficient, the mitochondria were identified (0.247 ± 0.04 µm -1 ) presenting average dimensions of 0.90 ± 0.20 µm in length and 0.63 ± 0.12 µm in width. The internal mitochondria structure was successfully identified by reaching up the limit of spatial resolution of 35 nm. The internal mitochondrial membranes invagination (cristae) complexity was calculated by the mitochondrial complexity index (MCI) providing quantitative and morphological information of mitochondria larger than 0.90 mm in length. The segmentation to visualize the cristae invaginations into the mitochondrial matrix was possible in mitochondria with MCI ≥ 7. Altogether, we demonstrated that the MCI is a valuable quantitative morphological parameter to evaluate cristae modelling and can be applied to compare healthy and disease state associated to mitochondria morphology.

3-D reconstruction↗
DOE OSTI2020

Generation, analysis, and transformation of macro-chloroplast Potato (Solanum tuberosum) lines for chloroplast biotechnology

Abstract Chloroplast biotechnology is a route for novel crop metabolic engineering. The potential bio-confinement of transgenes, the high protein expression and the possibility to organize genes into operons represent considerable advantages that make chloroplasts valuable targets in agricultural biotechnology. In the last 3 decades, chloroplast genomes from a few economically important crops have been successfully transformed. The main bottlenecks that prevent efficient transformation in a greater number of crops include the dearth of proven selectable marker gene-selection combinations and tissue culture methods for efficient regeneration of transplastomic plants. The prospects of increasing organelle size are attractive from several perspectives, including an increase in the surface area of potential targets. As a proof-of-concept, we generated Solanum tuberosum (potato) macro-chloroplast lines overexpressing the tubulin-like GTPase protein gene FtsZ1 from Arabidopsis thaliana . Macro-chloroplast lines exhibited delayed growth at anthesis; however, at the time of harvest there was no significant difference in height between macro-chloroplast and wild-type lines. Macro-chloroplasts were successfully transformed by biolistic DNA-delivery and efficiently regenerated into homoplasmic transplastomic lines. We also demonstrated that macro-chloroplasts accumulate the same amount of heterologous protein than wild-type organelles, confirming efficient usage in plastid engineering. Advantages and limitations of using enlarge compartments in chloroplast biotechnology are discussed.

Occhialini, Alessandro (ORCID:000000021162798X)↗
DOE OSTI2025

Interior soft x-ray tomography with sparse global sampling

To investigate the feasibility of interior imaging reconstruction in soft X-ray tomography for higher-resolution cellular imaging, including whole-cell imaging, we develop an alignment and reconstruction algorithm that combines a small number of sparse whole-cell images with a high-resolution local interior scan. Based on numerical simulations, we demonstrate that combined reconstructions mitigate the depth-of-field limitation in high-resolution scans, enable radiation dose optimization, and yield quantitative X-ray absorption values with sparse sampling. We further validate our numerical approach using experimental data from two different cell types and show that the combined reconstruction reliably provides high spatial resolution within an interior region of interest of a whole cell. The resulting sparse reconstruction framework offers robust, faithful visualization of cellular organelles in soft X-ray tomography. This mesoscale imaging strategy allows one to ‘scout’ and zoom into selected subcellular volumes of interest, enabling increased spatial resolution without sacrificing larger-volume imaging and providing information on the relative positions of all organelles within a cell.

3D imaging↗
DOE OSTI2022

SEED LIPID DROPLET PROTEIN1, SEED LIPID DROPLET PROTEIN2, and LIPID DROPLET PLASMA MEMBRANE ADAPTOR mediate lipid droplet–plasma membrane tethering

Membrane contact sites (MCSs) are interorganellar connections that allow for the direct exchange of molecules, such as lipids or Ca 2+ between organelles, but can also serve to tether organelles at specific locations within cells. Here, we identified and characterized three proteins of Arabidopsis thaliana that form a lipid droplet (LD)–plasma membrane (PM) tethering complex in plant cells, namely LD-localized SEED LD PROTEIN (SLDP) 1 and SLDP2 and PM-localized LD-PLASMA MEMBRANE ADAPTOR (LIPA). Using proteomics and different protein–protein interaction assays, we show that both SLDPs associate with LIPA. Disruption of either SLDP1 and SLDP2 expression, or that of LIPA, leads to an aberrant clustering of LDs in Arabidopsis seedlings. Ectopic co-expression of one of the SLDPs with LIPA is sufficient to reconstitute LD–PM tethering in Nicotiana tabacum pollen tubes, a cell type characterized by dynamically moving LDs in the cytosolic streaming. Furthermore, confocal laser scanning microscopy revealed both SLDP2.1 and LIPA to be enriched at LD–PM contact sites in seedlings. These and other results suggest that SLDP and LIPA interact to form a tethering complex that anchors a subset of LDs to the PM during post-germinative seedling growth in Arabidopsis.

59 BASIC BIOLOGICAL SCIENCES↗