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All sourcesNASA NTRSDOE OSTI
DOE OSTI2026

Hg Accumulation by Single-Cell Sulfate-Reducing Bacteria Methylating Mercury

Methylmercury (MeHg) is a potent neurotoxin that poses risks to ecosystems and human health. MeHg is produced by microbes following saturating-like kinetics. We hypothesize that this saturation reflects a limited intracellular mercury (Hg) accumulation. Here, in this study, we investigated Hg accumulation in Pseudodesulfovibrio hydrargyri BerOc1, a sulfate-reducing model strain able to methylate Hg. Cells were incubated with 0.5 and 2 μM of mercury (HgCl 2 ), and mercury localization was studied using synchrotron-based nano-X-ray fluorescence and high-resolution analytical electron microscopy. For both concentrations, Hg was detected in the bacterial cytosol, in addition to extracellular (Hg, S)-containing nanoparticles. Intracellular Hg levels were slightly higher at 2 μM than at 0.5 μM (1.61 vs 1.40 pg.mm –2 ), suggesting a regulated accumulation. However, the population exhibited heterogeneity in Hg accumulation, particularly at the highest Hg exposure, with some cells being Hg hyperaccumulators. Correlative imaging between Hg localization and cell viability revealed that these hyperaccumulating cells were non-active. Our results suggest that active cells regulate Hg accumulation. From an analytical perspective, a minor subpopulation of hyperaccumulating cells can bias bulk measurements and should be considered in interpreting Hg accumulation in microorganisms. Environmentally, these cells can impact Hg cycling by acting as a metal sink.

Intracellular accumulation↗
DOE OSTI2025

A fluorescent-protein spin qubit

Quantum bits (qubits) are two-level quantum systems that support initialization, readout and coherent control1. Optically addressable spin qubits form the foundation of an emerging generation of nanoscale sensors. The engineering of these qubits has mainly focused on solid-state systems. However, fluorescent proteins, rather than exogenous fluorescent probes, have become the gold standard for in vivo microscopy because of their genetic encodability. Although fluorescent proteins possess a metastable triplet state, they have not been investigated as qubits. Here we realize an optically addressable spin qubit in enhanced yellow fluorescent protein. A near-infrared laser pulse enables triggered readout of the triplet state with up to 20% spin contrast. Using coherent microwave control of the enhanced-yellow-fluorescent-protein spin at liquid-nitrogen temperatures, we measure a (16 ± 2) μs coherence time under Carr–Purcell–Meiboom–Gill decoupling. We express the qubit in mammalian cells, maintaining contrast and coherent control despite the complex intracellular environment. Finally, we demonstrate optically detected magnetic resonance in bacterial cells at room temperature with contrast up to 8%. Our results introduce fluorescent proteins as a powerful qubit platform that paves the way for applications in the life sciences, such as nanoscale field sensing and spin-based imaging modalities.

Feder, Jacob S. [Univ. of Chicago, IL (United Stat↗
NASA NTRS2014

A Novel Protocol for Decoating and Permeabilizing Bacterial Spores for Epifluorescent Microscopy

Based on previously reported procedures for permeabilizing vegetative bacterial cells, and numerous trial-and-error attempts with bacterial endospores, a protocol was developed for effectively permeabilizing bacterial spores, which facilitated the applicability of fluorescent in situ hybridization (FISH) microscopy. Bacterial endospores were first purified from overgrown, sporulated suspensions of B. pumilus SAFR-032. Purified spores at a concentration of approx equals 10 million spores/mL then underwent proteinase-K treatment, in a solution of 468.5 μL of 100 mM Tris-HCl, 30 μL of 10% SDS, and 1.5 microL of 20 mg/mL proteinase-K for ten minutes at 35 ºC. Spores were then harvested by centrifugation (15,000 g for 15 minutes) and washed twice with sterile phosphate-buffered saline (PBS) solution. This washing process consisted of resuspending the spore pellets in 0.5 mL of PBS, vortexing momentarily, and harvesting again by centrifugation. Treated and washed spore pellets were then resuspended in 0.5 mL of decoating solution, which consisted of 4.8 g urea, 3 mL Milli-Q water, 1 mL 0.5M Tris, 1 mL 1M dithiothreitol (DTT), and 2 mL 10% sodium-dodecylsulfate (SDS), and were incubated at 65 ºC for 15 minutes while being shaken at 165 rpm. Decoated spores were then, once again, washed twice with sterile PBS, and subjected to lysozyme/mutanolysin treatment (7 mg/mL lysozyme and 7U mutanolysin) for 15 minutes at 35 C. Spores were again washed twice with sterile PBS, and spore pellets were resuspended in 1-mL of 2% SDS. This treatment, facilitating inner membrane permeabilization, lasted for ten minutes at room temperature. Permeabilized spores were washed two final times with PBS, and were resuspended in 200 mkcroL of sterile PBS. At this point, the spores were permeable and ready for downstream processing, such as oligonucleotideprobe infiltration, hybridization, and microscopic evaluation. FISH-microscopic imagery confirmed the effective and efficient (≈50% successful permeabilization and recovery) permeabilization of numerous spore preparations. The novelty of the technology developed here is in its applicability to bacterial endospores. While protocols abound for the effective permeabilization of bacterial, archaeal, and eukaryotic vegetative cells, there are no such reliable methods for decoating and permeabilizing bacterial endospores in a manner that is amenable to downstream FISH microscopic analyses. This innovation enables the direct visualization and enumeration of spores via FISH-based microscopic techniques, circumventing the complications that accompany previously required germination regimes. The synergistic enzymatic weakening of the many spore layers facilitates a structural compromise that is just enough to render the spores permeable without degrading the spore to a level, which precludes it from recognition.

LaDuc, Myron T.↗
NASA NTRS1995

Physiological assessment of bacteria using fluorochromes

This minireview focuses on the application of fluorogenic compounds in the detection of bacteria with particular emphasis on the assessment of physiological activity using epifluorescence microscopy. Microbiological applications of several related methods will also be reviewed.

NASA Discipline Environmental Health↗
NASA NTRS2001

Deep UV Native Fluorescence Imaging of Antarctic Cryptoendolithic Communities

An interdisciplinary team at the Jet Propulsion Laboratory Center for Life Detection has embarked on a project to provide in situ chemical and morphological characterization of Antarctic cryptoendolithic microbial communities. We present here in situ deep ultraviolet (UV) native fluorescence and environmental scanning electron microscopy images transiting 8.5 mm into a sandstone sample from the Antarctic Dry Valleys. The deep ultraviolet imaging system employs 224.3, 248.6, and 325 nm lasers to elicit differential fluorescence and resonance Raman responses from biomolecules and minerals. The 224.3 and 248.6 nm lasers elicit a fluorescence response from the aromatic amino and nucleic acids. Excitation at 325 nm may elicit activity from a variety of biomolecules, but is more likely to elicit mineral fluorescence. The resultant fluorescence images provide in situ chemical and morphological maps of microorganisms and the associated organic matrix. Visible broadband reflectance images provide orientation against the mineral background. Environmental scanning electron micrographs provided detailed morphological information. The technique has made possible the construction of detailed fluorescent maps extending from the surface of an Antarctic sandstone sample to a depth of 8.5 mm. The images detect no evidence of microbial life in the superficial 0.2 mm crustal layer. The black lichen component between 0.3 and 0.5 mm deep absorbs all wavelengths of both laser and broadband illumination. Filamentous deep ultraviolet native fluorescent activity dominates in the white layer between 0.6 mm and 5.0 mm from the surface. These filamentous forms are fungi that continue into the red (iron-rich) region of the sample extending from 5.0 to 8.5 mm. Using differential image subtraction techniques it is possible to identify fungal nuclei. The ultraviolet response is markedly attenuated in this region, apparently from the absorption of ultraviolet light by iron-rich particles coating the filaments. Below 8.5 mm the filamentous morphology of the upper layers gives way to punctate 1-2 micron particles evidencing fluorescent activity following excitation at both deep ultraviolet wavelengths.

Storrie-Lombardi, M. C.↗
NASA NTRS1999

Identification of a calmodulin-regulated Ca2+-ATPase in the endoplasmic reticulum

A unique subfamily of calmodulin-dependent Ca2+-ATPases was recently identified in plants. In contrast to the most closely related pumps in animals, plasma membrane-type Ca2+-ATPases, members of this new subfamily are distinguished by a calmodulin-regulated autoinhibitor located at the N-terminal instead of a C-terminal end. In addition, at least some isoforms appear to reside in non-plasma membrane locations. To begin delineating their functions, we investigated the subcellular localization of isoform ACA2p (Arabidopsis Ca2+-ATPase, isoform 2 protein) in Arabidopsis. Here we provide evidence that ACA2p resides in the endoplasmic reticulum (ER). In buoyant density sucrose gradients performed with and without Mg2+, ACA2p cofractionated with an ER membrane marker and a typical "ER-type" Ca2+-ATPase, ACA3p/ECA1p. To visualize its subcellular localization, ACA2p was tagged with a green fluorescence protein at its C terminus (ACA2-GFPp) and expressed in transgenic Arabidopsis. We collected fluorescence images from live root cells using confocal and computational optical-sectioning microscopy. ACA2-GFPp appeared as a fluorescent reticulum, consistent with an ER location. In addition, we observed strong fluorescence around the nuclei of mature epidermal cells, which is consistent with the hypothesis that ACA2p may also function in the nuclear envelope. An ER location makes ACA2p distinct from all other calmodulin-regulated pumps identified in plants or animals.

NASA Discipline Plant Biology↗
NASA NTRS2016

Biomolecular Analysis Capability for Cellular and Omics Research on the International Space Station

International Space Station (ISS) assembly complete ushered a new era focused on utilization of this state-of-the-art orbiting laboratory to advance science and technology research in a wide array of disciplines, with benefits to Earth and space exploration. ISS enabling capability for research in cellular and molecular biology includes equipment for in situ, on-orbit analysis of biomolecules. Applications of this growing capability range from biomedicine and biotechnology to the emerging field of Omics. For example, Biomolecule Sequencer is a space-based miniature DNA sequencer that provides nucleotide sequence data for entire samples, which may be used for purposes such as microorganism identification and astrobiology. It complements the use of WetLab-2 SmartCycler"TradeMark", which extracts RNA and provides real-time quantitative gene expression data analysis from biospecimens sampled or cultured onboard the ISS, for downlink to ground investigators, with applications ranging from clinical tissue evaluation to multigenerational assessment of organismal alterations. And the Genes in Space-1 investigation, aimed at examining epigenetic changes, employs polymerase chain reaction to detect immune system alterations. In addition, an increasing assortment of tools to visualize the subcellular distribution of tagged macromolecules is becoming available onboard the ISS. For instance, the NASA LMM (Light Microscopy Module) is a flexible light microscopy imaging facility that enables imaging of physical and biological microscopic phenomena in microgravity. Another light microscopy system modified for use in space to image life sciences payloads is initially used by the Heart Cells investigation ("Effects of Microgravity on Stem Cell-Derived Cardiomyocytes for Human Cardiovascular Disease Modeling and Drug Discovery"). Also, the JAXA Microscope system can perform remotely controllable light, phase-contrast, and fluorescent observations. And upcoming confocal microscopy capability will allow for optical sectioning of biological tissues to determine microanatomical localization of biomarkers. Furthermore, NASA's geneLAB effort addresses integration of genomic, epigenomic, transcriptomic, proteomic and metabolomic datasets, by applying an innovative open source science platform for multi-investigator high throughput utilization of the ISS. In sum, the expanding ISS capability for analysis of biomolecules is enabling innovative research in a broad spectrum of areas such as cellular and molecular biology, biotechnology, tissue engineering, biomedicine, and Omics, providing manifold benefits for humanity.

Guinart-Ramirez, Y.↗
NASA NTRS2014

Locating Noctiluca Miliaris in the Arabian Sea: An Optical Proxy Approach

Coincident with shifting monsoon weather patterns over India, the phytoplankter Noctiluca miliaris has recently been observed to be dominating phytoplankton blooms in the northeastern Arabian Sea during the winter monsoons. Identifying the exact environmental and/or ecological conditions that favor this species has been hampered by the lack of concurrent environmental and biological observations on time and space scales relevant to ecologic and physiologic processes. We present a bio-optical proxy for N. miliaris measured on highly resolved depth scales coincident with hydrographic observations with the goal to identify conducive hydrographic conditions for the bloom. The proxy is derived from multichannel excitation chlorophyll a fluorescence and is validated with microscopy, pigment composition, and spectral absorption. Phytoplankton populations dominated by either diatoms or other dinoflagellates were additionally discerned. N. miliaris populations in full bloom were identified offshore in low-nutrient and low-N : P ratio surface waters within a narrow temperature and salinity range. These populations transitioned to high-biomass diatom-dominated coastal upwelling populations. A week later, the N. miliaris blooms were observed in declining phase, transitioning to very-low-biomass populations of non-N. miliaris dinoflagellates. There were no clear hydrographic conditions uniquely associated with the N. miliaris populations, although N. miliaris was not found in the upwelling or extremely oligotrophic waters. Taxonomic transitions were not discernible in the spatial structure of the bloom as identified by the ocean color Chl imagery, indicating that in situ observations may be necessary to resolve community structure, particularly for populations below the surface.

Arabian Sea↗
DOE OSTI2026

Mechanistic transition of stainless steel 304L corrosion in MgCl 2 phase change material under thermal cycling

Magnesium chloride (MgCl 2 )-based molten salt phase change materials (PCMs) are promising candidates for thermal energy storage (TES) owing to their advantageous thermophysical properties, however, their high corrosivity in the molten state limits material compatibility. In this study, the corrosion behavior of 304?L stainless steel (SS304L) exposed to MgCl2 PCM was investigated as a function of thermal cycling (up to 9000 cycles) relevant to PCM-based TES systems. Microstructural changes and elemental redistribution at the alloy-salt interface under thermal cycling conditions were characterized using synchrotron-based X-ray fluorescence (XRF) and scanning electron microscopy coupled with energy dispersive X-ray spectroscopy (SEM-EDS). Results show that corrosion is governed by selective Cr-dissolution and elemental diffusion near the interface and grain boundaries. A clear transition in the dominant corrosion mechanism is observed with increasing thermal cycles. Surface corrosion is the dominant mechanism up to 1000 cycles, whereas intergranular corrosion becomes the primary degradation mode beyond 2000 cycles. These mechanistic changes are interpreted in terms of evolving interfacial chemistry, microstructure and the diffusion-driven redistribution of alloying elements.

36 MATERIALS SCIENCE↗
DOE OSTI2024

Dynamic Surface Incorporation of Pb 2+ Ions at the Actively Dissolving Calcite (104) Surface

The reaction of dissolved Pb 2+ with calcite surfaces at near-equilibrium conditions involves adsorption of Pb 2+ and precipitation of secondary heteroepitaxial Pb-carbonate minerals. A more complex behavior is observed under far-from-equilibrium conditions, including strong inhibition of calcite dissolution, development of microtopography, and near-surface incorporation of multiple monolayers (ML) of Pb 2+ without precipitation of secondary phases [where 1 ML ≡ 1 Ca/20.2 Å 2 , the crystallographic site density of the calcite (104) lattice plane]. However, the mechanistic controls governing far-from-equilibrium reactivity are not well understood. Here, in this study, we observe the interfacial incorporation of dissolved Pb 2+ during the dissolution of calcite (104) surfaces at pH ~3.7 in a flow-through reaction cell, revealing the formation of a ~1 nm thick Pb-rich calcite layer with a total Pb coverage of ~1.4 ML. These observations of the sorbed Pb distribution used resonant anomalous X-ray reflectivity, X-ray fluorescence, and nanoinfrared atomic force microscopy. We propose that this altered surface layer represents a novel sorption mode that is stabilized by conditions of sustained disequilibrium. This behavior may significantly impact the transport of dissolved metals during disequilibrium processes occurring in acid mine drainage and subsurface CO 2 injection and, if appropriately accounted for, could improve the predictive capability of geochemical reactive-transport models.

37 INORGANIC, ORGANIC, PHYSICAL, AND ANALYTICAL CH↗
DOE OSTI2025

Three-dimensional characterization of modifications in sapphire exposed to laser-induced damage using multimodal spectral microimaging

Sapphire (Al 2 O 3 ) is a commonly used dielectric material with many applications in lasers and optical systems. Owing to its high resistivity to laser induced damage, it is particularly suitable for use in high power laser systems. This work focuses on developing techniques to characterize material modifications in sapphire. These techniques were applied following localized laser induced ablation, commonly referred to as laser-damage, resulting from exposure to single 100-ps and 6-ns pulses. Measurements of fluorescence-based piezospectroscopy and confocal Raman microscopy were performed with spatial resolution on the order of 1 μ m. Raman microscopy reveals that the relaxation of material exposed to the rapid laser heating, elastic and viscoplastic deformation, melting, and solidification leads to the formation of a polycrystalline material phase. In addition, narrowband fluorescence lines, referred to as R 1 and R 2 , exhibit pressure-sensitive changes to their spectral profiles, allowing 3D internal stresses to be recorded with spatial resolution of the order of a few micrometers.

Laser-damage↗
DOE OSTI2026

Artificial Soiling Replication of Field Losses on Commercial Photovoltaic Modules

Here, this study demonstrates the capabilities of an indoor artificial soiling approach developed to closely replicate the natural, cyclic soil accumulation processes in the field-dust suspension, deposition, and sedimentation/cementation-for a subtropical climate. In this work, a near-field environment is replicated in an artificial soiling cubic chamber through controlled regulation of humidity, temperature, dust type, and dust concentration, based on site-specific historical climate data. Two different models (MA and MB) of full-size commercial photovoltaic modules from a single manufacturer, installed side by side in the mid-Atlantic United States, were retrieved and subjected to artificial soiling experiments and various characterization measurements, including short-circuit current, colorimetry, reflectance, X-ray fluorescence, laser diffraction, and optical microscopy. Both in the field and in our improved field-representative artificial soiling tests, the MA model experienced roughly twice the soiling loss as the MB model. To closely replicate the field soiling losses for a site-specific climate, it is critical to include: 1) The use of field-collected dust with identical dust chemistry and particle distribution instead of standardized ISO 12103 Arizona Road dusts, 2) the use of only a small amount of field-collected dust inside the chamber during the deposition process (e.g., 0.15 g), and 3) the preconditioning of the surface coating for the partial/full dose of UV stress as experienced in the field during sunlight exposure and the abrasion as experienced in the field during regular module cleaning activities, if/as needed. The field-representative artificial soiling method developed here could potentially be adopted for rank ordering of various antisoiling coatings developed by researchers and industry stakeholders.

14 SOLAR ENERGY↗
DOE OSTI2025

High-resolution analysis of clay minerals and amorphous materials in martian analog environments

Mudrocks and mud-sized sediments (i.e., silt to clay) dominate the surface of Earth and Mars. These fine-grained sediments preserve a rich history of sedimentary processes from source to sink and shed light on ancient climates. However, both the physical and chemical nature of these materials make them difficult to fully characterize with traditional laboratory techniques. Here, we explore a cross-disciplinary and high-resolution approach using synchrotron radiation for X-ray diffraction, pair distribution function analysis, and submicrometer-scale X-ray fluorescence, combined with transmission electron microscopy, to better understand the nanostructure and composition of mud-sized sediments from a glacio-fluvial watershed in southwest Iceland. Our results demonstrate that sediments in the cold and wet climate of Iceland are more altered than previously thought, as evidenced by the identification of kaolinite and mixed-layer kaolinite-smectite. Additionally, sediments are enriched in amorphous materials and nanocrystalline phases, as determined from grain morphologies and compositions consistent with allophane, hisingerite, ferrihydrite, and halloysite. These alteration products are present as intimate mixtures that vary across depositional sites, demonstrating the dynamic nature of the secondary assemblage from source to sink. This work has implications for Mars, where, for example, basalt-sourced sedimentary rocks from Gale crater are abundant in clay minerals and amorphous materials. Finally, this work underpins the importance of using high-resolution techniques, a coordinated methodology, and developing innovative approaches for future planetary sample return missions (e.g., Mars sample return).

36 MATERIALS SCIENCE↗
NASA NTRS1985

Diffusion in Immiscible Melts

The objective of this program is to measure the diffusion coefficients for molten Pb in Zn in the immiscible liquid-phase region. Diffusion couples of pure Pb and Zn were prepared using a shear cell. These have been placed in graphite crucibles and encapsulated in stainless steel cartridges and are awaiting the next Materials Experiment Assembly (MEA) flight opportunity. In flight, one couple will be soaked for 40 minutes at 440 deg C (just above the monotectic temperature) and the second couple will be soaked for 40 minutes 820 deg C (just above the consolute temperature). After the soak both samples will be rapidly quenched by flowing He to minimize redistribution of the immiscible phases. Post flight compositional analysis will be accomplished using X-ray fluorescence in the scanning electron microscopy.

Pond, R. B.↗
NASA NTRS2005

Two-Photon Fluorescence Microscope for Microgravity Research

A two-photon fluorescence microscope has been developed for the study of biophysical phenomena. Two-photon microscopy is a novel form of laser-based scanning microscopy that enables three-dimensional imaging without many of the problems inherent in confocal microscopy. Unlike one-photon optical microscopy, two-photon microscopy utilizes the simultaneous nonlinear absorption of two near-infrared photons. However, the efficiency of two-photon absorption is much lower than that of one-photon absorption, so an ultra-fast pulsed laser source is typically employed. On the other hand, the critical energy threshold for two-photon absorption leads to fluorophore excitation that is intrinsically localized to the focal volume. Consequently, two-photon microscopy enables optical sectioning and confocal performance without the need for a signal-limiting pinhole. In addition, there is a reduction (relative to one-photon optical microscopy) in photon-induced damage because of the longer excitation wavelength. This reduction is especially advantageous for in vivo studies. Relative to confocal microscopy, there is also a reduction in background fluorescence, and, because of a reduction in Rayleigh scattering, there is a 4 increase of penetration depth. The prohibitive cost of a commercial two-photon fluorescence-microscope system, as well as a need for modularity, has led to the construction of a custom-built system (see Figure 1). This system includes a coherent mode-locked titanium: sapphire laser emitting 120-fs-duration pulses at a repetition rate of 80 MHz. The pulsed laser has an average output power of 800 mW and a wavelength tuning range of 700 to 980 nm, enabling the excitation of a variety of targeted fluorophores. The output from the laser is attenuated, spatially filtered, and then directed into a confocal scanning head that has been modified to provide for side entry of the laser beam. The laser output coupler has been replaced with a dichroic filter that reflects the longer-wavelength excitation light and passes the shorter-wavelength fluorescence light. Also, the confocal pinhole has been removed to increase the signal strength. The laser beam is scanned by a twoperpendicular- axis pair of galvanometer mirrors through a pupil transfer lens into the side port of an inverted microscope. Finally, the beam is focused by a 63-magnification, 1.3-numerical- aperture oil-immersion objective lens onto a specimen. The pupil transfer lens serves to match the intermediate image planes of the scanning head and the microscope, and its location is critical. In order to maximize the quality of the image, (that is, the point spread function of the objective lens for all scan positions), the entire system was modeled in optical-design software, and the various free design parameters (the parameters of the spatial-filter components as well as the separations of all of the system components) were determined through an iterative optimization process. A modular design was chosen to facilitate access to the optical train for future fluorescence correlation spectroscopy and fluorescence-lifetime experiments.

Fischer, David G.↗
NASA NTRS2001

Changes in root cap pH are required for the gravity response of the Arabidopsis root

Although the columella cells of the root cap have been identified as the site of gravity perception, the cellular events that mediate gravity signaling remain poorly understood. To determine if cytoplasmic and/or wall pH mediates the initial stages of root gravitropism, we combined a novel cell wall pH sensor (a cellulose binding domain peptide-Oregon green conjugate) and a cytoplasmic pH sensor (plants expressing pH-sensitive green fluorescent protein) to monitor pH dynamics throughout the graviresponding Arabidopsis root. The root cap apoplast acidified from pH 5.5 to 4.5 within 2 min of gravistimulation. Concomitantly, cytoplasmic pH increased in columella cells from 7.2 to 7.6 but was unchanged elsewhere in the root. These changes in cap pH preceded detectable tropic growth or growth-related pH changes in the elongation zone cell wall by 10 min. Altering the gravity-related columella cytoplasmic pH shift with caged protons delayed the gravitropic response. Together, these results suggest that alterations in root cap pH likely are involved in the initial events that mediate root gravity perception or signal transduction.

NASA Discipline Plant Biology↗
NASA NTRS2003

Furrow microtubules and localized exocytosis in cleaving Xenopus laevis embryos

In dividing Xenopus eggs, furrowing is accompanied by expansion of a new domain of plasma membrane in the cleavage plane. The source of the new membrane is known to include a store of oogenetically produced exocytotic vesicles, but the site where their exocytosis occurs has not been described. Previous work revealed a V-shaped array of microtubule bundles at the base of advancing furrows. Cold shock or exposure to nocodazole halted expansion of the new membrane domain, which suggests that these microtubules are involved in the localized exocytosis. In the present report, scanning electron microscopy revealed collections of pits or craters, up to approximately 1.5 micro m in diameter. These pits are evidently fusion pores at sites of recent exocytosis, clustered in the immediate vicinity of the deepening furrow base and therefore near the furrow microtubules. Confocal microscopy near the furrow base of live embryos labeled with the membrane dye FM1-43 captured time-lapse sequences of individual exocytotic events in which irregular patches of approximately 20 micro m(2) of unlabeled membrane abruptly displaced pre-existing FM1-43-labeled surface. In some cases, stable fusion pores, approximately 2 micro m in diameter, were seen at the surface for up to several minutes before suddenly delivering patches of unlabeled membrane. To test whether the presence of furrow microtubule bundles near the surface plays a role in directing or concentrating this localized exocytosis, membrane expansion was examined in embryos exposed to D(2)O to induce formation of microtubule monasters randomly under the surface. D(2)O treatment resulted in a rapid, uniform expansion of the egg surface via random, ectopic exocytosis of vesicles. This D(2)O-induced membrane expansion was completely blocked with nocodazole, indicating that the ectopic exocytosis was microtubule-dependent. Results indicate that exocytotic vesicles are present throughout the egg subcortex, and that the presence of microtubules near the surface is sufficient to mobilize them for exocytosis at the end of the cell cycle.

Non-NASA Center↗
DOE OSTI2024

Self-Sorting vs Coassembly in Peptide Amphiphile Supramolecular Nanostructures

The functionality of supramolecular nanostructures can be expanded if systems containing multiple components are designed to either self-sort or mix into coassemblies. This is critical to gain the ability to craft self-assembling materials that integrate functions, and our understanding of this process is in its early stages. Here, in this work, we have utilized three different peptide amphiphiles with the capacity to form β-sheets within supramolecular nanostructures and found binary systems that self-sort and others that form coassemblies. This was measured using atomic force microscopy to reveal the nanoscale morphology of assemblies and confocal laser scanning microscopy to determine the distribution of fluorescently labeled monomers. We discovered that PA assemblies with opposite supramolecular chirality self-sorted into chemically distinct nanostructures. In contrast, the PA molecules that formed a mixture of right-handed, left-handed, and flat nanostructures on their own were able to coassemble with the other PA molecules. We attribute this phenomenon to the energy barrier associated with changing the handedness of a β-sheet twist in a coassembly of two different PA molecules. This observation could be useful for designing biomolecular nanostructures with dual bioactivity or interpenetrating networks of PA supramolecular assemblies.

37 INORGANIC, ORGANIC, PHYSICAL, AND ANALYTICAL CH↗