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At least 55 records · Page 3
All sourcesNASA NTRSDOE OSTI
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 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 OSTI2022

Soft X-ray tomography to map and quantify organelle interactions at the mesoscale

Inter-organelle interactions are a vital part of normal cellular function; however, these have proven difficult to quantify due to the range of scales encountered in cell biology and the throughput limitations of traditional imaging approaches. Here, we demonstrate that soft X-ray tomography (SXT) can be used to rapidly map ultrastructural reorganization and inter-organelle interactions in intact cells. SXT takes advantage of the naturally occurring, differential X-ray absorption of the carbon-rich compounds in each organelle. Specifically, we use SXT to map the spatiotemporal evolution of insulin vesicles and their co-localization and interaction with mitochondria in pancreatic β cells during insulin secretion and in response to different stimuli. We quantify changes in the morphology, biochemical composition, and relative position of mitochondria and insulin vesicles. These findings highlight the importance of a comprehensive and unbiased mapping at the mesoscale to characterize cell reorganization that would be difficult to detect with other existing methodologies.

3D cell mapping↗
DOE OSTI2023

CryoET reveals organelle phenotypes in huntington disease patient iPSC-derived and mouse primary neurons

Huntington’s disease (HD) is caused by an expanded CAG repeat in the huntingtin gene, yielding a Huntingtin protein with an expanded polyglutamine tract. While experiments with patient-derived induced pluripotent stem cells (iPSCs) can help understand disease, defining pathological biomarkers remains challenging. Here, we used cryogenic electron tomography to visualize neurites in HD patient iPSC-derived neurons with varying CAG repeats, and primary cortical neurons from BACHD, deltaN17-BACHD, and wild-type mice. In HD models, we discovered sheet aggregates in double membrane-bound organelles, and mitochondria with distorted cristae and enlarged granules, likely mitochondrial RNA granules. We used artificial intelligence to quantify mitochondrial granules, and proteomics experiments reveal differential protein content in isolated HD mitochondria. Knockdown of Protein Inhibitor of Activated STAT1 ameliorated aberrant phenotypes in iPSC- and BACHD neurons. We show that integrated ultrastructural and proteomic approaches may uncover early HD phenotypes to accelerate diagnostics and the development of targeted therapeutics for HD.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2025

A hidden cysteine in Fis1 targeted to prevent excessive mitochondrial fission and dysfunction under oxidative stress

Fis1-mediated mitochondrial localization of Drp1 and excessive mitochondrial fission occur in human pathologies associated with oxidative stress. However, it is not known how Fis1 detects oxidative stress and what structural changes in Fis1 enable mitochondrial recruitment of Drp1. We find that conformational change involving α1 helix in Fis1 exposes its only cysteine, Cys41. In the presence of oxidative stress, the exposed Cys41 in activated Fis1 forms a disulfide bridge and the Fis1 covalent homodimers cause increased mitochondrial fission through increased Drp1 recruitment to mitochondria. Our discovery of a small molecule, SP11, that binds only to activated Fis1 by engaging Cys41, and data from genetically engineered cell lines lacking Cys41 strongly suggest a role of Fis1 homodimerization in Drp1 recruitment to mitochondria and excessive mitochondrial fission. The structure of activated Fis1-SP11 complex further confirms these insights related to Cys41 being the sensor for oxidative stress. Importantly, SP11 preserves mitochondrial integrity and function in cells during oxidative stress and thus may serve as a candidate molecule for the development of treatment for diseases with underlying Fis1-mediated mitochondrial fragmentation and dysfunction.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2021

A glossary of plant cell structures: Current insights and future questions

Abstract In this glossary of plant cell structures, we asked experts to summarize a present-day view of plant organelles and structures, including a discussion of outstanding questions. In the following short reviews, the authors discuss the complexities of the plant cell endomembrane system, exciting connections between organelles, novel insights into peroxisome structure and function, dynamics of mitochondria, and the mysteries that need to be unlocked from the plant cell wall. These discussions are focused through a lens of new microscopy techniques. Advanced imaging has uncovered unexpected shapes, dynamics, and intricate membrane formations. With a continued focus in the next decade, these imaging modalities coupled with functional studies are sure to begin to unravel mysteries of the plant cell.

Biochemistry & Molecular Biology↗
DOE OSTI2021

DeepLearnMOR: a deep-learning framework for fluorescence image-based classification of organelle morphology

Abstract The proper biogenesis, morphogenesis, and dynamics of subcellular organelles are essential to their metabolic functions. Conventional techniques for identifying, classifying, and quantifying abnormalities in organelle morphology are largely manual and time-consuming, and require specific expertise. Deep learning has the potential to revolutionize image-based screens by greatly improving their scope, speed, and efficiency. Here, we used transfer learning and a convolutional neural network (CNN) to analyze over 47,000 confocal microscopy images from Arabidopsis wild-type and mutant plants with abnormal division of one of three essential energy organelles: chloroplasts, mitochondria, or peroxisomes. We have built a deep-learning framework, DeepLearnMOR (Deep Learning of the Morphology of Organelles), which can rapidly classify image categories and identify abnormalities in organelle morphology with over 97% accuracy. Feature visualization analysis identified important features used by the CNN to predict morphological abnormalities, and visual clues helped to better understand the decision-making process, thereby validating the reliability and interpretability of the neural network. This framework establishes a foundation for future larger-scale research with broader scopes and greater data set diversity and heterogeneity.

Plant Sciences↗
DOE OSTI2022

Identification of a secretory heme‐binding protein from Nocardia seriolae involved in cell apoptosis

Abstract According to the whole‐genome bioinformatics analysis, a heme‐binding protein from Nocardia seriolae (HBP) was found. HBP was predicted to be a bacterial secretory protein, located at mitochondrial membrane in eukaryotic cells and have a similar protein structure with the heme‐binding protein of Mycobacterium tuberculosis , Rv0203. In this study, HBP was found to be a secretory protein and co‐localized with mitochondria in FHM cells. Quantitative analysis of mitochondrial membrane potential value, caspase‐3 activity, and transcription level of apoptosis‐related genes suggested that overexpression of HBP protein can induce cell apoptosis. In conclusion, HBP was a secretory protein which may target to mitochondria and involve in cell apoptosis in host cells. This research will promote the function study of HBP and deepen the comprehension of the virulence factors and pathogenic mechanisms of N. seriolae .

Wen, Yiming↗
DOE OSTI2023

Structural analysis of platelet fragments and extracellular vesicles produced by apheresis platelets during storage

Platelets (PLTs) for transfusion can be stored for up to 7 days at room temperature (RT). The quality of apheresis PLTs decreases over storage time, which affects PLT hemostatic functions. Here, we characterized the membranous particles produced by PLT storage lesion (PSLPs), including degranulated PLTs, PLT ghosts, membrane fragments, and extracellular membrane vesicles (PEVs). The PSLPs generated in apheresis platelet units were analyzed on days 1, 3, 5, and 7 of RT storage. A differential centrifugation and a sucrose density gradient were used to separate PSLP populations. PSLPs were characterized using scanning and transmission electron microscopy (EM), flow cytometry (FC), and nanoparticle tracking analysis (NTA). PSLPs have different morphologies and a broad size distribution; FC and NTA showed that the concentration of small and large PSLPs increases with storage time. The density gradient separated 3 PSLP populations: (1) degranulated PLTs, PLT ghosts, and large PLT fragments; (2) PEVs originated from PLT activation and organelles released by necrotic PLTs; and (3) PEV ghosts. Most PSLPs expressed phosphatidyl serine and induced thrombin generation in the plasma. PSLPs contained extracellular mitochondria and some had the autophagosome marker LC3. PSLPs encompass degranulated PLTs, PLT ghosts, large PLT fragments, large and dense PEVs, and low-density PEV ghosts. The activation-related PSLPs are released, particularly during early stage of storage (days 1-3), and the release of apoptosis- and necrosis-related PSLPs prevails after that. No elevation of LC3- and TOM20-positive PSLPs indicates that the increase of extracellular mitochondria during later-stage storage is not associated with PLT mitophagy.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2026

Data for Protoplast Fusion as a Strategy to Increase Ploidy in Rhodotorula toruloides for Strain Development

Rhodotorula toruloides is a red oleaginous yeast with growing commercial interest because of its hardiness and exceptional lipid production capacity. Because it is a basidiomycete yeast with a complex life cycle, many of the classical breeding methods used with ascomycetes are unavailable for strain improvement. However, we have been able to construct polyploid yeast by fusing protoplasts of parents with the same mating type. Fusing of Y-6985 (A2) and Y-48190 (A2), which had been transformed with complementary antibiotic markers, led to the recovery of two diploids and one triploid. The stability of the fusion yeasts was tested by plating them on non-selective medium after several growth cycles under antibiotics and then testing five colonies per strain for nuclear DNA contents using flow cytometry and standard cell cycle analysis: the triploid and one diploid were stable. Fusants inherited their mitochondria from a single parent, which was demonstrated using restriction fragment length polymorphism (RFLP) of mitochondrial DNA. The phenotypic properties of the parents and fusants were compared in glucose fed-batch bioreactor studies and cellulosic sugar batch cultures. The final lipid titers for the fed-batch cultures were 24.9–39.7 g/L with Y-6985 and the diploid and triploid performing the best and worst, respectively. The fusants demonstrated intermediate hardiness for growth on hydrolysate prepared with dilute-acid pretreated switchgrass and were outperformed by Y-48190. Unlike one of the haploid parents, the fusants grew in 70% v/v concentrated hydrolysate. However, they did not grow as fast as the other haploid. In this study, a modernized protoplast fusion method is resurrected a useful tool for strain development in this yeast, which is complementary with other available methods.

FOS: Biological sciences↗
DOE OSTI2021

Arabidopsis thaliana EARLY RESPONSIVE TO DEHYDRATION 7 Localizes to Lipid Droplets via Its Senescence Domain

Lipid droplets (LDs) are neutral-lipid-containing organelles found in all kingdoms of life and are coated with proteins that carry out a vast array of functions. Compared to mammals and yeast, relatively few LD proteins have been identified in plants, particularly those associated with LDs in vegetative (non-seed) cell types. Thus, to better understand the cellular roles of LDs in plants, a more comprehensive inventory and characterization of LD proteins is required. Here, we performed a proteomics analysis of LDs isolated from drought-stressed Arabidopsis leaves and identified EARLY RESPONSIVE TO DEHYDRATION 7 (ERD7) as a putative LD protein. mCherry-tagged ERD7 localized to both LDs and the cytosol when ectopically expressed in plant cells, and the protein’s C-terminal senescence domain (SD) was both necessary and sufficient for LD targeting. Phylogenetic analysis revealed that ERD7 belongs to a six-member family in Arabidopsis that, along with homologs in other plant species, is separated into two distinct subfamilies. Notably, the SDs of proteins from each subfamily conferred targeting to either LDs or mitochondria. Further, the SD from the ERD7 homolog in humans, spartin, localized to LDs in plant cells, similar to its localization in mammals; although, in mammalian cells, spartin also conditionally localizes to other subcellular compartments, including mitochondria. Disruption of ERD7 gene expression in Arabidopsis revealed no obvious changes in LD numbers or morphology under normal growth conditions, although this does not preclude a role for ERD7 in stress-induced LD dynamics. Consistent with this possibility, a yeast two-hybrid screen using ERD7 as bait identified numerous proteins involved in stress responses, including some that have been identified in other LD proteomes. Collectively, these observations provide new insight to ERD7 and the SD-containing family of proteins in plants and suggest that ERD7 may be involved in functional aspects of plant stress response that also include localization to the LD surface.

54 ENVIRONMENTAL 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 OSTI2022

Legionella pneumophila modulates host energy metabolism by ADP-ribosylation of ADP/ATP translocases

The intracellular pathogen Legionella pneumophila delivers more than 330 effectors into host cells by its Dot/Icm type IV secretion system. Those effectors are essential for the biogenesis of the Legionella-containing vacuole (LCV) that permits its intracellular survival and replication. It has long been documented that the LCV is associated with mitochondria and a number of Dot/Icm effectors have been shown to target to this organelle. Yet, the biochemical function and host cell target of most of these effectors remain unknown. Here, we found that the Dot/Icm substrate Ceg3 (Lpg0080) is a mono-ADP-ribosyltransferase that localizes to the mitochondria in host cells where it attacks ADP/ATP translocases (ANTs) by ADP-ribosylation and blunts their ADP/ATP exchange activity. The modification occurs on the second arginine residue in the -RRRMMM- element, which is conserved among all known ADP/ATP carriers from different organisms. Our results reveal modulation of host energy metabolism as a novel virulence mechanism for L. pneumophila.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2020

Mitochondrial apoptotic sensor

Mitochondria are central to the intrinsic apoptotic pathway of programmed cell death. Commitment to cell death occurs upon mitochondrial outer membrane permeabilization, and dysregulation of this vital apoptotic control point is implicated in various pathologies. Provided herein are novel sensors which can detect, in real time and with great sensitivity, the release of protons from intact mitochondria caused by mitochondrial outer membrane permeabilization. Embodiments include graphene based ion-sensitive field effect transistors.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2021

Identification of protein/mRNA network involving the PSORS1 locus gene CCHCR1 and the PSORS4 locus gene HAX1

Highlights: • CCHCR1 and HAX1 proteins directly interact. • CCHCR1 is a psoriasis candidate gene located on PSORS1 locus. • HAX1 gene is located on PSORS4 locus and is overexpressed in psoriasis. • CCHCR1 and HAX1 co-localize with and affect specific mRNAs in vesicles. • Novel protein/mRNA networks involving CCHCR1 and HAX1 could play a role in psoriasis. CCHCR1 (Coiled-Coil alpha-Helical Rod 1), maps to chromosomal region 6p21.3, within the major psoriasis susceptibility locus PSORS1. CCHCR1 itself is a plausible psoriasis candidate gene, however its role in psoriasis pathogenesis remains unclear. We previously demonstrated that CCHCR1 protein acts as a cytoplasmic docking site for RNA polymerase II core subunit 3 (RPB3) in cycling cells, suggesting a role for CCHCR1 in vesicular trafficking between cellular compartments. Here, we report a novel interaction between CCHCR1 and the RNA binding protein HAX1. HAX1 maps to chromosomal region 1q21.3 within the PSORS4 locus and is over-expressed in psoriasis. Both CCHCR1 and HAX1 share subcellular co-localization with mitochondria, nuclei and cytoplasmic vesicles as P-bodies. By a series of ribonucleoprotein immunoprecipitation (RIP) assays, we isolated a pool of mRNAs complexed with HAX1 and/or CCHCR1 proteins. Among the mRNAs complexed with both CCHCR1 and HAX1 proteins, there are Vimentin mRNA, previously described to be bound by HAX1, and CAMP/LL37 mRNA, whose gene product is over-expressed in psoriasis.

60 APPLIED LIFE SCIENCES↗
DOE OSTI2021

Cellular metabolic stress responses via organelles

Metabolite fluctuations following nutrient metabolism or environmental stresses impact various intracellular signaling networks and stress responses to maintain cellular and organismal homeostasis. It has been shown that subcellular organelles, such as the endoplasmic reticulum, the Golgi apparatus, lysosomes and mitochondria serve as crucial hubs linking alterations in metabolite levels to cellular responses. This role is coordinated by molecular machineries that are associated with the lipid membranes of organelles, which sense the fluctuations in specific metabolites and activate the appropriate signaling and effector molecules. Moreover, recent studies have demonstrated that membraneless organelles, such as the nucleolus and stress granules, are involved in the metabolic stress response. Metabolite-induced post-translational modifications appear to play an important role in this process. Here, we review the molecular mechanisms of metabolite sensing and metabolite-mediated stress responses through membrane-bound and membraneless organelles in mammalian cells.

60 APPLIED LIFE SCIENCES↗
DOE OSTI2021

Mitoferrin 2 deficiency prevents mitochondrial iron overload-induced endothelial injury and alleviates atherosclerosis

Endothelial dysfunction is an early step in the development of atherosclerotic cardiovascular disease. Iron overload can lead to excessive mitochondrial reactive oxygen species (mtROS) production, resulting in mitochondrial dysfunction and vascular endothelial cell (EC) damage. Mitoferrin 2 (Mfrn2) is an iron transporter in the inner mitochondrial membrane. This study aimed to assess whether Mfrn2 and mitochondrial iron overload were involved in atherosclerosis progression and to explore the potential mechanism. We observed significant upregulation of Mfrn2 in the arteries of high-fat diet (HFD)-fed Apolipoprotein E{sup −/-} (ApoE{sup −/-}) mice and in TNF-α-induced mouse aortic endothelial cells (MAECs). Mfrn2 gene silencing inhibited mitochondrial iron overload, stabilized mitochondrial membrane potential and improved mitochondrial function in TNF-α-induced MAECs. Vascular EC-specific knockdown of Mfrn2 in ApoE{sup −/-} mice markedly decreased atherosclerotic lesion formation and the levels of ICAM-1 in aortas and reduced monocyte infiltration into the vascular wall. Furthermore, TNF-α increased the binding of 14-3-3 epsilon (ε) and Mfrn2, preventing Mfrn2 degradation and leading to mitochondrial iron overload in ECs, while 14-3-3ε overexpression increased Mfrn2 stability by inhibiting its ubiquitination. Together, our results reveal that Mfrn2 deficiency attenuates endothelial dysfunction by decreasing iron levels within the mitochondria and mitochondrial dysfunction. These findings may provide new insights into preventive and therapeutic strategies against vascular endothelial dysfunction in atherosclerotic disease.

60 APPLIED LIFE SCIENCES↗