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Results for “cellular structures and organelles”

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 19 records
All sourcesNASA NTRSDOE OSTI
DOE OSTI2022

An intensity-based post-processing tool for 3D instance segmentation of organelles in soft X-ray tomograms

Investigating the 3D structures and rearrangements of organelles within a single cell is critical for better characterizing cellular function. Imaging approaches such as soft X-ray tomography have been widely applied to reveal a complex subcellular organization involving multiple inter-organelle interactions. However, 3D segmentation of organelle instances has been challenging despite its importance in organelle characterization. Here we propose an intensity-based post-processing tool to identify and separate organelle instances. Our tool separates sphere-like (insulin vesicle) and columnar-shaped organelle instances (mitochondrion) based on the intensity of raw tomograms, semantic segmentation masks, and organelle morphology. We validate our tool using synthetic tomograms of organelles and experimental tomograms of pancreatic β -cells to separate insulin vesicle and mitochondria instances. As compared to the commonly used connected regions labeling, watershed, and watershed + Gaussian filter methods, our tool results in improved accuracy in identifying organelles in the synthetic tomograms and an improved description of organelle structures in β -cell tomograms. In addition, under different experimental treatment conditions, significant changes in volumes and intensities of both insulin vesicle and mitochondrion are observed in our instance results, revealing their potential roles in maintaining normal β -cell function. Our tool is expected to be applicable for improving the instance segmentation of other images obtained from different cell types using multiple imaging modalities.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2025

Soft X-Ray Tomography Has Evolved into a Powerful Tool for Revealing Cell Structures

Over the past three decades, soft X-ray tomography (SXT) has rapidly evolved from a proof-of-concept microscopy method into a high-throughput quantitative imaging modality. This advancement enables researchers to address central questions in cell biology. Despite its relatively short developmental period compared to light and electron microscopy, SXT has emerged as a powerful imaging technology. It enables measuring chemical changes in cellular organelles, analyzing three-dimensional structures of whole cells and creating digital cellular models to study cell motility. We discuss the unique nature of SXT to visualize cells without fixation or labeling, enabling quantitative analyses of organelle chemical composition. We explore SXT microscopes available worldwide, SXT segmentation software, and the diverse cell types studied using this technique. We conclude with emerging directions in SXT imaging, including a brief discussion of recent discoveries that are highly influential and likely to become integral to cell biology textbooks.

Weinhardt, Venera↗
DOE OSTI2024

Correlative single-cell hard X-ray computed tomography and X-ray fluorescence imaging

Abstract X-ray computed tomography (XCT) and X-ray fluorescence (XRF) imaging are two non-invasive imaging techniques to study cellular structures and chemical element distributions, respectively. However, correlative X-ray computed tomography and fluorescence imaging for the same cell have yet to be routinely realized due to challenges in sample preparation and X-ray radiation damage. Here we report an integrated experimental and computational workflow for achieving correlative multi-modality X-ray imaging of a single cell. The method consists of the preparation of radiation-resistant single-cell samples using live-cell imaging-assisted chemical fixation and freeze-drying procedures, targeting and labeling cells for correlative XCT and XRF measurement, and computational reconstruction of the correlative and multi-modality images. With XCT, cellular structures including the overall structure and intracellular organelles are visualized, while XRF imaging reveals the distribution of multiple chemical elements within the same cell. Our correlative method demonstrates the feasibility and broad applicability of using X-rays to understand cellular structures and the roles of chemical elements and related proteins in signaling and other biological processes.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2024

Time-resolved cryogenic electron tomography for the study of transient cellular processes

Cryogenic electron tomography (cryo-ET) is the highest resolution imaging technique applicable to the life sciences, enabling sub-nanometer visualization of specimens preserved in their near native states. The rapid plunge freezing process used to prepare samples lends itself to time-resolved studies, which researchers have pursued for in vitro samples for decades. Here, we focus on developing a freezing apparatus for time-resolved studies in situ. The device mixes cellular samples with solution-phase stimulants before spraying them directly onto an electron microscopy grid that is transiting into cryogenic liquid ethane. By varying the flow rates of cell and stimulant solutions within the device, we can control the reaction time from tens of milliseconds to over a second prior to freezing. In a proof-of-principle demonstration, the freezing method is applied to a model bacterium, Caulobacter crescentus, mixed with an acidic buffer. Through cryo-ET we resolved structural changes throughout the cell, including surface-layer protein dissolution, outer membrane deformation, and cytosolic rearrangement, all within 1.5 seconds of reaction time. This new approach, Time-Resolved cryo-ET (TR-cryo-ET), enhances the capabilities of cryo-ET by incorporating a sub-second temporal axis and enables the visualization of induced structural changes at the molecular, organelle, or cellular level.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2020

The plant endoplasmic reticulum: an organized chaos of tubules and sheets with multiple functions

The endoplasmic reticulum is a fascinating organelle at the core of the secretory pathway. It is responsible for the synthesis of one third of the cellular proteome and, in plant cells, it produces receptors and transporters of hormones as well as the proteins responsible for the biosynthesis of critical components of a cellulosic cell wall. The endoplasmic reticulum structure resembles a spider-web network of interconnected tubules and cisternae that pervades the cell. The study of the dynamics and interaction of this organelles with other cellular structures such as the plasma membrane, the Golgi apparatus and the cytoskeleton, have been permitted by the implementation of fluorescent protein and advanced confocal imaging. In this review,we report on the findings that contributed towards the understanding of the endoplasmic reticulum morphology and function with the aid of fluorescent proteins, focusing on the contributions provided by pioneering work from the lab of the late Professor Chris Hawes.

59 BASIC BIOLOGICAL SCIENCES↗
NASA NTRS1985

A morphometric analysis of cellular differentiation in caps of primary and lateral roots of Helianthus annuus

In order to determine if patterns of cell differentiation are similar in primary and lateral roots, I performed a morphometric analysis of the ultrastructure of calyptrogen, columella, and peripheral cells in primary and lateral roots of Helianthus annuus. Each cell type is characterized by a unique ultrastructure, and the ultrastructural changes characteristic of cellular differentiation in root caps are organelle specific. No major structural differences exist in the structures of the composite cell types, or in patterns of cell differentiation in caps of primary vs. lateral roots.

Non-NASA Center↗
NASA NTRS2003

Molecular Control of Cell Growth During Gravity Responses of Maize Seedlings

Gravity influences plants in many ways via its physical effects on the convective flows of gases and liquids, the buoyancy and sedimentation of cellular organelles, and the distribution of mechanical stresses in weight-bearing structures. These physical effects lead to a variety of reactions and adaptive developmental responses in plants. Perhaps the best-studied plant gravity response is gravitropism - the "homing in" of growing organs towards a particular angle with respect to gravity. Most plants respond to gravity by gravitropic bending of roots downwards and stems upwards. Such gravitropic bending arises from differential cell growth on the two sides of the bending organ. For this project we hypothesized that such growth differences arise from differences in expansin activity, which come about because of organ-level asymmetries of H+ efflux and expansin export to the wall.

Cosgrove, Daniel J.↗
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↗
NASA NTRS1986

A morphometric analysis of the redistribution of organelles in columella cells of horizontally-oriented roots of Zea mays

In order to determine what structural changes in graviperceptive cells are associated with onset of root gravicurvature, the redistribution of organelles in columella cells of horizontally-oriented, graviresponding roots of Zea mays has been quantified. Root gravicurvature began by 15 min after reorientation, and did not involve significant changes in the (i) volume of individual columella cells or amyloplasts, (ii) relative volume of any cellular organelle, (iii) number of amyloplasts per columella cell, or (iv) surface area of cellular location of endoplasmic reticulum. Sedimentation of amyloplasts began within 1 to 2 min after reorientation, and was characterized by an intensely staining area of cytoplasm adjacent to the sedimenting amyloplasts. By 5 min after reorientation, amyloplasts were located in the lower distal corner of columella cells, and, by 15 min after reorientation, overlaid the entire length of the lower cell wall. No consistent contact between amyloplasts and any cellular structure was detected at any stage of gravicurvature. Centrally-located nuclei initially migrated upward in columella cells of horizontally-oriented roots, after which they moved to the proximal ends of the cells by 15 min after reorientation. No significant pattern of redistribution of vacuoles, mitochondria, dictyosomes, or hyaloplasm was detected that correlated with the onset of gravicurvature. These results indicate that amyloplasts and nuclei are the only organelles whose movements correlate positively with the onset of gravicurvature by primary roots of this cultivar of Zea mays.

NASA Discipline Plant Biology↗
DOE OSTI2021

Assembly and Analysis of Cell-Scale Membrane Envelopes

The march toward exascale computing will enable routine molecular simulation of larger and more complex systems, for example, simulation of entire viral particles, on the scale of approximately billions of atoms-a simulation size commensurate with a small bacterial cell. Anticipating the future hardware capabilities that will enable this type of research and paralleling advances in experimental structural biology, efforts are currently underway to develop software tools, procedures, and workflows for constructing cell-scale structures. Herein, we describe our efforts in developing and implementing an efficient and robust workflow for construction of cell-scale membrane envelopes and embedding membrane proteins into them. A new approach for construction of massive membrane structures that are stable during the simulations is built on implementing a subtractive assembly technique coupled with the development of a structure concatenation tool (fastmerge), which eliminates overlapping elements based on volumetric criteria rather than adding successive molecules to the simulation system. Using this approach, we have constructed two "protocells" consisting of MARTINI coarse-grained beads to represent cellular membranes, one the size of a cellular organelle and another the size of a small bacterial cell. The membrane envelopes constructed here remain whole during the molecular dynamics simulations performed and exhibit water flux only through specific proteins, demonstrating the success of our methodology in creating tight cell-like membrane compartments. Extended simulations of these cell-scale structures highlight the propensity for nonspecific interactions between adjacent membrane proteins leading to the formation of protein microclusters on the cell surface, an insight uniquely enabled by the scale of the simulations. We anticipate that the experiences and best practices presented here will form the basis for the next generation of cell-scale models, which will begin to address the addition of soluble proteins, nucleic acids, and small molecules essential to the function of a cell.

37 INORGANIC, ORGANIC, PHYSICAL, AND ANALYTICAL CH↗
NASA NTRS1987

Influence of microgravity on cellular differentiation in root caps of Zea mays

We launched imbibed seeds of Zea mays into outer space aboard the space shuttle Columbia to determine the influence of microgravity on cellular differentiation in root caps. The influence of microgravity varied with different stages of cellular differentiation. Overall, microgravity tended to 1) increase relative volumes of hyaloplasm and lipid bodies, 2) decrease the relative volumes of plastids, mitochondria, dictyosomes, and the vacuome, and 3) exert no influence on the relative volume of nuclei in cells comprising the root cap. The reduced allocation of dictyosomal volume in peripheral cells of flight-grown seedlings correlated positively with their secretion of significantly less mucilage than peripheral cells of Earth-grown seedlings. These results indicate that 1) microgravity alters the patterns of cellular differentiation and structures of all cell types comprising the root cap, and 2) the influence of microgravity on cellular differentiation in root caps of Zea mays is organelle specific.

NASA Program Space Biology↗
DOE OSTI2020

Understanding the Structure and Dynamics of Complex Biomembrane Interactions by Neutron Scattering Techniques

The membrane is one of the key structural materials of biology at the cellular level. Composed predominantly of a bilayer of lipids with embedded and bound proteins, it defines the boundaries of the cell and many organelles essential to life and therefore is involved in almost all biological processes. Membrane-specific interactions, such as drug binding to a membrane receptor or the interactions of an antimicrobial compound with the lipid matrix of a pathogen membrane, are of interest across the scientific disciplines. Herein we present a review, aimed at nonexperts, of the major neutron scattering techniques used in membrane studies: small-angle neutron scattering, neutron membrane diffraction, neutron reflectometry, quasielastic neutron scattering, and neutron spin echo. Neutron scattering techniques are well suited to studying biological membranes. The nondestructive nature of cold neutrons means that samples can be measured for long periods without fear of beam damage from ultraviolet, electron, or X-ray radiation, and neutron beams are highly penetrating, thus offering flexibility in samples and sample environments. Most important is the strong difference in neutron scattering lengths between the two most abundant forms of hydrogen, protium and deuterium. Changing the relative amounts of protium/deuterium in a sample allows the production of a series of neutron scattering data sets, enabling the observation of differing components within complex membrane architectures. This approach can be as simple as using the naturally occurring neutron contrast between different biomolecules to study components in a complex by changing the solution H 2 O/D 2 O ratio or as complex as selectively labeling individual components with hydrogen isotopes. This review presents an overview of each experimental technique with the neutron instrument configuration, related sample preparation and sample environment, and data analysis, highlighted by a special emphasis on using prominent neutron contrast to understand structure and dynamics. This review gives researchers a practical introduction to the often enigmatic suite of neutron beamlines, thereby lowering the barrier to taking advantage of these large-facility techniques to achieve new understandings of membranes and their interactions with other molecules.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2024

Functional in vitro diversity of an intrinsically disordered plant protein during freeze–thawing is encoded by its structural plasticity

Intrinsically disordered late embryogenesis abundant (LEA) proteins play a central role in the tolerance of plants and other organisms to dehydration brought upon, for example, by freezing temperatures, high salt concentration, drought or desiccation, and many LEA proteins have been found to stabilize dehydration-sensitive cellular structures. Their conformational ensembles are highly sensitive to the environment, allowing them to undergo conformational changes and adopt ordered secondary and quaternary structures and to participate in formation of membraneless organelles. In an interdisciplinary approach, we discovered how the functional diversity of the Arabidopsis thaliana LEA protein COR15A found in vitro is encoded in its structural repertoire, with the stabilization of membranes being achieved at the level of secondary structure and the stabilization of enzymes accomplished by the formation of oligomeric complexes. We provide molecular details on intra- and inter-monomeric helix–helix interactions, demonstrate how oligomerization is driven by an α-helical molecular recognition feature (α-MoRF) and provide a rationale that the formation of noncanonical, loosely packed, right-handed coiled-coils might be a recurring theme for homo- and hetero-oligomerization of LEA proteins.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2024

Packaging “vegetable oils”: Insights into plant lipid droplet proteins

Abstract Plant neutral lipids, also known as “vegetable oils”, are synthesized within the endoplasmic reticulum (ER) membrane and packaged into subcellular compartments called lipid droplets (LDs) for stable storage in the cytoplasm. The biogenesis, modulation, and degradation of cytoplasmic LDs in plant cells are orchestrated by a variety of proteins localized to the ER, LDs, and peroxisomes. Recent studies of these LD-related proteins have greatly advanced our understanding of LDs not only as steady oil depots in seeds but also as dynamic cell organelles involved in numerous physiological processes in different tissues and developmental stages of plants. In the past 2 decades, technology advances in proteomics, transcriptomics, genome sequencing, cellular imaging and protein structural modeling have markedly expanded the inventory of LD-related proteins, provided unprecedented structural and functional insights into the protein machinery modulating LDs in plant cells, and shed new light on the functions of LDs in nonseed plant tissues as well as in unicellular algae. Here, we review critical advances in revealing new LD proteins in various plant tissues, point out structural and mechanistic insights into key proteins in LD biogenesis and dynamic modulation, and discuss future perspectives on bridging our knowledge gaps in plant LD biology.

Cai, Yingqi (ORCID:0000000203575809)↗
DOE OSTI2024

Subcellular Feature-Based Classification of α and β Cells Using Soft X-ray Tomography

The dysfunction of α and β cells in pancreatic islets can lead to diabetes. Many questions remain on the subcellular organization of islet cells during the progression of disease. Existing three-dimensional cellular mapping approaches face challenges such as time-intensive sample sectioning and subjective cellular identification. To address these challenges, we have developed a subcellular feature-based classification approach, which allows us to identify α and β cells and quantify their subcellular structural characteristics using soft X-ray tomography (SXT). We observed significant differences in whole-cell morphological and organelle statistics between the two cell types. Additionally, we characterize subtle biophysical differences between individual insulin and glucagon vesicles by analyzing vesicle size and molecular density distributions, which were not previously possible using other methods. These sub-vesicular parameters enable us to predict cell types systematically using supervised machine learning. We also visualize distinct vesicle and cell subtypes using Uniform Manifold Approximation and Projection (UMAP) embeddings, which provides us with an innovative approach to explore structural heterogeneity in islet cells. This methodology presents an innovative approach for tracking biologically meaningful heterogeneity in cells that can be applied to any cellular system.

3D cell mapping↗
DOE OSTI2023

A chloroplast protein atlas reveals punctate structures and spatial organization of biosynthetic pathways

Chloroplasts are eukaryotic photosynthetic organelles that drive the global carbon cycle. Despite their importance, our understanding of their protein composition, function, and spatial organization remains limited. Here, we determined the localizations of 1,034 candidate chloroplast proteins using fluorescent protein tagging in the model alga Chlamydomonas reinhardtii. The localizations provide insights into the functions of poorly characterized proteins; identify novel components of nucleoids, plastoglobules, and the pyrenoid; and reveal widespread protein targeting to multiple compartments. We discovered and further characterized cellular organizational features, including eleven chloroplast punctate structures, cytosolic crescent structures, and unexpected spatial distributions of enzymes within the chloroplast. We also used machine learning to predict the localizations of other nuclear-encoded Chlamydomonas proteins. The strains and localization atlas developed here will serve as a resource to accelerate studies of chloroplast architecture and functions.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2025

Automated segmentation of soft X-ray tomography: Native cellular structure with submicron resolution at high-throughput for whole-cell quantitative imaging in yeast

Soft X-ray tomography (SXT) is an invaluable tool for quantitatively analyzing cellular structures at suboptical isotropic resolution. However, it has traditionally depended on manual segmentation, limiting its scalability for large datasets. Here, we leverage a deep learning-based autosegmentation pipeline to segment and label cellular structures in hundreds of cells across three Saccharomyces cerevisiae strains. This task-based pipeline uses manual iterative refinement to improve segmentation accuracy for key structures, including the cell body, nucleus, vacuole, and lipid droplets, enabling high-throughput and precise phenotypic analysis. Using this approach, we quantitatively compared the three-dimensional (3D) whole-cell morphometric characteristics of wild-type, VPH1-GFP, and vac14 strains, uncovering detailed strain-specific cell and organelle size and shape variations. We show the utility of SXT data for precise 3D curvature analysis of entire organelles and cells and detection of fine morphological features using surface meshes. Our approach facilitates comparative analyses with high spatial precision and statistical throughput, uncovering subtle morphological features at the single-cell and population level. This workflow significantly enhances our ability to characterize cell anatomy and supports scalable studies on the mesoscale, with applications in investigating cellular architecture, organelle biology, and genetic research across diverse biological contexts.

Chen, Jianhua [Lawrence Berkeley National Laborato↗