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At least 73 records · Page 4

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NASA NTRS2005

How actin crosslinking and bundling proteins cooperate to generate an enhanced cell mechanical response

Actin-crosslinking proteins organize actin filaments into dynamic and complex subcellular scaffolds that orchestrate important mechanical functions, including cell motility and adhesion. Recent mutation studies have shown that individual crosslinking proteins often play seemingly non-essential roles, leading to the hypothesis that they have considerable redundancy in function. We report live-cell, in vitro, and theoretical studies testing the mechanical role of the two ubiquitous actin-crosslinking proteins, alpha-actinin and fascin, which co-localize to stress fibers and the basis of filopodia. Using live-cell particle tracking microrheology, we show that the addition of alpha-actinin and fascin elicits a cell mechanical response that is significantly greater than that originated by alpha-actinin or fascin alone. These live-cell measurements are supported by quantitative rheological measurements with reconstituted actin filament networks containing pure proteins that show that alpha-actinin and fascin can work in concert to generate enhanced cell stiffness. Computational simulations using finite element modeling qualitatively reproduce and explain the functional synergy of alpha-actinin and fascin. These findings highlight the cooperative activity of fascin and alpha-actinin and provide a strong rationale that an evolutionary advantage might be conferred by the cooperative action of multiple actin-crosslinking proteins with overlapping but non-identical biochemical properties. Thus the combination of structural proteins with similar function can provide the cell with unique properties that are required for biologically optimal responses.

Non-NASA Center↗

NASA NTRS2005

Pre-biotic stage of life origin under non-photosynthetic conditions

Spontaneous assembling of a simplest bacterial cell even if all necessary molecules are present in a solution seems to be extremely rare event and from the scientific standpoint has to be considered as impossible. Therefore, a predecessor of a living cell has to be very simple for providing its self-assembling and at the same time it should be able of progressive increase in complexity. Now phase-separated particles, first of all micelles, are put forward as possible predecessors of living cell. According to the offered working concept only phase-separated particles possessing autocatalytic properties can be considered as predecessors of living cells. The first stage of evolution of these phase-separated autocatalytic systems is the appearance of pre-biotic metabolism providing synthesis of amphiphiles for formation of capsules of these systems. This synthesis is maintained by the energy of a base reaction being a component of a planet-chemical cycle. Catalytic system providing functioning of pre-biotic metabolism is based on multivariate oligomeric autocatalyst, which reproduces itself from monomers, penetrating the particles from the outside. Since the autocatalyst realizes random polymerization then a collection of other oligomers possessing different catalytic functions is produced. In the paper the functioning of multivariate oligomeric autocatalyst in flow reactor is analyzed. c2005 Published by Elsevier Ltd on behalf of COSPAR.

Biogenesis↗

DOE OSTI2024

Non-Invasive Biophysical Techniques to Monitor the Structural Plasticity of the Photosynthetic Machinery of Live Diatom Cells

The photosynthetic performance of diatoms depends largely on the organization and structural flexibility of their thylakoid membranes, the densely packed, highly organized membrane vesicles in which light reactions of photosynthesis occur. Different regulatory mechanisms that fine tune the photosynthetic functions affect the organization of the photosynthetic machinery at different levels of structural complexity, from the level of individual protein complexes to the macroarray of membrane proteins and the remodeling of the entire thylakoid membrane system. To monitor these reorganizations, non-invasive techniques are of special value. In this chapter, we focus our attention on three of these techniques, which have been demonstrated to provide unique and useful information on the structure and structural and functional plasticity of live diatom cells: (i) circular dichroism (CD) spectroscopy, which has provided unique information on the chiral (macro-)organization of protein complexes and on their rapid, reversible reorganizations, fine-tuning the light-harvesting processes, as well as on variations in the short-range excitonic interactions in the antenna complexes; (ii) small-angle neutron scattering (SANS), which has been used to determine the periodic organization of the thylakoid membranes and to monitor reversible ultrastructural changes on the time-scale of minutes, induced by variations in the environmental conditions such as changes in temperature or light intensity; and (iii) electrochromic shift absorbance transients (ΔA ECS ), a spectroscopic tool which has been shown to be capable of identifying distinct functional groups of the light-harvesting carotenoid fucoxanthin in different diatoms and in cells exposed to different light intensities. Future use of these techniques will most certainly contribute to the deeper understanding of key regulatory mechanisms of photosynthesis in diatoms.

Szabo, Milan↗

DOE OSTI2020

Switchable resolution in soft x-ray tomography of single cells

The diversity of living cells, in both size and internal complexity, calls for imaging methods with adaptable spatial resolution. Soft x-ray tomography (SXT) is a three-dimensional imaging technique ideally suited to visualizing and quantifying the internal organization of single cells of varying sizes in a near-native state. The achievable resolution of the soft x-ray microscope is largely determined by the objective lens, but switching between objectives is extremely time-consuming and typically undertaken only during microscope maintenance procedures. Since the resolution of the optic is inversely proportional to the depth of focus, an optic capable of imaging the thickest cells is routinely selected. This unnecessarily limits the achievable resolution in smaller cells and eliminates the ability to obtain high-resolution images of regions of interest in larger cells. Here, we describe developments to overcome this shortfall and allow selection of microscope optics best suited to the specimen characteristics and data requirements. We demonstrate that switchable objective capability advances the flexibility of SXT to enable imaging cells ranging in size from bacteria to yeast and mammalian cells without physically modifying the microscope, and we demonstrate the use of this technology to image the same specimen with both optics.

59 BASIC BIOLOGICAL SCIENCES↗

NASA NTRS2016

Chapter 7: Fluorescence Imaging of the Cytoskeleton in Plant Roots

During the past two decades the use of live cytoskeletal probes has increased dramatically due to the introduction of the green fluorescent protein. However, to make full use of these live cell reporters it is necessary to implement simple methods to maintain plant specimens in optimal growing conditions during imaging. To image the cytoskeleton in living Arabidopsis roots, we rely on a system involving coverslips coated with nutrient supplemented agar where the seeds are directly germinated. This coverslip system can be conveniently transferred to the stage of a confocal microscope with minimal disturbance to the growth of the seedling. For roots with a larger diameter such as Medicago truncatula, seeds are first germinated in moist paper, grown vertically in between plastic trays, and roots mounted on glass slides for confocal imaging. Parallel with our live cell imaging approaches, we routinely process fixed plant material via indirect immunofluorescence. For these methods we typically use non-embedded vibratome-sectioned and whole mount permeabilized root tissue. The clearly defined developmental regions of the root provide us with an elegant system to further understand the cytoskeletal basis of plant development.

Actin↗

DOE OSTI2024

Macroscale structural changes of thylakoid architecture during high light acclimation in Chlamydomonas reinhardtii

Abstract Photoprotection mechanisms are ubiquitous among photosynthetic organisms. The photoprotection capacity of the green alga Chlamydomonas reinhardtii is correlated with protein levels of stress-related light-harvesting complex (LHCSR) proteins, which are strongly induced by high light (HL). However, the dynamic response of overall thylakoid structure during acclimation to growth in HL has not been fully understood. Here, we combined live-cell super-resolution microscopy and analytical membrane subfractionation to investigate macroscale structural changes of thylakoid membranes during HL acclimation in Chlamydomonas . Subdiffraction-resolution live-cell imaging revealed that the overall thylakoid structures became thinned and shrunken during HL acclimation. The stromal space around the pyrenoid also became enlarged. Analytical density-dependent membrane fractionation indicated that the structural changes were partly a consequence of membrane unstacking. The analysis of both an LHCSR loss-of-function mutant , npq4 lhcsr1 , and a regulatory mutant that over-expresses LHCSR, spa1-1 , showed that structural changes occurred independently of LHCSR protein levels, demonstrating that LHCSR was neither necessary nor sufficient to induce the thylakoid structural changes associated with HL acclimation. In contrast, stt7-9 , a mutant lacking a kinase of major light-harvesting antenna proteins, had a slower thylakoid structural response to HL relative to all other lines tested but still showed membrane unstacking. These results indicate that neither LHCSR- nor antenna-phosphorylation-dependent HL acclimation are required for the observed macroscale structural changes of thylakoid membranes in HL conditions.

59 BASIC BIOLOGICAL SCIENCES↗

DOE OSTI2021

A Palette of Minimally Tagged Sucrose Analogues for Real-Time Raman Imaging of Intracellular Plant Metabolism

Sucrose is the main saccharide used for longdistance transport in plants and plays an essential role in energy metabolism; however, there are no analogues for real-time imaging in live cells. We have optimised a synthetic approach to prepare sucrose analogues including very small ( 50 Da or less) Raman tags in the fructose moiety. Spectroscopic analysis identified the alkyne-tagged compound 6 as a sucrose analogue recognised by endogenous transporters in live cells and with higher Raman intensity than other sucrose derivatives. Herein, we demonstrate the application of compound 6 as the first optical probe to visualise real-time uptake and intracellular localisation of sucrose in live plant cells using Raman microscopy.

60 APPLIED LIFE SCIENCES↗

DOE OSTI2023

Engineered molecular sensors for quantifying cell surface crowding

Cells mediate interactions with the extracellular environment through a crowded assembly of transmembrane proteins, glycoproteins and glycolipids on their plasma membrane. The extent to which surface crowding modulates the biophysical interactions of ligands, receptors, and other macromolecules is poorly understood due to the lack of methods to quantify surface crowding on native cell membranes. In this work, we demonstrate that physical crowding on reconstituted membranes and live cell surfaces attenuates the effective binding affinity of macromolecules such as IgG antibodies in a surface crowding-dependent manner. We combine experiment and simulation to design a crowding sensor based on this principle that provides a quantitative readout of cell surface crowding. Our measurements reveal that surface crowding decreases IgG antibody binding by 2 to 20 fold in live cells compared to a bare membrane surface. Our sensors show that sialic acid, a negatively charged monosaccharide, contributes disproportionately to red blood cell surface crowding via electrostatic repulsion, despite occupying only ~1% of the total cell membrane by mass. We also observe significant differences in surface crowding for different cell types and find that expression of single oncogenes can both increase and decrease crowding, suggesting that surface crowding may be an indicator of both cell type and state. Our high-throughput, single-cell measurement of cell surface crowding may be combined with functional assays to enable further biophysical dissection of the cell surfaceome.

60 APPLIED LIFE SCIENCES↗

NASA NTRS1977

The development of a potassium-sulfide glass fiber cell and studies on impurities in alkali metal-sulfur cells

Potassium sulfur rechargeable cells, having as the electrolyte the thin walls of hollow glass fibers made from permeable glass, were developed. The cells had short lives, probably due to the construction materials and impurities in the potassium. The effect of the impurities in the analogous NA-S system was studied. Calcium, potassium, and NaOH/oxide impurities caused increased resistance or corrosion of the glass fibers. For long lived cell operation, the Na must contain less than 1 ppm Ca and less than a few ppm of hydroxide/oxide. Up to 150 ppm K can be tolerated. After purification of the Na anolyte, cell lifetimes in excess of 1000 deep charge-discharge cycles or over 8 months on continuous cycling at 10-30 percent depth of discharge were obtained.

Tsang, F. Y.↗

DOE OSTI2025

Investigating the FLASH Effect in a Rat Brain Organotypic Model With a Novel High-Energy Electron Beam

Ultrahigh dose rate (FLASH) radiation therapy is reported to reduce normal tissue toxicity while maintaining tumor control; however, mechanism(s) remain obscure. To study FLASH mechanisms in brain tissue, we developed a novel experimental platform featuring a specialized high-energy electron linear accelerator, High Intensity Gamma Ray Source (HIGS), paired with an organotypic ex vivo brain metastasis model. We varied interpulse spacing to modulate the mean dose rate (MDR) of our unique 35 MeV electron beam, while maintaining extremely high instantaneous dose rate (IDR). We characterized dosimetry and targeting accuracy of the FLASH beam with film dosimetry. We combined this FLASH beam with an organotypic rat brain slice/breast carcinoma coculture model of brain metastasis to assess effects on normal and neoplastic tissues. Live-cell and bioluminescence imaging demonstrated cancer cell growth effects, whereas normal tissue responses and immune activation were assessed using live-cell imaging, cytokine profiles, and confocal microscopy. Here, we performed comparison experiments with 20 MeV electrons from a Varian clinical linear accelerator (VCLA) using conventional dose rates. The highest IDR of the FLASH beam to date was 20.7 ± 0.6 MGy/s, with maximum MDR of 20.7 MGy/s delivered in 1 pulse of 1 µs duration. Beam targeting was accurate to <1 mm and reproducible. HIGS-FLASH and VCLA dose rates equivalently decreased cancer cell growth. HIGS-FLASH irradiation significantly increased tumor necrosis factor α and fractalkine levels and confocal microscopy revealed distinct changes in microglial morphology slices suggesting microglia activation. Our novel experimental platform produces extremely high dose rates and rapid normal/neoplastic tissue readouts for mechanistic research into the effects of FLASH radiation in the brain. HIGS-FLASH irradiation induces comparable cancer cell growth inhibition but differential effects on cytokines and microglial morphology, suggesting that acute innate immune responses may be involved in FLASH normal tissue effects in the brain.

Kay, Tyler V. [Duke University, Durham, NC (United↗

NASA NTRS1998

In search of cellular control: signal transduction in context

The field of molecular cell biology has experienced enormous advances over the last century by reducing the complexity of living cells into simpler molecular components and binding interactions that are amenable to rigorous biochemical analysis. However, as our tools become more powerful, there is a tendency to define mechanisms by what we can measure. The field is currently dominated by efforts to identify the key molecules and sequences that mediate the function of critical receptors, signal transducers, and molecular switches. Unfortunately, these conventional experimental approaches ignore the importance of supramolecular control mechanisms that play a critical role in cellular regulation. Thus, the significance of individual molecular constituents cannot be fully understood when studied in isolation because their function may vary depending on their context within the structural complexity of the living cell. These higher-order regulatory mechanisms are based on the cell's use of a form of solid-state biochemistry in which molecular components that mediate biochemical processing and signal transduction are immobilized on insoluble cytoskeletal scaffolds in the cytoplasm and nucleus. Key to the understanding of this form of cellular regulation is the realization that chemistry is structure and hence, recognition of the the importance of architecture and mechanics for signal integration and biochemical control. Recent work that has unified chemical and mechanical signaling pathways provides a glimpse of how this form of higher-order cellular control may function and where paths may lie in the future.

Non-NASA Center↗

NASA NTRS2002

Mechanics of vimentin intermediate filaments

It is increasingly evident that the cytoskeleton of living cells plays important roles in mechanical and biological functions of the cells. Here we focus on the contribution of intermediate filaments (IFs) to the mechanical behaviors of living cells. Vimentin, a major structural component of IFs in many cell types, is shown to play an important role in vital mechanical and biological functions such as cell contractility, migration, stiffness, stiffening, and proliferation.

Review↗

DOE OSTI2026

Programmed synthesis of mesoporous protein crystals in cellular reactors

Protein crystals are naturally derived mesoporous materials with versatile structures and physicochemical properties. Here we introduce an intracellular synthesis platform that enables controllable and programmable protein crystallization. In live cells, we show that, after initial nucleation, steady protein expression governs crystal growth, yielding predictable, tunable dynamics in live cells. Exploiting this feature, we combined HaloTag and click chemistries to achieve modular, programmable immobilization of diverse guest materials with spatial patterning down to ~100 nm resolution. We further demonstrated the sequential release of immobilized materials in physiologically relevant fluids. As a proof of concept, we programmed particles to carry human fibroblast growth factors in distinct layers, which elicited designed oscillatory Akt signalling patterns in cell culture. Finally, this work outlines a programmable method for producing mesoporous materials, with possible applications in catalysis and biomedicine.

Yang, Hongru [Johns Hopkins Univ., Baltimore, MD (↗

DOE OSTI2021

Immunotargeting of Nanocrystals by SpyCatcher Conjugation of Engineered Antibodies

Inorganic nanocrystals such as quantum dots (QDs) and upconverting nanoparticles (UCNPs) are uniquely suited for quantitative live-cell imaging and are typically functionalized with ligands to study specific receptors or cellular targets. Antibodies (Ab) are among the most useful targeting reagents owing to their high affinities and specificities, but common nanocrystal labeling methods may orient Ab incorrectly, be reversible or denaturing, or lead to Ab-NP complexes too large for some applications. Here, we report that SpyCatcher proteins, which bind and spontaneously form covalent isopeptide bonds with cognate SpyTag peptides, can conjugate engineered Ab to nanoparticle surfaces with control over stability, orientation, and stoichiometry. Compact SpyCatcher-functionalized QDs and UCNPs may be labeled with short-chain variable fragment Ab (scFv) engineered to bind urokinase-type plasminogen activator receptors (uPAR) that are overexpressed in many human cancers. Confocal imaging of anti-uPAR scFv-QD conjugates shows the antibody mediates specific binding and internalization by breast cancer cells expressing uPAR. Time-lapse imaging of photostable scFv-UCNP conjugates shows that Ab binding causes uPAR internalization with a ~20 min half-life on the cell surface, and uPAR is internalized to endolysosomal compartments distinct from general membrane stains and without significant recycling to the cell surface. The controlled and stable conjugation of engineered Ab to NPs enables targeting of diverse receptors for live-cell study of their distribution, trafficking, and physiology.

60 APPLIED LIFE SCIENCES↗

NASA NTRS2013

Microbial Burden Approach : New Monitoring Approach for Measuring Microbial Burden

Advantages of new approach for differentiating live cells/ spores from dead cells/spores. Four examples of Salmonella outbreaks leading to costly destruction of dairy products. List of possible collaboration activities between JPL and other industries (for future discussion). Limitations of traditional microbial monitoring approaches. Introduction to new approach for rapid measurement of viable (live) bacterial cells/spores and its areas of application. Detailed example for determining live spores using new approach (similar procedure for determining live cells). JPL has developed a patented approach for measuring amount of live and dead cells/spores. This novel "molecular" method takes less than 5 to 7 hrs. compared to the seven days required using conventional techniques. Conventional "molecular" techniques can not discriminate live cells/spores among dead cells/spores. The JPL-developed novel method eliminates false positive results obtained from conventional "molecular" techniques that lead to unnecessary delay in the processing and to unnecessary destruction of food products.

microbial monitoring↗

DOE OSTI2020

Design, characterization, and intracellular trafficking of biofunctionalized chitosan nanomicelles

The hydrophobically modified glycol chitosan (HGC) nanomicelle has received increasing attention as a promising platform for the delivery of chemotherapeutic drugs. To improve the tumor selectivity of HGC, here an avidin and biotin functionalization strategy was applied. The hydrodynamic diameter of the biotin-avidin-functionalized HGC (cy5.5-HGC-B4F) was observed to be 104.7 nm, and the surface charge was +3.1 mV. Confocal and structured illumination microscopy showed that at 0.1 mg/ml, cy5.5-HGC-B4F nanomicelles were distributed throughout the cytoplasm of MDA-MB-231 breast cancer cells after 2 h of exposure without significant cytotoxicity. To better understand the intracellular fate of the nanomicelles, entrapment studies were performed and demonstrated that some cy5.5-HGC-B4F nanomicelles were capable of escaping endocytic vesicles, likely via the proton sponge effect. Quantitative analysis of the movements of endosomes in living cells revealed that the addition of HGC greatly enhanced the motility of endosomal compartments, and the nanomicelles were transported by early and late endosomes from cell periphery to the perinuclear region. Our results validate the importance of using live-cell imaging to quantitatively assess the dynamics and mechanisms underlying the complex endocytic pathways of nanosized drug carriers.

Li, Weiyi↗

DOE OSTI2025

Precision Labeling of Native Antibodies with Lock Coupling

The formation of stable protein complexes enables much of biotechnology, but even high-affinity complexes can dissociate, limiting potential applications in biomaterials, bioimaging, nanomedicine, and other protein-based technologies. Here, in this study, we describe lock coupling, a simple and selective one-step reaction between interfacial lysine and glutamate or aspartate side chains to form stable isopeptide bonds and be used for the precise labeling of native antibodies. We identify conditions in which short-lived activated esters formed by the aqueous carbodiimide EDC promote isopeptide bond formation specifically at preassociated amine-acid pairs. Indiscriminate cross-linking is minimized by formation of protein complexes before addition of catalyst, use of acidic pH to suppress exposed Lys reactivity, and limiting the aqueous stability of activated esters. For native antibody (Ab) labeling, we show that the small IgG-binding protein GB1 can be covalently attached to the Ab Fc domain and that introduction of Cys into GB1 loops allows for facile conjugation of fluorophores, micelles, or inorganic nanocrystals for imaging in live cells and animals. By varying Cys substituents and protein stoichiometry, a defined number of probes can be uniformly attached without the need for extensive purification. In live-cell confocal microscopy, labeled GB1 serves as a stable replacement for secondary Abs, enabling simple multicolor immunostaining and imaging. Lock coupling requires just a single reagent in aqueous buffer and leverages both the innate ability of proteins to form high-affinity complexes and the widespread presence of Lys-Glu/Asp pairs at their interfaces, with the potential for precision synthesis of protein-based probes for imaging, biomaterials, biophysics, and medicine.

antibody↗

DOE OSTI2023

Sneaking in SpyCatcher using cell penetrating peptides for in vivo imaging

Abstract In vivo imaging of protein complexes is a powerful method for understanding the underlying biological function of these key biomolecules. Though the engineering of small, high affinity nanobodies have become more prevalent, the off-rates of these tags may result in incomplete or partial labeling of proteins in live cells. The SpyCatcher003 and SpyTag split protein system allow for irreversible, covalent binding to a short target peptide unlike nanobody-affinity based probes. However, delivering these tags into a cell without disrupting its normal function is a key challenge. Cell penetrating peptides (CPPs) are short peptide sequences that facilitate the transduction of otherwise membrane-impermeable ‘cargo’ , such as proteins, into cells. Here we report on our efforts to engineer and characterize CPP-SpyCatcher003 fusions as modular imaging probes. We selected three CPPs, CUPID, Pentratin, and pVEC, to engineer fusion protein probes for superresolution microscopy, with the aim to eliminate prior permeabilization treatments that could introduce imaging artifacts. We find that fusing the CPP sequences to SpyCatcher003 resulted in dimer and multimer formation as determined by size exclusion chromatography, dynamic light scattering, and SDS resistant dimers on SDS-PAGE gels. By isolating and labeling the monomeric forms of the engineered protein, we show these constructs retained their ability to bind SpyTag and all three CPP sequences remain membrane active, as assessed by CD spectroscopy in the presence of SDS detergent. Using fluorescence and super resolution Lattice structured illumination microscopy (Lattice SIM) imaging we show that the CPPs did not enhance uptake of SpyCatcher by E. coli, however with Caulobacter crescentus cells, we show that Penetratin, and to a lesser degree CUPID, does enhance uptake. Our results demonstrate the ability of the CPP-SpyCatcher003 to label targets within living cells, providing the groundwork for using split protein systems for targeted in vivo imaging.

59 BASIC BIOLOGICAL SCIENCES↗