Plasma membranes (PMs) are highly dynamic structures where lipids and proteins can theoretically diffuse freely. However, reports indicate that PM proteins do not freely diffuse within their planes but are constrained by cytoskeleton networks, though the mechanisms for how the cytoskeleton restricts lateral diffusion of plant PM proteins are unclear. Through single-molecule tracking, we investigated the dynamics of six Arabidopsis (Arabidopsis thaliana) PM proteins with diverse structures and found distinctions in sizes and dynamics among these proteins. Moreover, we showed that the cytoskeleton, particularly microtubules, limits the diffusion of PM proteins, including transmembrane and membrane-anchoring proteins. Interestingly, the microfilament skeleton regulates intracellular transport of endocytic cargo. Therefore, these findings indicate that the cytoskeleton controls signal transduction by limiting diffusion of PM proteins in specific membrane compartments and participating in transport of internalized cargo vesicles, thus actively regulating plant signal transduction.

Contactin 2 (CNTN2) is a cell adhesion molecule involved in axon guidance, neuronal migration, and fasciculation. The ectodomains of CNTN1-CNTN6 are composed of six Ig domains (Ig1-Ig6) and four FN domains. Here, we show that CNTN2 forms transient homophilic interactions (KD ∼200 nM). Cryo-EM structures of full-length CNTN2 and CNTN2_Ig1-Ig6 reveal a T-shaped homodimer formed by intertwined, parallel monomers. Unexpectedly, the horseshoe-shaped Ig1-Ig4 headpieces extend their Ig2-Ig3 tips outwards on either side of the homodimer, while Ig4, Ig5, Ig6, and the FN domains form a central stalk. Cross-linking mass spectrometry and cell-based binding assays confirm the 3D assembly of the CNTN2 homodimer. The interface mediating homodimer formation differs between CNTNs, as do the homophilic versus heterophilic interaction mechanisms. The CNTN family thus encodes a versatile molecular platform that supports a very diverse portfolio of protein interactions and that can be leveraged to strategically guide neural circuit development.

Single-particle cryo-electron microscopy (cryo-EM) has become an essential structural determination technique with recent hardware developments making it possible to reach atomic resolution, at which individual atoms, including hydrogen atoms, can be resolved. In this study, we used the enzyme involved in the penultimate step of riboflavin biosynthesis as a test specimen to benchmark a recently installed microscope and determine if other protein complexes could reach a resolution of 1.5 Å or better, which so far has only been achieved for the iron carrier ferritin. Using state-of-the-art microscope and detector hardware as well as the latest software techniques to overcome microscope and sample limitations, a 1.42 Å map of Aquifex aeolicus lumazine synthase (AaLS) was obtained from a 48 h microscope session. In addition to water molecules and ligands involved in the function of AaLS, we can observe positive density for ∼50% of the hydrogen atoms. A small improvement in the resolution was achieved by Ewald sphere correction which was expected to limit the resolution to ∼1.5 Å for a molecule of this diameter. Our study confirms that other protein complexes can be solved to near-atomic resolution. Future improvements in specimen preparation and protein complex stabilization may allow more flexible macromolecules to reach this level of resolution and should become a priority of study in the field.

Nanosized ultrafine particles (UFPs) from natural and anthropogenic sources are widespread and pose serious health risks when inhaled by humans. However, tracing the inhaled UFPs in vivo is extremely difficult, and the distribution, translocation, and metabolism of UFPs remain unclear. Here, we report a label-free, machine learning-aided single-particle inductively coupled plasma mass spectrometry (spICP-MS) approach for tracing the exposure pathways of airborne magnetite nanoparticles (MNPs), including external emission sources, and distribution and translocation in vivo using a mouse model. Our results provide quantitative analysis of different metabolic pathways in mice exposed to MNPs, revealing that the spleen serves as the primary site for MNP metabolism (84.4%), followed by the liver (11.4%). The translocation of inhaled UFPs across different organs alters their particle size. This work provides novel insights into the in vivo fate of UFPs as well as a versatile and powerful platform for nanotoxicology and risk assessment.

Lipid-based nanomedicines (LBNMs), including liposomes, lipid nanoparticles (LNPs) and extracellular vesicles (EVs), are recognized as one of the most clinically acceptable nano-formulations. However, the bench-to-bedside translation efficiency is far from satisfactory, mainly due to the lack of in-depth understanding of their physical and biochemical attributes at the single-particle level. In this review, we first give a brief introduction of LBNMs, highlighting some milestones and related scientific and clinical achievements in the past several decades, as well as the grand challenges in the characterization of LBNMs. Next, we present an overview of each category of LBNMs as well as the core properties that largely dictate their biological characteristics and clinical performance, such as size distribution, particle concentration, morphology, drug encapsulation and surface properties. Then, the recent applications of several analytical techniques including electron microscopy, atomic force microscopy, fluorescence microscopy, Raman microscopy, nanoparticle tracking analysis, tunable resistive pulse sensing and flow cytometry on the single-particle characterization of LBNMs are thoroughly discussed. Particularly, the comparative advantages of the newly developed nano-flow cytometry that enables quantitative analysis of both the physical and biochemical characteristics of LBNMs smaller than 40 nm with high throughput and statistical robustness are emphasized. The overall aim of this review article is to illustrate the importance, challenges and achievements associated with single-particle characterization of LBNMs.

BACKGROUND: Detection of elements in individual cells by inductively coupled plasma (ICP) spectrometry has recently attracted significant interest in biological research, due to the unique ability of ICP spectrometry for trace element analysis. However, performing single-cell analysis using ICP optical emission spectrometry (ICP-OES) remains a challenge due to the small size and discrete nature of cells. This is while ICP-OES can serve as a cost-effective and label-free method for this purpose. Therefore, it is necessary to improve the current ICP-OES technique to facilitate the detection of elements in single cells, thereby unlocking novel applications.
RESULTS: A new conical ICP torch, which has been illustrated to offer better analytical performance than the conventional ones, was applied to achieve the detection of calcium in single micro-sized cells. A new heated chamber was designed and coupled with a high-efficiency nebulizer as the sample introduction system. For the detection of single SiO2 particles, the number of particle events obtained by the new sample introduction system was found to be up to 9 times higher than that of the conventional system without sacrificing the signal intensity. Subsequently, calcium in human breast cancer cells (MDA-MB-231), mice breast cancer cells (Py8119), and mice osteocytes (MLO-Y4) was successfully detected using the new ICP-OES system. The cell detection efficiency turned out to be around 2%-3% which is much higher than that the reported values in previous single-cell ICP-OES research. Finally, as a new application, the effect of Yoda1, a recently identified activator of Piezo1 calcium channel, on osteocytes was investigated. The calcium content in Yoda1-treated MLO-Y4 cells was seen increase by 36% compared to the control sample.
CONCLUSIONS: This research reveals the capability of ICP-OES in single-cell analysis for micro-sized cells which was made possible by the new conical ICP torch and the new sample introduction system. The ability to detect calcium in single mammalian cells enables the first ever application of this technique to assess the impact of the Yoda1 activator on the calcium level in osteocytes.

The 21st amino acid, selenocysteine (Sec), is synthesized on its dedicated transfer RNA (tRNASec). In bacteria, Sec is synthesized from Ser-tRNA[Ser]Sec by Selenocysteine Synthase (SelA), which is a pivotal enzyme in the biosynthesis of Sec. The structural characterization of bacterial SelA is of paramount importance to decipher its catalytic mechanism and its role in the regulation of the Sec-synthesis pathway. Here, we present a comprehensive single-particle cryo-electron microscopy (SPA cryoEM) structure of the bacterial SelA with an overall resolution of 2.69 Å. Using recombinant Escherichia coli SelA, we purified and prepared samples for single-particle cryoEM. The structural insights from SelA, combined with previous in vivo and in vitro knowledge, underscore the indispensable role of decamerization in SelA\'s function. Moreover, our structural analysis corroborates previous results that show that SelA adopts a pentamer of dimers configuration, and the active site architecture, substrate binding pocket, and key K295 catalytic residue are identified and described in detail. The differences in protein architecture and substrate coordination between the bacterial enzyme and its counterparts offer compelling structural evidence supporting the independent molecular evolution of the bacterial and archaea/eukarya Ser-Sec biosynthesis present in the natural world.

The pH of atmospheric aerosols is a key characteristic that profoundly influences their impacts on climate change, human health, and ecosystems. Despite widely performed aerosol pH research, determining the pH levels of individual atmospheric aerosol particles has been a challenge. This study presents a novel analytical technique that utilizes surface-enhanced Raman spectroscopy to assess the pH of individual ambient PM2.5-10 aerosol particles in conjunction with examining their hygroscopic behavior, morphology, and elemental compositions. The results revealed a substantial pH variation among simultaneously collected aerosol particles, ranging from 3.3 to 5.7. This variability is likely related to each particle\'s unique reaction and aging states. The extensive particle-to-particle pH variability suggests that atmospheric aerosols present at the same time and location can exhibit diverse reactivities, reaction pathways, phase equilibria, and phase separation properties. This pioneering study paves the way for in-depth investigations into particle-to-particle variability, size dependency, and detailed spatial and temporal variations of aerosol pH, thus deepening our understanding of atmospheric chemistry and its environmental implications.

Structural insights into macromolecular and protein complexes provide key clues about the molecular basis of the function. Cryogenic electron microscopy (cryo-EM) has emerged as a powerful structural biology method for studying protein and macromolecular structures at high resolution in both native and near-native states. Despite the ability to get detailed structural insights into the processes underlying protein function using cryo-EM, there has been hesitancy amongst plant biologists to apply the method for biomolecular interaction studies. This is largely evident from the relatively fewer structural depositions of proteins and protein complexes from plant origin in electron microscopy databank. Even though the progress has been slow, cryo-EM has significantly contributed to our understanding of the molecular biology processes underlying photosynthesis, energy transfer in plants, besides viruses infecting plants. This chapter introduces sample preparation for both negative-staining electron microscopy (NSEM) and cryo-EM for plant proteins and macromolecular complexes and data analysis using single particle analysis for beginners.

In addressing the challenges faced by laboratories and universities with limited (or no) cryo-electron microscopy (cryo-EM) infrastructure, the ESRF, in collaboration with the Grenoble Institute for Structural Biology (IBS), has implemented the cryo-EM Solution-to-Structure (SOS) pipeline. This inclusive process, spanning grid preparation to high-resolution data collection, covers single-particle analysis and cryo-electron tomography (cryo-ET). Accessible through a rolling access route, proposals undergo scientific merit and technical feasibility evaluations. Stringent feasibility criteria demand robust evidence of sample homogeneity. Two distinct entry points are offered: users can either submit purified protein samples for comprehensive processing or initiate the pipeline with already vitrified cryo-EM grids. The SOS pipeline integrates negative stain imaging (exclusive to protein samples) as a first quality step, followed by cryo-EM grid preparation, grid screening and preliminary data collection for single-particle analysis, or only the first two steps for cryo-ET. In both cases, if the screening steps are successfully completed, high-resolution data collection will be carried out using a Titan Krios microscope equipped with a latest-generation direct electron counting detector coupled to an energy filter. The SOS pipeline thus emerges as a comprehensive and efficient solution, further democratizing access to cryo-EM research.