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Much evidence has shown that reactive oxygen species (ROS) regulate several plant hormone signaling cascades, but little is known about the real-time kinetics and the underlying molecular mechanisms of the target proteins in the brassinosteroid (BR) signaling pathway. In this study, we used single-molecule techniques to investigate the true signaling timescales of the major BR signaling components BRI1-EMS-SUPPRESSOR 1 (BES1) and BRASSINOSTEROID INSENSITIVE 2 (BIN2) of Arabidopsis thaliana. The rate constants of BIN2 associating with ATP and phosphorylating BES1 were determined to be 0.7 ± 0.4 mM-1 s-1 and 2.3 ± 1.4 s-1, respectively. Interestingly, we found that the interaction of BIN2 and BES1 was oxygen-dependent, and oxygen can directly modify BIN2. The activity of BIN2 was switched on via modification of specific cysteine (Cys) residues, including C59, C95, C99 and C162. The mutation of these Cys residues inhibited the BR signaling outputs. These findings demonstrate the power of using single-molecule techniques to study the dynamic interactions of signaling components, which is difficult to be discovered by conventional physiological and biochemical methods.

The yeast prion protein Sup35 is a translation termination factor, whose activity is modulated by sequestration into a self-perpetuating amyloid. The prion-determining domain, NM, consists of two distinct regions: an amyloidogenic N terminus domain (N) and a charged solubilizing middle region (M). To gain insight into prion conversion, we used single-molecule fluorescence resonance energy transfer (SM-FRET) and fluorescence correlation spectroscopy to investigate the structure and dynamics of monomeric NM. Low protein concentrations in these experiments prevented the formation of obligate on-pathway oligomers, allowing us to study early folding intermediates in isolation from higher-order species. SM-FRET experiments on a dual-labeled amyloid core variant (N21C/S121C, retaining wild-type prion behavior) indicated that the N region of NM adopts a collapsed form similar to \"burst-phase\" intermediates formed during the folding of many globular proteins, even though it lacks a typical hydrophobic core. The mean distance between residues 21 and 121 was [almost equal to]43 Å. This increased with denaturant in a noncooperative fashion to [almost equal to]63 Å, suggesting a multitude of interconverting species rather than a small number of discrete monomeric conformers. Fluorescence correlation spectroscopy analysis of singly labeled NM revealed fast conformational fluctuations on the 20- to 300-ns time scale. Quenching from proximal and distal tyrosines resulted in distinct fast and slower fluctuations. Our results indicate that native monomeric NM is composed of an ensemble of structures, having a collapsed and rapidly fluctuating N region juxtaposed with a more extended M region. The stability of such ensembles is likely to play a key role in prion conversion.

The photophysical properties of single-molecule emitters are altered by nanophotonic structures such as single plasmonic nanoparticles. The intensity and spectral properties of plasmon-coupled emitters have been studied extensively, but little is known about the effect of plasmon coupling on emission polarization. Here, we examine how particle-emitter coupling modifies the polarization of single fluorophores in both experiment and simulation. We quantify degree of linear polarization using Stokes polarimetry with a polarization-sensitive camera and quantify the Stokes parameters with a single-shot acquisition without requiring additional optics in the detection path. We then perform polarization-resolved measurements of plasmon-coupled fluorescence from single-molecule emitters using an approach based on DNA-PAINT. We quantify the effect of the setup and associated noise sources on the measured Stokes parameters. We then quantify the angle of linear polarization (AoLP) and the degree of linear polarization (DoLP) for thousands of single molecules. We compare our results to a numerical model that propagates the plasmon-coupled single-molecule emission through the optical setup to yield the polarized point spread function in the camera plane. Simulations and experiments are in good agreement and shed new light on the polarization of antenna-coupled fluorophores, while it establishes single-shot polarimetry as a promising and straightforward method to quantify polarization properties at the single-molecule level.

To advance the development of novel and efficient electrochemical systems, it is crucial to dynamically image electrochemical reaction processes in real‐time at the single‐particle or single‐molecule level. Single‐molecule fluorescence microscopy has emerged as a powerful tool for in situ imaging of dynamic reaction processes, which is extensively utilized in the field of electrochemical reactions. In this perspective, we provide a concise summary of the recent applications of single‐molecule fluorescence microscopy and super‐resolution fluorescence microscopy within energy electrochemistry. This paper offers insights and evidence regarding electron transfer, surface adsorption, and desorption of reactants, as well as the kinetic processes and mechanisms involved in energy‐related electrochemical reactions. Finally, several remaining challenges are outlined based on the vision for the expanded application of single‐molecule fluorescence microscopy across a broader spectrum of energy‐related fields, including carbon dioxide reduction, methanol electrooxidation, nitric acid electroreduction, furfural electrooxidation reaction, etc. Single‐molecule fluorescence microscopy, characterized by its high spatial and temporal resolution, has been extensively employed to investigate the kinetics of electrochemical reaction processes as well as the distribution of active sites in electrocatalysts. Gaining a profound understanding of the mechanisms underlying electrochemical reactions can provide valuable inspiration for the rational design of high‐performance electrocatalysts in energy‐related fields.

Single‐molecule techniques have been used for only a subset of biological problems because of difficulties in studying proteins that require cofactors or post‐translational modifications. Here, we present a new method integrating single‐molecule fluorescence microscopy and immunopurification to study protein complexes. We used this method to investigate Lin28‐mediated microRNA uridylation by TUT4 (terminal uridylyl transferase 4, polyU polymerase), which regulates let‐7 microRNA biogenesis. Our real‐time analysis of the uridylation by the TUT4 immunoprecipitates suggests that Lin28 functions as a processivity factor of TUT4. Our new technique, SIMPlex (single‐molecule approach to immunoprecipitated protein complexes), provides a universal tool to analyse complex proteins at the single‐molecule level. This study describes a single‐molecule immunoprecipitation method used to investigate the mechanisms of Lin28‐mediated miRNA uridylation. The method expands the scope of single‐molecule approaches to new and complex protein systems.

Trafficking of AMPA receptors in and out of synapses is crucial for synaptic plasticity. Previous studies have focused on the role of endo/exocytosis processes or that of lateral diffusion of extra‐synaptic receptors. We have now directly imaged AMPAR movements inside and outside synapses of live neurons using single‐ molecule fluorescence microscopy. Inside individual synapses, we found immobile and mobile receptors, which display restricted diffusion. Extra‐synaptic receptors display free diffusion. Receptors could also exchange between these membrane compartments through lateral diffusion. Glutamate application increased both receptor mobility inside synapses and the fraction of mobile receptors present in a juxtasynaptic region. Block of inhibitory transmission to favor excitatory synaptic activity induced a transient increase in the fraction of mobile receptors and a decrease in the proportion of juxtasynaptic receptors. Altogether, our data show that rapid exchange of receptors between a synaptic and extra‐synaptic localization occurs through regulation of receptor diffusion inside synapses.

MicroRNAs (miRNAs) associate with components of the RNA‐induced silencing complex (RISC) to assemble on mRNA targets and regulate protein expression in higher eukaryotes. Here we describe a method for the intracellular single‐molecule, high‐resolution localization and counting (iSHiRLoC) of miRNAs. Microinjected, singly fluorophore‐labelled, functional miRNAs were tracked within diffusing particles, a majority of which contained single such miRNA molecules. Mobility and mRNA‐dependent assembly changes suggest the existence of two kinetically distinct pathways for miRNA assembly, revealing the dynamic nature of this important gene regulatory pathway. iSHiRLOC achieves an unprecedented resolution in the visualization of functional miRNAs, paving the way to understanding RNA silencing through single‐molecule systems biology. This study reports a new method—iSHiRLOC—that allows an unprecedented resolution in the visualization of functional small RNAs. Its use has revealed the existence of both a time‐dependent and an mRNA‐dependent pathway for miRNA assembly.

Circular RNAs (circRNAs), a novel RNA type generated by back‐splicing, are key regulators of gene expression, with deregulated expression and established involvement in leukemia. The products of BCL2 and its homologs, including BAX and BCL2L12, are implicated in chronic lymphocytic leukemia (CLL). However, to the best of our knowledge, nothing is known about circRNAs produced by these two genes and their role in CLL. We sought to further elucidate the contribution of BAX and BCL2L12 in CLL by unraveling the identity, localization, and potential role of their circRNAs. Therefore, total RNA from the EHEB cell line and peripheral blood mononuclear cells (PBMCs) of CLL patients and non‐leukemic blood donors was extracted and reverse‐transcribed using random hexamers. Next, nested PCRs with divergent primers were performed and the purified PCR products were subjected to 3rd generation nanopore sequencing. Nested PCRs were also applied to first‐strand cDNAs synthesized from total RNA extracts of PBMCs from CLL patients and non‐leukemic blood donors. Lastly, a single‐molecule resolution fluorescent in situ hybridization method called circFISH was used to visualize the circRNA distribution in EHEB cells. We discovered several novel circRNAs produced by BAX and BCL2L12, which were characterized by great exon structure diversity. In addition, intriguing findings regarding their formation emerged. Interestingly, visualization of the most abundant circRNAs showed distinct intracellular localization. Moreover, a complex BAX and BCL2L12 circRNA expression pattern was revealed in CLL patients and non‐leukemic blood donors. Our data suggest a multifaceted role of BAX and BCL2L12 circRNAs in B‐cell CLL. Novel circular RNAs (circRNAs) deriving from BAX and BCL2L12 were discovered using nanopore sequencing. These circRNAs exhibit diverse exon structures, distinct intracellular localization, and may interact with microRNAs with a proven role in chronic lymphocytic leukemia (CLL). A complex circRNA expression pattern in CLL patients and non‐leukemic blood donors was found as well. Our findings suggest a multifaceted role of these circRNAs in B‐cell CLL.

Atomically precise metal clusters have attracted increasing interest owing to their unique size-dependent properties; however, little has been known about the effect of size on the catalytic properties of metal clusters at the single-cluster level. Here, by real-time monitoring with single-molecule fluorescence microscopy the size-dependent catalytic process of individual Au clusters at single-turnover resolution, we study the size-dependent catalytic behaviors of gold (Au) clusters at the single-cluster level, and then observe the strong size effect on the catalytic properties of individual Au clusters, in both catalytic product formation and dissociation processes. Surprisingly, indicated by both experiments and density functional theory (DFT) calculations, due to such a unique size effect, besides observing the different product dissociation behaviors on different-sized Au clusters, we also observe that small Au clusters [i.e., Au15(MPA)13; here, MPA denotes 3-mercaptopropionic acid] catalyze the product formation through a competitive Langmuir–Hinshelwood mechanism, while those relatively larger Au clusters [e.g., Au18(MPA)14 and Au25(MPA)18] or nanoparticles catalyze the same process through a noncompetitive Langmuir–Hinshelwood mechanism. Such a size effect on the nanocatalysis could be attributed intrinsically to the size-dependent electronic structure of Au clusters. Further analysis of dynamic activity fluctuation of Au clusters reveals more different catalytic properties between Au clusters and traditional Au nanoparticles due to their different size-dependent structures.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is a beta-CoV that recently emerged as a human pathogen and is the causative agent of the COVID-19 pandemic. A molecular framework of how the virus manipulates host cellular machinery to facilitate infection remains unclear. Here, we focus on SARS-CoV-2 NSP1, which is proposed to be a virulence factor that inhibits protein synthesis by directly binding the human ribosome. We demonstrate biochemically that NSP1 inhibits translation of model human and SARS-CoV-2 messenger RNAs (mRNAs). NSP1 specifically binds to the small (40S) ribosomal subunit, which is required for translation inhibition. Using single-molecule fluorescence assays to monitor NSP1–40S subunit binding in real time, we determine that eukaryotic translation initiation factors (eIFs) allosterically modulate the interaction of NSP1 with ribosomal preinitiation complexes in the absence of mRNA. We further elucidate that NSP1 competes with RNA segments downstream of the start codon to bind the 40S subunit and that the protein is unable to associate rapidly with 80S ribosomes assembled on an mRNA. Collectively, our findings support a model where NSP1 proteins from viruses in at least two subgenera of beta-CoVs associate with the open head conformation of the 40S subunit to inhibit an early step of translation, by preventing accommodation of mRNA within the entry channel.