The molecular architecture of the nuclear basket.

The nuclear pore complex (NPC) is the sole mediator of nucleocytoplasmic transport. Despite great advances in understanding its conserved core architecture, the peripheral regions can exhibit considerable variation within and between species. One such structure is the cage-like nuclear basket. Despite its crucial roles in mRNA surveillance and chromatin organization, an architectural understanding has remained elusive. Using in-cell cryo-electron tomography and subtomogram analysis, we explored the NPC's structural variations and the nuclear basket across fungi (yeast; S. cerevisiae), mammals (mouse; M. musculus), and protozoa (T. gondii). Using integrative structural modeling, we computed a model of the basket in yeast and mammals that revealed how a hub of nucleoporins (Nups) in the nuclear ring binds to basket-forming Mlp/Tpr proteins: the coiled-coil domains of Mlp/Tpr form the struts of the basket, while their unstructured termini constitute the basket distal densities, which potentially serve as a docking site for mRNA preprocessing before nucleocytoplasmic transport.

Molecular mechanisms of stress-induced reactivation in mumps virus condensates.

Negative-stranded RNA viruses can establish long-term persistent infection in the form of large intracellular inclusions in the human host and cause chronic diseases. Here, we uncover how cellular stress disrupts the metastable host-virus equilibrium in persistent infection and induces viral replication in a culture model of mumps virus. Using a combination of cell biology, whole-cell proteomics, and cryo-electron tomography, we show that persistent viral replication factories are dynamic condensates and identify the largely disordered viral phosphoprotein as a driver of their assembly. Upon stress, increased phosphorylation of the phosphoprotein at its interaction interface with the viral polymerase coincides with the formation of a stable replication complex. By obtaining atomic models for the authentic mumps virus nucleocapsid, we elucidate a concomitant conformational change that exposes the viral genome to its replication machinery. These events constitute a stress-mediated switch within viral condensates that provide an environment to support upregulation of viral replication.

ER-to-Golgi protein delivery through an interwoven, tubular network extending from ER.

Cellular versatility depends on accurate trafficking of diverse proteins to their organellar destinations. For the secretory pathway (followed by approximately 30% of all proteins), the physical nature of the vessel conducting the first portage (endoplasmic reticulum [ER] to Golgi apparatus) is unclear. We provide a dynamic 3D view of early secretory compartments in mammalian cells with isotropic resolution and precise protein localization using whole-cell, focused ion beam scanning electron microscopy with cryo-structured illumination microscopy and live-cell synchronized cargo release approaches. Rather than vesicles alone, the ER spawns an elaborate, interwoven tubular network of contiguous lipid bilayers (ER exit site) for protein export. This receptacle is capable of extending microns along microtubules while still connected to the ER by a thin neck. COPII localizes to this neck region and dynamically regulates cargo entry from the ER, while COPI acts more distally, escorting the detached, accelerating tubular entity on its way to joining the Golgi apparatus through microtubule-directed movement.

A Structured Tumor-Immune Microenvironment in Triple Negative Breast Cancer Revealed by Multiplexed Ion Beam Imaging.

The immune system is critical in modulating cancer progression, but knowledge of immune composition, phenotype, and interactions with tumor is limited. We used multiplexed ion beam imaging by time-of-flight (MIBI-TOF) to simultaneously quantify in situ expression of 36 proteins covering identity, function, and immune regulation at sub-cellular resolution in 41 triple-negative breast cancer patients. Multi-step processing, including deep-learning-based segmentation, revealed variability in the composition of tumor-immune populations across individuals, reconciled by overall immune infiltration and enriched co-occurrence of immune subpopulations and checkpoint expression. Spatial enrichment analysis showed immune mixed and compartmentalized tumors, coinciding with expression of PD1, PD-L1, and IDO in a cell-type- and location-specific manner. Ordered immune structures along the tumor-immune border were associated with compartmentalization and linked to survival. These data demonstrate organization in the tumor-immune microenvironment that is structured in cellular composition, spatial arrangement, and regulatory-protein expression and provide a framework to apply multiplexed imaging to immune oncology.

The Fly Brain Atlas.

The brain's synaptic networks endow an animal with powerfully adaptive biological behavior. Maps of such synaptic circuits densely reconstructed in those model brains that can be examined and manipulated by genetic means offer the best prospect for understanding the underlying biological bases of behavior. That prospect is now technologically feasible and a scientifically enabling possibility in neurobiology, much as genomics has been in molecular biology and genetics. In Drosophila, two major advances are in electron microscopic technology, using focused ion beam-scanning electron microscopy (FIB-SEM) milling to capture and align digital images, and in computer-aided reconstruction of neuron morphologies. The last decade has witnessed enormous progress in detailed knowledge of the actual synaptic circuits formed by real neurons. Advances in various brain regions that heralded identification of the motion-sensing circuits in the optic lobe are now extending to other brain regions, with the prospect of encompassing the fly's entire nervous system, both brain and ventral nerve cord.

In Situ Architecture and Cellular Interactions of PolyQ Inclusions.

Expression of many disease-related aggregation-prone proteins results in cytotoxicity and the formation of large intracellular inclusion bodies. To gain insight into the role of inclusions in pathology and the in situ structure of protein aggregates inside cells, we employ advanced cryo-electron tomography methods to analyze the structure of inclusions formed by polyglutamine (polyQ)-expanded huntingtin exon 1 within their intact cellular context. In primary mouse neurons and immortalized human cells, polyQ inclusions consist of amyloid-like fibrils that interact with cellular endomembranes, particularly of the endoplasmic reticulum (ER). Interactions with these fibrils lead to membrane deformation, the local impairment of ER organization, and profound alterations in ER membrane dynamics at the inclusion periphery. These results suggest that aberrant interactions between fibrils and endomembranes contribute to the deleterious cellular effects of protein aggregation. VIDEO ABSTRACT.

In situ architecture of Opa1-dependent mitochondrial cristae remodeling.

Cristae membrane state plays a central role in regulating mitochondrial function and cellular metabolism. The protein Optic atrophy 1 (Opa1) is an important crista remodeler that exists as two forms in the mitochondrion, a membrane-anchored long form (l-Opa1) and a processed short form (s-Opa1). The mechanisms for how Opa1 influences cristae shape have remained unclear due to lack of native three-dimensional views of cristae. We perform in situ cryo-electron tomography of cryo-focused ion beam milled mouse embryonic fibroblasts with defined Opa1 states to understand how each form of Opa1 influences cristae architecture. In our tomograms, we observe a variety of cristae shapes with distinct trends dependent on s-Opa1:l-Opa1 balance. Increased l-Opa1 levels promote cristae stacking and elongated mitochondria, while increased s-Opa1 levels correlated with irregular cristae packing and round mitochondria shape. Functional assays indicate a role for l-Opa1 in wild-type apoptotic and calcium handling responses, and show a compromised respiratory function under Opa1 imbalance. In summary, we provide three-dimensional visualization of cristae architecture to reveal relationships between mitochondrial ultrastructure and cellular function dependent on Opa1-mediated membrane remodeling.

Airy-beam holographic sonogenetics for advancing neuromodulation precision and flexibility.

Advancing our understanding of brain function and developing treatments for neurological diseases hinge on the ability to modulate neuronal groups in specific brain areas without invasive techniques. Here, we introduce Airy-beam holographic sonogenetics (AhSonogenetics) as an implant-free, cell type-specific, spatially precise, and flexible neuromodulation approach in freely moving mice. AhSonogenetics utilizes wearable ultrasound devices manufactured using 3D-printed Airy-beam holographic metasurfaces. These devices are designed to manipulate neurons genetically engineered to express ultrasound-sensitive ion channels, enabling precise modulation of specific neuronal populations. By dynamically steering the focus of Airy beams through ultrasound frequency tuning, AhSonogenetics is capable of modulating neuronal populations within specific subregions of the striatum. One notable feature of AhSonogenetics is its ability to flexibly stimulate either the left or right striatum in a single mouse. This flexibility is achieved by simply switching the acoustic metasurface in the wearable ultrasound device, eliminating the need for multiple implants or interventions. AhSonogentocs also integrates seamlessly with in vivo calcium recording via fiber photometry, showcasing its compatibility with optical modalities without cross talk. Moreover, AhSonogenetics can generate double foci for bilateral stimulation and alleviate motor deficits in Parkinson's disease mice. This advancement is significant since many neurological disorders, including Parkinson's disease, involve dysfunction in multiple brain regions. By enabling precise and flexible cell type-specific neuromodulation without invasive procedures, AhSonogenetics provides a powerful tool for investigating intact neural circuits and offers promising interventions for neurological disorders.

Graphene sandwich-based biological specimen preparation for cryo-EM analysis.

High-quality specimen preparation plays a crucial role in cryo-electron microscopy (cryo-EM) structural analysis. In this study, we have developed a reliable and convenient technique called the graphene sandwich method for preparing cryo-EM specimens. This method involves using two layers of graphene films that enclose macromolecules on both sides, allowing for an appropriate ice thickness for cryo-EM analysis. The graphene sandwich helps to mitigate beam-induced charging effect and reduce particle motion compared to specimens prepared using the traditional method with graphene support on only one side, therefore improving the cryo-EM data quality. These advancements may open new opportunities to expand the use of graphene in the field of biological electron microscopy.

Direct visualization of proton beam irradiation effects in liquids by MRI.

The main advantage proton beams offer over photon beams in radiation therapy of cancer patients is the dose maximum at their finite range, yielding a reduction in the dose deposited in healthy tissues surrounding the tumor. Since no direct method exists to measure the beam's range during dose delivery, safety margins around the tumor are applied, compromising the dose conformality and reducing the targeting accuracy. Here, we demonstrate that online MRI can visualize the proton beam and reveal its range during irradiation of liquid-filled phantoms. A clear dependence on beam energy and current was found. These results stimulate research into novel MRI-detectable beam signatures and already find application in the geometric quality assurance for magnetic resonance-integrated proton therapy systems currently under development.

Optical dipole-induced anisotropic growth of semiconductors: A facile strategy toward chiral and complex nanostructures.

Chiral nanostructures based on semiconductors exhibit pronounced properties of chiral luminescence and optoelectronic responses, which are fundamental for chiroptoelectronic devices. However, the state-of-the-art techniques of generating semiconductors with chiral configurations are poorly developed, most of which are complicated or of low yield, rendering low compatibility to the platform of optoelectronic devices. Here we show polarization-directed oriented growth of platinum oxide/sulfide nanoparticles based on optical dipole interactions and near-field-enhanced photochemical deposition. By rotating the polarization during the irradiation or employing vector beam, both three dimensional and planar chiral nanostructures can be obtained, which is extendable to cadmium sulfide. These chiral superstructures exhibit broadband optical activity with a g-factor of ~0.2 and a luminescence g-factor of ~0.5 in the visible, making them promising candidate for chiroptoelectronic devices.

Quantification of gallium cryo-FIB milling damage in biological lamellae.

Cryogenic electron microscopy (cryo-EM) can reveal the molecular details of biological processes in their native, cellular environment at atomic resolution. However, few cells are sufficiently thin to permit imaging with cryo-EM. Thinning of frozen cells to <500 nm lamellae by focused-ion-beam (FIB) milling has enabled visualization of cellular structures with cryo-EM. FIB milling represents a significant advance over prior approaches because of its ease of use, scalability, and lack of large-scale sample distortions. However, the amount of damage it causes to a thinned cell section has not yet been determined. We recently described an approach for detecting and identifying single molecules in cryo-EM images of cells using 2D template matching (2DTM). 2DTM is sensitive to small differences between a molecular model (template) and the detected structure (target). Here, we use 2DTM to demonstrate that under the standard conditions used for machining lamellae of biological samples, FIB milling introduces a layer of variable damage that extends to a depth of 60 nm from each lamella surface. This layer of damage limits the recovery of information for in situ structural biology. We find that the mechanism of FIB milling damage is distinct from radiation damage during cryo-EM imaging. By accounting for both electron scattering and FIB milling damage, we estimate that FIB milling damage with current protocols will negate the potential improvements from lamella thinning beyond 90 nm.

Selective binding and periodic arrangement of magic boron clusters on monolayer borophene.

The synthesis and characterization of small boron clusters with unique size and regular arrangement are crucial for boron chemistry and two-dimensional borophene materials. In this study, together with theoretical calculations, the joint molecular beam epitaxy and scanning tunneling microscopy experiments achieve the formation of unique B5 clusters on monolayer borophene (MLB) on a Cu(111) surface. The B5 clusters tend to selectively bind to specific sites of MLB with covalent boron-boron bonds in the periodic arrangement, which can be ascribed to the charge distribution and electron delocalization character of MLB and also prohibits nearby co-adsorption of B5 clusters. Furthermore, the close-packed adsorption of B5 clusters would facilitate the synthesis of bilayer borophene, exhibiting domino effect-like growth mode. The successful growth and characterization of uniform boron clusters on a surface enrich the boron-based nanomaterials and reveal the essential role of small clusters during the growth of borophene.

Targeted volume correlative light and electron microscopy of an environmental marine microorganism.

Photosynthetic microalgae are responsible for an important fraction of CO2 fixation and O2 production on Earth. Three-dimensional (3D) ultrastructural characterization of these organisms in their natural environment can contribute to a deeper understanding of their cell biology. However, the low throughput of volume electron microscopy (vEM) methods along with the complexity and heterogeneity of environmental samples pose great technical challenges. In the present study, we used a workflow based on a specific electron microscopy sample preparation method compatible with both light and vEM imaging in order to target one cell among a complex natural community. This method revealed the 3D subcellular landscape of a photosynthetic dinoflagellate, which we identified as Ensiculifera tyrrhenica, with quantitative characterization of multiple organelles. We show that this cell contains a single convoluted chloroplast and show the arrangement of the flagellar apparatus with its associated photosensitive elements. Moreover, we observed partial chromatin unfolding, potentially associated with transcription activity in these organisms, in which chromosomes are permanently condensed. Together with providing insights in dinoflagellate biology, this proof-of-principle study illustrates an efficient tool for the targeted ultrastructural analysis of environmental microorganisms in heterogeneous mixes.

Spectroscopy in Nanoscopic Cavities: Models and Recent Experiments.

The ability of nanophotonic cavities to confine and store light to nanoscale dimensions has important implications for enhancing molecular, excitonic, phononic, and plasmonic optical responses. Spectroscopic signatures of processes that are ordinarily exceedingly weak such as pure absorption and Raman scattering have been brought to the single-particle limit of detection, while new emergent polaritonic states of optical matter have been realized through coupling material and photonic cavity degrees of freedom across a wide range of experimentally accessible interaction strengths. In this review, we discuss both optical and electron beam spectroscopies of cavity-coupled material systems in weak, strong, and ultrastrong coupling regimes, providing a theoretical basis for understanding the physics inherent to each while highlighting recent experimental advances and exciting future directions.

Single-Cell Imaging Maps Inflammatory Cell Subsets to Pulmonary Arterial Hypertension Vasculopathy.

Rationale: Unraveling immune-driven vascular pathology in pulmonary arterial hypertension (PAH) requires a comprehensive understanding of the immune cell landscape. Although patients with hereditary (H)PAH and bone morphogenetic protein receptor type 2 (BMPR2) mutations have more severe pulmonary vascular pathology, it is not known whether this is related to specific immune cell subsets. Objectives: This study aims to elucidate immune-driven vascular pathology by identifying immune cell subtypes linked to severity of pulmonary arterial lesions in PAH. Methods: We used cutting-edge multiplexed ion beam imaging by time of flight to compare pulmonary arteries (PAs) and adjacent tissue in PAH lungs (idiopathic [I]PAH and HPAH) with unused donor lungs, as controls. Measurements and Main Results: We quantified immune cells' proximity and abundance, focusing on those features linked to vascular pathology, and evaluated their impact on pulmonary arterial smooth muscle cells (SMCs) and endothelial cells. Distinct immune infiltration patterns emerged between PAH subtypes, with intramural involvement independently linked to PA occlusive changes. Notably, we identified monocyte-derived dendritic cells within PA subendothelial and adventitial regions, influencing vascular remodeling by promoting SMC proliferation and suppressing endothelial gene expression across PAH subtypes. In patients with HPAH, pronounced immune dysregulation encircled PA walls, characterized by heightened perivascular inflammation involving T cell immunoglobulin and mucin domain-3 (TIM-3)+ T cells. This correlated with an expanded DC subset expressing indoleamine 2,3-dioxygenase 1, TIM-3, and SAM and HD domain-containing deoxynucleoside triphosphate triphosphohydrolase 1, alongside increased neutrophils, SMCs, and alpha-smooth muscle actin (ACTA2)+ endothelial cells, reinforcing the heightened severity of pulmonary vascular lesions. Conclusions: This study presents the first architectural map of PAH lungs, connecting immune subsets not only with specific PA lesions but also with heightened severity in HPAH compared with IPAH. Our findings emphasize the therapeutic potential of targeting monocyte-derived dendritic cells, neutrophils, cellular interactions, and immune responses to alleviate severe vascular pathology in IPAH and HPAH.

Unveiling the Anomalous Hall Response of the Magnetic Structure Changes in the Epitaxial MnBi2Te4 Films.

Recently discovered as an intrinsic antiferromagnetic topological insulator, MnBi2Te4 has attracted tremendous research interest, as it provides an ideal platform to explore the interplay between topological and magnetic orders. MnBi2Te4 displays distinct exotic topological phases that are inextricably linked to the different magnetic structures of the material. In this study, we conducted electrical transport measurements and systematically investigated the anomalous Hall response of epitaxial MnBi2Te4 films when subjected to an external magnetic field sweep, revealing the different magnetic structures stemming from the interplay of applied fields and the material's intrinsic antiferromagnetic (AFM) ordering. Our results demonstrate that the nonsquare anomalous Hall loop is a consequence of the distinct reversal processes within individual septuple layers. These findings shed light on the intricate magnetic structures in MnBi2Te4 and related materials, offering insights into understanding their transport properties and facilitating the implementation of AFM topological electronics.

Dynamic Spatial-Selective Metasurface with Multiple-Beam Interference.

The emergence of the metasurface has provided a versatile platform for the manipulation of light at the nanoscale. Recent research in metasurfaces has explored a plethora of dynamic control and switching of multifunctionalities, paving the way for innovative applications in fields such as imaging, sensing, and communication. However, current dynamic multifunctional metasurfaces face challenges in terms of functional scalability and selective activation. In this work, we introduce and experimentally demonstrate a strategy that utilizes multiple plane waves to create arbitrary periodic patterns on the metasurface, thus enabling the dynamic and arbitrary spatial-selective activation of its embedded multiplexed functionalities. Furthermore, our strategy facilitates dynamic light control through mechanical translation, as demonstrated by a high-speed, dynamically switchable beam deflection scenario. Our method effectively overcomes the limitations associated with traditional spatially multiplexing techniques, offering greater flexibility and selectivity for dynamic control in multifunctional metasurfaces.

Functionality Expansion of Guided Mode Radiation via On-Chip Metasurfaces.

On-chip metasurfaces play a crucial role in bridging the guided mode and free-space light, enabling full control over the wavefront of scattered free-space light in an optimally compact manner. Recently, researchers have introduced various methods and on-chip metasurfaces to engineer the radiation of guided modes, but the total functions that a single metasurface can achieve are still relatively limited. In this work, we propose a novel on-chip metasurface design that can multiplex up to four distinct functions. We can efficiently control the polarization state, phase, angular momentum, and beam profile of the radiated waves by tailoring the geometry of V-shaped nanoantennas integrated on a slab waveguide. We demonstrate several innovative on-chip metasurfaces for switchable focusing/defocusing and for multifunctional generators of orbital angular momentum beams. Our on-chip metasurface design is expected to advance modern integrated photonics, offering applications in optical data storage, optical interconnection, augmented reality, and virtual reality.

Strain Solitons in an Epitaxially Strained van der Waals-like Material.

Strain solitons are quasi-dislocations that form in van der Waals materials to relieve the energy associated with lattice or rotational mismatch. Novel electronic properties of strain solitons were predicted and observed. To date, strain solitons have been observed only in exfoliated crystals or mechanically strained crystals. The lack of a scalable approach toward the generation of strain solitons poses a significant challenge in the study of and use of their properties. Here, we report the formation of strain solitons with epitaxial growth of bismuth on InSb(111)B by molecular beam epitaxy. The morphology of the strain solitons for films of varying thickness is characterized with scanning tunneling microscopy, and the local strain state is determined from atomic resolution images. Bending in the solitons is attributed to interactions with the interface, and large angle bending is associated with edge dislocations. Our results enable the scalable generation of strain solitons.