Micron-scale Domains Research Articles

Transcription-dependent mobility of single genes and genome-wide motions in live human cells

The human genome is highly dynamic across all scales. At the gene level, chromatin is persistently remodeled and rearranged during active processes such as transcription, replication and DNA repair. At the genome level, chromatin moves in micron-scale domains that break up and re-form over seconds, but the origin of these coherent motions is unknown. Here, we investigate the connection between genomic motions and gene-level activity. Simultaneous mapping of single-gene and genome-wide motions shows that the coupling of gene transcriptional activity to flows of the nearby genome is modulated by chromatin compaction. A motion correlation analysis suggests that a single active gene drives larger-scale motions in low-compaction regions, but high-compaction chromatin drives gene motion regardless of its activity state. By revealing unexpected connections among gene activity, spatial heterogeneities of chromatin and its emergent genome-wide motions, these findings uncover aspects of the genome’s spatiotemporal organization that directly impact gene regulation and expression.

INDIVIDUAL FORAMINIFERAL ANALYSES: A REVIEW OF CURRENT AND EMERGING GEOCHEMICAL TECHNIQUES

Abstract The trace element (TE) and isotopic composition of calcareous foraminifera has been invaluable in advancing our understanding of environmental change throughout the geological record. Whereas “bulk” geochemical techniques, typically requiring the dissolution of tens to hundreds of foraminiferal tests for a single analysis, have been used for decades to reconstruct past ocean-climate conditions, recent technological advances have increased our ability to investigate foraminiferal geochemistry from an individual test to a micron-scale domain level. Here we review current and emerging techniques and approaches to studying the trace element and stable isotope geochemistry of individual foraminifera (i.e., individual foraminiferal analyses or “IFA”), covering spatial scales including whole-test analysis, intratest spot analysis, and cross-sectional chemical mapping techniques. Our discussion of each technique provides an overview of how the specific analytical tool works, the history of its usage in foraminiferal studies, its applications, considerations, and limitations, and potential directions for future study. Lastly, we describe potential applications of combining multiple IFA techniques to resolve key questions related to paleoceanography, (paleo)ecology, and biomineralization, and provide recommendations for the storage, dissemination, and transparency of the vast amounts of data produced through these methods. This review serves as a resource for budding and experienced foraminiferal geochemists to explore the wide array of cutting-edge approaches being used to study the geochemical composition of modern and fossil foraminifera.

Sterol-lipids enable large-scale, liquid-liquid phase separation in bilayer membranes of only 2 components.

A wide diversity of bilayer membranes, from those with hundreds of lipids (e.g., vacuoles of living yeast cells) to those with very few (e.g., artificial vesicles) phase separate into micron-scale liquid domains. The number of components required for liquid-liquid phase separation has been perplexing: only two should be necessary, but more are required except in extraordinary circumstances. What minimal set of molecular characteristics leads to liquid-liquid phase separation in bilayer membranes? This question inspired us to search for single, joined "sterol-lipid" molecules to replace both a sterol and a phospholipid in membranes undergoing liquid-liquid phase separation. By producing phase-separating membranes with only two components, we mitigate experimental challenges in determining tie-lines and in maintaining constant chemical potentials of lipids.

Multifractal detrended fluctuation analysis on the fracture surface of polycarbonate and acrylonitrile-butadiene-styrene alloy

Multifractal detrended fluctuation analysis (MF-DFA) has been carried out to evaluate the multifractal properties of the fracture morphology for Polycarbonate (PC) and Acrylonitrile-butadiene-styrene (ABS) alloy, and the relationship between the long-range correlation parameter of the samples fracture - Hurst exponent and fractal dimension was explored and analyzed. The results suggest that the existence of multifractal properties within the micrometer scale domain we investigated for the fracture surface of PC/ABS alloy samples. The Hurst exponent and fractal dimension can both be used to measure and characterize the fracture surface morphology of the PC/ABS alloys. As the content of compatibilizer PP-g-MAH increased, the tensile strength of PC/ABS alloy decreased, the fracture surface peaks tend to dominate, the Hurst exponent increased, the fractal dimension decreased, and its strength retention rate increases, displaying persistence.

Mixed-Material Feedstocks for Cold Spray Additive Manufacturing of Metal–Polymer Composites

High-performance polymers such as poly(ether ether ketone) (PEEK) are appealing as composite components for a wide variety of industrial and medical applications due to their excellent thermomechanical properties. However, conventional PEEK metallization methods can often lead to poor quality control, low deposition rate, and high cost. Cold spray is a promising potential alternative to produce polymer–metal composites rapidly and inexpensively due to its relatively mild operating conditions and high throughput. In this study, we investigated the deposition characteristics of metal–polymer composite feedstock, composed of PEEK powder and copper flake in varying ratios, onto a PEEK substrate. Copper-PEEK powder blends were prepared by both hand-mixing and cryogenic milling (cryomilling), which predominantly creates composite particles with micron-scale copper domains coating PEEK particle surfaces. This process non-monotonically affects the relative dominance and length scales of the multiple contributing deposition mechanisms present in mixed-material cold spray. In low-pressure cold spray, deposits showed significant changes in deposition efficiency and composition as a result of milling, with improvements in these characteristics most dramatic at lower Cu fractions. Deposits of a cryomilled blend of nominally 30 vol.% copper in PEEK exhibited minimal porosity under scanning electron microscopy, complete retention of powder composition, and the highest deposition efficiency among all samples tested. Notably, neither neat PEEK nor neat Cu meaningfully deposited at the same mild conditions as this 30 vol.% Cu blend, indicating a synergistic departure from linear mixing rules driven by the relative balance of local deposition interactions (e.g., hard–soft, soft–soft, etc.). Intentional powder and process design toward optimizing this balance may facilitate cold spray metallization applications.

Reversible, large-scale, liquid-liquid phase separation in living yeast membranes.

Liquid-liquid phase separation in living biological membranes is usually described as occurring on sub-micron length scales. A stunning counterexample occurs in S. cerevisiae. When the yeast shift from the log stage of growth to the stationary stage, huge, micron-scale liquid domains appear in the membranes of the vacuole, an endosomal organelle. These phases are functionally important, enabling yeast survival during periods of stress. This talk will review recent results showing: (1) This miscibility transition is reversible as would be expected from equilibrium thermodynamics, even though it occurs in a living system. (2) Yeast actively regulate this phase transition to hold the membrane transition ∼15°C above the yeast growth temperature. (3) Yeast significantly remodel their vacuole lipidomes in the shift from the log stage to the stationary stage.

Remodeling of yeast vacuole membrane lipidomes from the log (one phase) to stationary stage (two phases)

Upon nutrient limitation, budding yeast of Saccharomyces cerevisiae shift from fast growth (the log stage) to quiescence (the stationary stage). This shift is accompanied by liquid-liquid phase separation in the membrane of the vacuole, an endosomal organelle. Recent work indicates that the resulting micrometer-scale domains in vacuole membranes enable yeast to survive periods of stress. An outstanding question is which molecular changes might cause this membrane phase separation. Here, we conduct lipidomics of vacuole membranes in both the log and stationary stages. Isolation of pure vacuole membranes is challenging in the stationary stage, when lipid droplets are in close contact with vacuoles. Immuno-isolation has previously been shown to successfully purify log-stage vacuole membranes with high organelle specificity, but it was not previously possible to immuno-isolate stationary-stage vacuole membranes. Here, we develop Mam3 as a bait protein for vacuole immuno-isolation, and demonstrate low contamination by non-vacuolar membranes. We find that stationary-stage vacuole membranes contain surprisingly high fractions of phosphatidylcholine lipids (∼40%), roughly twice as much as log-stage membranes. Moreover, in the stationary stage, these lipids have higher melting temperatures, due to longer and more saturated acyl chains. Another surprise is that no significant change in sterol content is observed. These lipidomic changes, which are largely reflected on the whole-cell level, fit within the predominant view that phase separation in membranes requires at least three types of molecules to be present: lipids with high melting temperatures, lipids with low melting temperatures, and sterols.

Investigation of piezoelectric 0.65Pb(Mg1/3Nb2/3)O3–0.35PbTiO3 films in cross section using piezo-response force microscopy

Interest in the piezoelectric and ferroelectric properties of micro- and nanomaterials is increasing due to the advances being made in nanotechnology. However, there are only a few techniques that can detect functional properties at the nanoscale, and one of them is piezo-response force microscopy (PFM). So far, this technique has been mainly used to study surface properties of piezoelectric films. In this investigation, we develop a procedure to study films in the cross section by PFM and to investigate the relaxor-ferroelectric domain structure of pristine, screen-printed, and aerosol-deposited 0.65Pb(Mg1/3Nb2/3)O3–0.35PbTiO3 films in the cross section. Due to the different preparation methods used for two films, the grain size and, thus, the relaxor-ferroelectric domain structures differ. Micron-scale domains are observed in the screen-printed films, while sub micrometer-scale domains are found in the aerosol-deposited films. However, no change in the ferroelectric domain structures was observed across the thicknesses of the films.

Highly Anisotropic Tip-Induced Domain Growth in Polydomain Triglycine Sulfate

Ferroelectrics are widely used as modern functional materials for nonlinear optics, optoelectronics, nonvolatile data storage, and nanoelectronics. Most of these applications require creation of precise micron-scale domain patterns. We have studied the current limited wall motion regime driven by anisotropic bulk screening processes during local switching in polydomain triglycine sulfate single crystals. The field application using a conductive tip of a scanning probe microscope (SPM) in a dry atmosphere resulted in formation of a partially switched area, strongly elongated in the c crystallographic direction. At the same time, switching at high humidity led to a circular shape of the area. The calculation of the spatial distribution of the external field demonstrated the possibility of domain wall shift at distances up to hundreds of microns from the SPM tip. However, after external field switch-off the depolarization field in nonscreened areas led to reverse domain wall motion. Thus, the domain wall stabilization is possible only in the regions with sufficient screening of the depolarization field. We have shown that the growth of the area size with time obeys the same current-limited wall motion expressions that were derived previously for domain growth during local switching by a highly resistive electrode. The change in the area shape with humidity was attributed to the change in the governing screening mechanism from anisotropic bulk conductivity to isotropic surface conductivity through an adsorbed water layer. The obtained results demonstrate the essential role of the screening processes during periodical poling and pave the way for further improvement of the domain engineering methods.

New insight into anodization of aluminium with focused ion beam pre-patterning

The self-ordered anodic aluminium oxide (AAO) structure consists of micron-scale domains—defect-free areas with a hexagonal arrangement of pores. A substantial increase in domain size is possible solely by pre-patterning the aluminium surface in the form of a defect-free hexagonal array of concaves, which guide the pore growth during subsequent anodization. Among the numerous pre-patterning techniques, direct etching by focused gallium ion beam (Ga FIB) allows the preparation of AAO with a custom-made geometry through precise control of the irradiation positions, beam energy, and ion dosage. The main drawback of the FIB approach includes gallium contamination of the aluminium surface. Here, we propose a multi-step anodizing procedure to prevent gallium incorporation into the aluminium substrate. The suggested approach successfully covers a wide range of AAO interpore distances from 100 to 500 nm. In particular, anodization of FIB pre-patterned aluminium in 0.1 M phosphoric acid at 195 V to prepare AAO with the interpore distance of about 500 nm was demonstrated for the first time. The quantification of the degree of pore ordering reveals the fraction of pores in hexagonal coordination above 96% and the in-plane mosaicity below 3° over an area of about 1000 μm2. Large-scale defect-free AAO structures are promising for creating photonic crystals and hyperbolic metamaterials with distinct functional properties.

Magnetic Field-Induced Self-Assembly of Conjugated Block Copolymers and Nanoparticles at the Air-Water Interface.

Here, we report the magnetic field-induced self-assembly of a conjugated block copolymer, poly(3-hexylthiopene)-block-poly(ethylene glycol) (P3HT-b-PEG), and iron oxide nanoparticles (IONPs) at the air-water interface. Binary self-assembly of P3HT-b-PEG and IONPs at the interface results in nanoparticle-embedded P3HT-b-PEG nanowire arrays with a micrometer-scale domain size. Under the magnetic field, the field-induced magnetic interaction significantly improves the degree of order, generating long-range ordered, direction-controlled nanoarrays of P3HT-b-PEG and IONPs, where IONPs are aligned in the direction of the magnetic field over a sub-millimeter scale. The size of IONPs is an important factor for the formation of an ordered assembly structure at the nanometer scale, as it dictates the magnetic dipole interaction and the entropic interaction between nanoparticles and polymers. The consideration of magnetic dipole interactions suggests that the field-induced self-assembly occurs through the formation of intermediate magnetic subunits composed of short IONP strings along the semirigid P3HT nanowires, which can be aligned through the magnetic interactions, ultimately driving the long-range ordered self-assembly. This study demonstrates for the first time that the magnetic field-induced self-assembly can be used to generate macroscopically ordered polymer films with a nanometer-scale order in low fields.

Yeast cells actively tune their membranes to phase separate at temperatures that scale linearly with growth temperatures

Membranes of vacuoles, the lysosomal organelles in yeast, undergo extraordinary changes during the cell's normal growth cycle. The cycle begins with a stage of rapid cell growth. Then, as glucose becomes scarce, growth slows, and the vacuole membranes phase-separate into micron-scale liquid domains. Studies by other researchers suggest that these domains are important for yeast survival by laterally organizing membrane proteins that play a role in a central signaling pathway conserved among eukaryotes (TORC1).

Yeast cells actively tune their membranes to phase separate at temperatures that scale with growth temperatures

Membranes of vacuoles, the lysosomal organelles of Saccharomyces cerevisiae (budding yeast), undergo extraordinary changes during the cell's normal growth cycle. The cycle begins with a stage of rapid cell growth. Then, as glucose becomes scarce, growth slows, and vacuole membranes phase separate into micrometer-scale domains of two liquid phases. Recent studies suggest that these domains promote yeast survival by organizing membrane proteins that play key roles in a central signaling pathway conserved among eukaryotes (TORC1). An outstanding question in the field has been whether cells regulate phase transitions in response to new physical conditions and how this occurs. Here, we measure transition temperatures and find that after an increase of roughly 15 °C, vacuole membranes appear uniform, independent of growth temperature. Moreover, populations of cells grown at a single temperature regulate this transition to occur over a surprisingly narrow temperature range. Remarkably, the transition temperature scales linearly with the growth temperature, demonstrating that the cells physiologically adapt to maintain proximity to the transition. Next, we ask how yeast adjust their membranes to achieve phase separation. We isolate vacuoles from yeast during the rapid stage of growth, when their membranes do not natively exhibit domains. Ergosterol is the major sterol in yeast. We find that domains appear when ergosterol is depleted, contradicting the prevalent assumption that increases in sterol concentration generally cause membrane phase separation invivo, but in agreement with previous studies using artificial and cell-derived membranes.

Domain switching dynamics in relaxor ferroelectric Pb(Mg1/3Nb2/3)O3–PbTiO3 revealed by time-resolved high-voltage electron microscopy

Ferroelectric domain dynamics in Pb(Mg1/3Nb2/3)O3–0.3PbTiO3 single crystals have been studied by in situ biasing high-voltage transmission electron microscopy with a direct electron detection camera. We have achieved time-resolved recording of polarization switching in real space on a 2.5 ms time scale. The reversible response of micrometer-scale domains was observed by applying an electric field of 1 kV/mm. Detailed analyses on smaller sized domains 100–500 nm in size revealed that the domain switching initiated at a corner of a rectangular domain and propagated inward rapidly. The switching proceeded within 60 ms and the maximum switching rate, as fast as 6–8 μm/s, was observed. The domain switching kinetics was classified as two-dimensional nucleation and growth mode based on the Kolmogolov–Avrami–Ishibashi model.

Characterization of micron-scale protein-depleted plasma membrane domains in phosphatidylserine-deficient yeast cells.

Membrane phase separation to form micron-scale domains of lipids and proteins occurs in artificial membranes; however, a similar large-scale phase separation has not been reported in the plasma membrane of the living cells. We show here that a stable micron-scale protein-depleted region is generated in the plasma membrane of yeast mutants lacking phosphatidylserine at high temperatures. We named this region the 'void zone'. Transmembrane proteins and certain peripheral membrane proteins and phospholipids are excluded from the void zone. The void zone is rich in ergosterol, and requires ergosterol and sphingolipids for its formation. Such properties are also found in the cholesterol-enriched domains of phase-separated artificial membranes, but the void zone is a novel membrane domain that requires energy and various cellular functions for its formation. The formation of the void zone indicates that the plasma membrane in living cells has the potential to undergo phase separation with certain lipid compositions. We also found that void zones were frequently in contact with vacuoles, in which a membrane domain was also formed at the contact site.

Surface and bulk ferroelectric phase transition in super-tetragonal BiFeO3 thin films

The temperature-dependent ferroelectric properties of super-tetragonal ${\mathrm{BiFeO}}_{3}$ are investigated using surface-sensitive low-energy electron microscopy (LEEM). We use epitaxial oxide ${\mathrm{BiFeO}}_{3}/{\mathrm{Ca}}_{0.96}{\mathrm{Ce}}_{0.04}{\mathrm{MnO}}_{3}$ bilayers grown by pulsed laser deposition on ${\mathrm{YAlO}}_{3}$ substrates. Ferroelectric, micrometer-scale domains are written by piezoresponse force microscopy and subsequently observed by LEEM from room temperature up to about 950 K. Kelvin probe force microscopy and LEEM spectroscopy reveal that the surface potential is efficiently (>50%) screened by adsorbates that are only released after annealing above 873 $\ifmmode\pm\else\textpm\fi{}$ 50 K in ultrahigh vacuum. The surface structure and chemistry of the ferroelectric thin films are analyzed using scanning transmission electron microscopy, electron energy loss spectroscopy, and x-ray photoelectron spectroscopy, discarding the occurrence of a putative ``skin layer'' effect. While its magnetic and structural transitions were reported in the literature, the true, ferroelectric Curie temperature of super-tetragonal ${\mathrm{BiFeO}}_{3}$ has not been determined so far. Here, we measure a Curie temperature of 930 $\ifmmode\pm\else\textpm\fi{}$ 30 K for the super-tetragonal ${\mathrm{BiFeO}}_{3}$ surface and corroborate it with volume-sensitive, temperature-dependent x-ray diffraction measurements. These results suggest that LEEM can be used as a powerful tool to probe surface charge and ferroelectric transitions in ultrathin films.

Giant energy density and high efficiency achieved in silver niobate-based lead-free antiferroelectric ceramic capacitors via domain engineering

Dielectric materials have drawn increasing attention due to their high power density and fast charge-discharge speed. Although satisfactory energy storage performance has been achieved in lead-based ceramics, the exploration of suitable lead-free substitutions is highly desired since the rising environmental concerns caused by lead-based compounds. Herein, we demonstrate that a giant recoverable energy density of 7.01 J cm−3 together with high energy efficiency under 476 kV cm−1, as well as impressive frequency stability (maximum variation of recoverable energy density < ±3% at 1 − 100 Hz) and thermal stability (maximum variation < ±10% over 25−120 °C) under 320 kV cm−1, can be obtained in Lanthanum (La)-modified AgNbO3 ceramics. It is revealed that the long-range antiferroelectric order in AgNbO3 is interrupted by the incorporation of La, resulting in the transformation of micron-scale antiferroelectric domains into antiferroelectric nanodomains with concurrently improved energy density and efficiency. Additionally, the introduction of La content greatly suppresses the ferrielectricity of AgNbO3 and enhances the dielectric breakdown strength up to 494 kV cm−1 versus 178 kV cm−1 for the pure AgNbO3 ceramics. These results not only reveal the high potential of La-modified AgNbO3 ceramics for energy storage applications but also open up a feasible approach of domain engineering to develop new lead-free energy-storage dielectric materials.

Amyloid-β Interactions with Lipid Rafts in Biomimetic Systems: A Review of Laboratory Methods.

Biomimetic lipid bilayer systems are a useful tool for modeling specific properties of cellular membranes in order to answer key questions about their structure and functions. This approach has prompted scientists from all over the world to create more and more sophisticated model systems in order to decipher the complex lateral and transverse organization of cellular plasma membranes. Among a variety of existing biomembrane domains, lipid rafts are defined as small, dynamic, and ordered assemblies of lipids and proteins, enriched in cholesterol and sphingolipids. Lipid rafts appear to be involved in the development of Alzheimer's disease (AD) by affecting the aggregation of the amyloid-β (Aβ) peptide at neuronal membranes thereby forming toxic oligomeric species. In this review, we summarize the laboratory methods which allow to study the interaction of Aβ with lipid rafts. We describe step by step protocols to form giant (GUVs) and large unilamellar vesicles (LUVs) containing raft-mimicking domains surrounded by membrane nonraft regions. Using fluorescence microscopy GUV imaging protocols, one can design experiments to visualize micron-scale raft-like domains, to determine the micron-scale demixing temperature of a given lipid mixture, construct phase diagram, and photogenerate domains in order to assess the dynamics of raft formation and raft size distribution. LUV fluorescence spectroscopy protocols with proper data analysis can be used to measure molecular packing of raft/nonraft regions of the membrane, to report on nanoscale raft formation and determine nanoscale demixing temperature. Because handling of the Aβ requires dedicated laboratory experience, we present illustrated protocols for Aβ-stock aliquoting, Aβ aqueous solubilization, oligomer preparation, determination of the Aβ concentration before and after filtration. Thioflavin binding, dynamic light scattering, and transmission electron microscopy protocols are described as complementary methods to detect Aβ aggregation kinetics, aggregate sizes, and morphologies of observed aggregates.

Direct imaging of liquid domains in membranes by cryo-electron tomography

Images of micrometer-scale domains in lipid bilayers have provided the gold standard of model-free evidence to understand the domains' shapes, sizes, and distributions. Corresponding techniques to directly and quantitatively assess smaller (nanoscale and submicron) liquid domains have been limited. Researchers commonly seek to correlate activities of membrane proteins with attributes of the domains in which they reside; doing so hinges on identification and characterization of membrane domains. Although some features of membrane domains can be probed by indirect methods, these methods are often constrained by the limitation that data must be analyzed in the context of models that require multiple assumptions or parameters. Here, we address this challenge by developing and testing two methods of identifying submicron domains in biomimetic membranes. Both methods leverage cryo-electron tomograms of ternary membranes under vitrified, hydrated conditions. The first method is optimized for probe-free applications: Domains are directly distinguished from the surrounding membrane by their thickness. This technique quantitatively and accurately measures area fractions of domains, in excellent agreement with known phase diagrams. The second method is optimized for applications in which a single label is deployed for imaging membranes by both high-resolution cryo-electron tomography and diffraction-limited optical microscopy. For this method, we test a panel of probes, find that a trimeric mCherry label performs best, and specify criteria for developing future high-performance, dual-use probes. These developments have led to direct and quantitative imaging of submicron membrane domains in vitrified, hydrated vesicles.

An Autoinhibitory Clamp of Actin Assembly Promotes Synaptic Endocytosis

Synaptic membrane-remodeling events such as endocytosis require force-generating actin assembly. The membrane-cytoskeleton remodeling machinery controlling these events localizes to a micron-scale membrane domain called the ‘periactive zone’ (PAZ). However, endocytic events in the PAZ are paradoxically sparse in space and time given the widespread localization of actin and membrane-regulatory PAZ machinery. Here we describe a mechanism whereby autoinhibition clamps the PAZ machinery to limit actin assembly to discrete functional events. We found that collective interactions between the Drosophila PAZ proteins Nwk/FCHSD2, Dap160/Intersectin, and WASp relieve Nwk autoinhibition and promote robust membrane-coupled actin assembly in vitro. Using automated particle tracking to quantify synaptic actin dynamics in vivo, we discovered that Nwk-Dap160 interactions constrain spurious assembly of WASp-dependent actin structures. These interactions also promote synaptic endocytosis, suggesting that autoinhibition both clamps and primes the synaptic endocytic machinery, thereby constraining actin assembly to drive productive membrane remodeling in response to physiological cues.