Single-molecule Visualization Research Articles

Single-molecule live-cell RNA imaging with CRISPR-Csm.

High-resolution, real-time imaging of RNA is essential for understanding the diverse, dynamic behaviors of individual RNA molecules in single cells. However, single-molecule live-cell imaging of unmodified endogenous RNA has not yet been achieved. Here, we present single-molecule live-cell fluorescence in situ hybridization (smLiveFISH), a robust approach that combines the programmable RNA-guided, RNA-targeting CRISPR-Csm complex with multiplexed guide RNAs for efficient, direct visualization of single RNA molecules in a range of cell types, including primary cells. Using smLiveFISH, we tracked individual endogenous NOTCH2 and MAP1B mRNA transcripts in living cells and identified two distinct localization mechanisms: co-translational translocation of NOTCH2 mRNA at the endoplasmic reticulum, and directional transport of MAP1B mRNA toward the cell periphery. This method has the potential to unlock principles governing the spatiotemporal organization of native transcripts in health and disease.

Single molecule visualization of tropomyosin isoform organization in the mammalian actin cytoskeleton.

The actin cytoskeleton is composed of both branched and unbranched actin filaments. In mammals, the unbranched actin filaments are primarily copolymers of actin and tropomyosin. Biochemical and imaging studies indicate that different tropomyosin isoforms are segregated to different actin filament populations in cells and tissues, providing isoform-specific functionality to the actin filament. Intrinsic to this model is the prediction that single-molecule imaging of tropomyosin isoforms would confirm homopolymer formation along the length of single actin filaments, a knowledge gap that remains unaddressed in the cellular environment. We combined chemical labeling of genetically engineered tropomyosin isoforms with electron tomography to locate individual tropomyosin molecules in fibroblasts. We find that the organization of two non-muscle tropomyosins, Tpm3.1 with Tpm4.2, can be distinguished from each other using light and electron microscopy. Visualization of single tropomyosin molecules associated with actin filaments supports the hypothesis that tropomyosins form continuous homopolymers, instead of heteropolymers, in the presence of all physiologically native actin-binding proteins. This is true for both isoforms tested. Furthermore, the data suggest that the tropomyosin molecules on one side of an actin filament may not be in register with those on the opposite side, indicating that each tropomyosin polymer may assembly independently.

Real-time single-molecule visualization using DNA curtains reveals the molecular mechanisms underlying DNA repair pathways

The demand for direct observation of biomolecular interactions provides new insights into the molecular mechanisms underlying many biological processes. Single-molecule imaging techniques enable real-time visualization of individual biomolecules, providing direct observations of protein machines. Various single-molecule imaging techniques have been developed and have contributed to breakthroughs in biological research. One such technique is the DNA curtain, a novel, high-throughput, single-molecule platform that integrates lipid fluidity, nano-fabrication, microfluidics, and fluorescence imaging. Many DNA metabolic reactions, such as replication, transcription, and chromatin dynamics, have been studied using DNA curtains. In particular, the DNA curtain platform has been intensively applied in investigating the molecular details of DNA repair processes. This article reviews DNA curtain techniques and their applications for imaging DNA repair proteins.

Single-molecule visualization of twin-supercoiled domains generated during transcription.

Transcription-coupled supercoiling of DNA is a key factor in chromosome compaction and the regulation of genetic processes in all domains of life. It has become common knowledge that, during transcription, the DNA-dependent RNA polymerase (RNAP) induces positive supercoiling ahead of it (downstream) and negative supercoils in its wake (upstream), as rotation of RNAP around the DNA axis upon tracking its helical groove gets constrained due to drag on its RNA transcript. Here, we experimentally validate this so-called twin-supercoiled-domain model with in vitro real-time visualization at the single-molecule scale. Upon binding to the promoter site on a supercoiled DNA molecule, RNAP merges all DNA supercoils into one large pinned plectoneme with RNAP residing at its apex. Transcription by RNAP in real time demonstrates that up- and downstream supercoils are generated simultaneously and in equal portions, in agreement with the twin-supercoiled-domain model. Experiments carried out in the presence of RNases A and H, revealed that an additional viscous drag of the RNA transcript is not necessary for the RNAP to induce supercoils. The latter results contrast the current consensus and simulations on the origin of the twin-supercoiled domains, pointing at an additional mechanistic cause underlying supercoil generation by RNAP in transcription.

Single-molecule visualization of human A2A adenosine receptor activation by a G protein and constitutively activating mutations

Mutations that constitutively activate G protein-coupled receptors (GPCRs), known as constitutively activating mutations (CAMs), modify cell signaling and interfere with drugs, resulting in diseases with limited treatment options. We utilize fluorescence imaging at the single-molecule level to visualize the dynamic process of CAM-mediated activation of the human A2A adenosine receptor (A2AAR) in real time. We observe an active-state population for all CAMs without agonist stimulation. Importantly, activating mutations significantly increase the population of an intermediate state crucial for receptor activation, notably distinct from the addition of a partner G protein. Activation kinetics show that while CAMs increase the frequency of transitions to the intermediate state, mutations altering sodium sensitivity increase transitions away from it. These findings indicate changes in GPCR function caused by mutations may be predicted based on whether they favor or disfavor formation of an intermediate state, providing a framework for designing receptors with altered functions or therapies that target intermediate states.

Chromogenic Visualization of Single RNA Molecules In Situ with Duplex Capability by Rolling Circle Amplification with Alkaline Phosphatase.

Visualizing spatial patterns of gene expression by optical microscopy at single-molecule resolution represents a long-standing challenge for imaging and molecular engineering technologies. In this study, we developed a method for visualizing mRNA with duplex capability by optical microscopy using rolling circle amplification with streptavidin-modified alkaline phosphatase (SA-ALP) to provide highly selective and sensitive RNA detection. ALP-based RNA detection provides comparable sensitivity and specificity to fluorescence-based in situ assays and similar performance to the current RNAscope technique for single-molecule RNA detection, but with improved ease of operation. This versatile and relatively user-friendly method of single-molecule RNA visualization can also overcome common problems of background interference. Our findings show that the red spots generated by the Fast Red staining in situ are readily quantified by image analysis, even in samples with heavy melanin deposition, supporting the clinical translation of this assay to improve diagnostic assays for recalcitrant tissues. This system was adaptable for duplex assays with multiple probes and multiple stains, which is ALP with horseradish peroxidase to produce red and brown signals to simultaneously visualize two different RNA targets. The duplex assay can be successfully applied to quantify mRNA expression from two genes in situ within single cells and multiple cell types. With the advantages of high sensitivity and low hardware requirements, this versatile and user-friendly method of RNA visualization may enable low-resource institutions to conduct previously inaccessible diagnostic or research questions about the in situ expression and distribution of RNAs at single-molecule resolution.

Single-molecule visualization of stalled replication-fork rescue by the Escherichia coli Rep helicase.

Genome duplication occurs while the template DNA is bound by numerous DNA-binding proteins. Each of these proteins act as potential roadblocks to the replication fork and can have deleterious effects on cells. In Escherichia coli, these roadblocks are displaced by the accessory helicase Rep, a DNA translocase and helicase that interacts with the replisome. The mechanistic details underlying the coordination with replication and roadblock removal by Rep remain poorly understood. Through real-time fluorescence imaging of the DNA produced by individual E. coli replisomes and the simultaneous visualization of fluorescently-labeled Rep, we show that Rep continually surveils elongating replisomes. We found that this association of Rep with the replisome is stochastic and occurs independently of whether the fork is stalled or not. Further, we visualize the efficient rescue of stalled replication forks by directly imaging individual Rep molecules as they remove a model protein roadblock, dCas9, from the template DNA. Using roadblocks of varying DNA-binding stabilities, we conclude that continuation of synthesis is the rate-limiting step of stalled replication rescue.

Visualization of Single Dynein Molecules in Mammalian Cells.

In vitro single-molecule imaging experiments have provided insight into the stepping behavior, force production, and activation of several molecular motors. However, due to the difficulty in visualizing single molecules of motor proteins in vivo, the physiological function and regulation of motors at the single-molecule level have not been studied widely. Here, we describe how highly inclined and laminated optical sheet (HILO) microscopy can be adapted to visualize single molecules of the motor protein cytoplasmic dynein-1 in mammalian cells with high signal-to-noise ratio and temporal resolution.

Stabilization of DNA fork junctions by Smc5/6 complexes revealed by single-molecule imaging.

SMC complexes play key roles in genome maintenance, where they ensure efficient genome replication and segregation. The SMC complex Smc5/6 is a crucial player in DNA replication and repair, yet many molecular features that determine its roles are unclear. Here, we use single-molecule microscopy to investigate Smc5/6's interaction with DNA. We find that Smc5/6 forms oligomers that dynamically redistribute on dsDNA by 1D diffusion and statically bind to ssDNA. Using combined force manipulation and single-molecule microscopy, we generate ssDNA-dsDNA junctions that mimic structures present in DNA repair intermediates or replication forks. We show that Smc5/6 accumulates at these junction sites, stabilizes the fork, and promotes the retention of RPA. Our observations provide a model for the complex's enrichment at sites of replication stress and DNA lesions from where it coordinates the recruitment and activation of downstream repair proteins.

Origin recognition complex harbors an intrinsic nucleosome remodeling activity

Eukaryotic DNA replication is initiated at multiple chromosomal sites known as origins of replication that are specifically recognized by the origin recognition complex (ORC) containing multiple ATPase sites. In budding yeast, ORC binds to specific DNA sequences known as autonomously replicating sequences (ARSs) that are mostly nucleosome depleted. However, nucleosomes may still inhibit the licensing of some origins by occluding ORC binding and subsequent MCM helicase loading. Using purified proteins and single-molecule visualization, we find here that the ORC can eject histones from a nucleosome in an ATP-dependent manner. The ORC selectively evicts H2A-H2B dimers but leaves the (H3-H4)2 tetramer on DNA. It also discriminates canonical H2A from the H2A.Z variant, evicting the former while retaining the latter. Finally, the bromo-adjacent homology (BAH) domain of the Orc1 subunit is essential for ORC-mediated histone eviction. These findings suggest that the ORC is a bona fide nucleosome remodeler that functions to create a local chromatin environment optimal for origin activity.

SMC complexes can traverse physical roadblocks bigger than their ring size.

Ring-shaped structural maintenance of chromosomes (SMC) complexes like condensin and cohesin extrude loops of DNA. It remains, however, unclear how they can extrude DNA loops in chromatin that is bound with proteins. Here, we use invitro single-molecule visualization to show that nucleosomes, RNA polymerase, and dCas9 pose virtually no barrier to loop extrusion by yeast condensin. We find that even DNA-bound nanoparticles as large as 200nm, much bigger than the SMC ring size, also translocate into DNA loops during extrusion by condensin and cohesin. This even occurs for a single-chain version of cohesin in which the ring-forming subunits are covalently linked and cannot open to entrap DNA. The data show that SMC-driven loop extrusion has surprisingly little difficulty in accommodating large roadblocks into the loop. The findings also show that the extruded DNA does not pass through the SMC ring (pseudo)topologically, hence pointing to a nontopological mechanism for DNA loop extrusion.

DNA Visualization Using Fluorescent Proteins.

DNA binding fluorescent proteins are a powerful tool for single-molecule visualization. In this chapter, we discuss a protocol for the synthesis of DNA binding fluorescent proteins and visualization of single DNA molecules. This chapter includes stepwise methods for molecular cloning, reversible staining, two-color staining, sequence-specific staining, and microscopic visualization of single DNA molecules in a microfluidic device. This content will be useful for DNA characterization using DNA binding fluorescent proteins and its visualization at the single-molecule level.

Nucleosome-directed replication origin licensing independent of a consensus DNA sequence

The numerous enzymes and cofactors involved in eukaryotic DNA replication are conserved from yeast to human, and the budding yeast Saccharomyces cerevisiae (S.c.) has been a useful model organism for these studies. However, there is a gap in our knowledge of why replication origins in higher eukaryotes do not use a consensus DNA sequence as found in S.c. Using in vitro reconstitution and single-molecule visualization, we show here that S.c. origin recognition complex (ORC) stably binds nucleosomes and that ORC-nucleosome complexes have the intrinsic ability to load the replicative helicase MCM double hexamers onto adjacent nucleosome-free DNA regardless of sequence. Furthermore, we find that Xenopus laevis nucleosomes can substitute for yeast ones in engaging with ORC. Combined with re-analyses of genome-wide ORC binding data, our results lead us to propose that the yeast origin recognition machinery contains the cryptic capacity to bind nucleosomes near a nucleosome-free region and license origins, and that this nucleosome-directed origin licensing paradigm generalizes to all eukaryotes.

Electron microscopic visualization of single molecules by tag-mediated metal particle labeling.

Genetically encoded tags have introduced extensive lines of application from purification of tagged proteins to their visualization at the single molecular, cellular, histological and whole-body levels. Combined with other rapidly developing technologies such as clustered regularly interspaced palindromic repeats (CRISPR)/CRISPR-associated protein 9 (Cas9) system, proteomics, super-resolution microscopy and proximity labeling, a large variety of genetically encoded tags have been developed in the last two decades. In this review, I focus on the current status of tag development for electron microscopic (EM) visualization of proteins with metal particle labeling. Compared with conventional immunoelectron microscopy using gold particles, tag-mediated metal particle labeling has several advantages that could potentially improve the sensitivity, spatial and temporal resolution, and applicability to a wide range of proteins of interest (POIs). It may enable researchers to detect single molecules in situ, allowing the quantitative measurement of absolute numbers and exact localization patterns of POI in the ultrastructural context. Thus, genetically encoded tags for EM could revolutionize the field as green fluorescence protein did for light microscopy, although we still have many challenges to overcome before reaching this goal.

Visualization and identification of single meteoritic organic molecules by atomic force microscopy.

Using high‐resolution atomic force microscopy (AFM) with CO‐functionalized tips, we atomically resolved individual molecules from Murchison meteorite samples. We analyzed powdered Murchison meteorite material directly, as well as processed extracts that we prepared to facilitate characterization by AFM. From the untreated Murchison sample, we resolved very few molecules, as the sample contained mostly small molecules that could not be identified by AFM. By contrast, using a procedure based on several trituration and extraction steps with organic solvents, we isolated a fraction enriched in larger organic compounds. The treatment increased the fraction of molecules that could be resolved by AFM, allowing us to identify organic constituents and molecular moieties, such as polycyclic aromatic hydrocarbons and aliphatic chains. The AFM measurements are complemented by high‐resolution mass spectrometry analysis of Murchison fractions. We provide a proof of principle that AFM can be used to image and identify individual organic molecules from meteorites and propose a method for extracting and preparing meteorite samples for their investigation by AFM. We discuss the challenges and prospects of this approach to study extraterrestrial samples based on single‐molecule identification.

Single-molecule visualization of conformation and dynamics of P-glycoprotein

P-glycoprotein (Pgp) is an ATP-binding cassette transporter which effluxes foreign bodies out of a cell. It non-selectively binds a variety of oncology drugs and is overexpressed in cancer cells, making it a leading cause of multi-drug resistance. Understanding Pgp’s mechanism of action is a critical research effort. Single-molecule atomic force microscopy (AFM) is a technique that directly visualizes membrane-external protrusions of proteins embedded in native lipid environments at physiologically relevant temperatures and ionic strength.

Direct R-Loop Visualization on Genomic DNA by Native Automated Electron Microscopy.

R-loop are physiologically present on genomic DNA of different organisms and play important roles in genome regulation. However, an increase in their abundance and/or size has been suggested to interfere with the DNA replication process, contributing to genome instability. Most available approaches to monitor R-loops are based on antibodies/enzymes that cannot effectively distinguish R-loops from DNA-RNA hybrids and assess R-loop size and frequency in a population of molecules. Electron microscopy has successfully allowed single-molecule visualization of DNA replication and repair intermediates, uncovering key architectural modifications in DNA, induced by genotoxic stress or by the associated cellular response. Here, we describe recent modifications of this visualization workflow to implement partial automation of image acquisition and analysis. Coupling this refined workflow with sample preparation procedures that protect R-loop stability allows for direct visualization of R-loop structures on genomic DNA, independently from probes. Combining single-molecule information and DNA content assessment, this approach provides direct estimations of R-loop frequency, size, and burden on genomic DNA.

Distributed synthesis of sarcolemmal and sarcoplasmic reticulum membrane proteins in cardiac myocytes

It is widely assumed that synthesis of membrane proteins, particularly in the heart, follows the classical secretory pathway with mRNA translation occurring in perinuclear regions followed by protein trafficking to sites of deployment. However, this view is based on studies conducted in less-specialized cells, and has not been experimentally addressed in cardiac myocytes. Therefore, we undertook direct experimental investigation of protein synthesis in cardiac tissue and isolated myocytes using single-molecule visualization techniques and a novel proximity-ligated in situ hybridization approach for visualizing ribosome-associated mRNA molecules for a specific protein species, indicative of translation sites. We identify here, for the first time, that the molecular machinery for membrane protein synthesis occurs throughout the cardiac myocyte, and enables distributed synthesis of membrane proteins within sub-cellular niches where the synthesized protein functions using local mRNA pools trafficked, in part, by microtubules. We also observed cell-wide distribution of membrane protein mRNA in myocardial tissue from both non-failing and hypertrophied (failing) human hearts, demonstrating an evolutionarily conserved distributed mechanism from mouse to human. Our results identify previously unanticipated aspects of local control of cardiac myocyte biology and highlight local protein synthesis in cardiac myocytes as an important potential determinant of the heart’s biology in health and disease.

Single-Molecule Insights Into the Dynamics of Replicative Helicases.

Helicases are molecular motors that translocate along single-stranded DNA and unwind duplex DNA. They rely on the consumption of chemical energy from nucleotide hydrolysis to drive their translocation. Specialized helicases play a critically important role in DNA replication by unwinding DNA at the front of the replication fork. The replicative helicases of the model systems bacteriophages T4 and T7, Escherichia coli and Saccharomyces cerevisiae have been extensively studied and characterized using biochemical methods. While powerful, their averaging over ensembles of molecules and reactions makes it challenging to uncover information related to intermediate states in the unwinding process and the dynamic helicase interactions within the replisome. Here, we describe single-molecule methods that have been developed in the last few decades and discuss the new details that these methods have revealed about replicative helicases. Applying methods such as FRET and optical and magnetic tweezers to individual helicases have made it possible to access the mechanistic aspects of unwinding. It is from these methods that we understand that the replicative helicases studied so far actively translocate and then passively unwind DNA, and that these hexameric enzymes must efficiently coordinate the stepping action of their subunits to achieve unwinding, where the size of each step is prone to variation. Single-molecule fluorescence microscopy methods have made it possible to visualize replicative helicases acting at replication forks and quantify their dynamics using multi-color colocalization, FRAP and FLIP. These fluorescence methods have made it possible to visualize helicases in replication initiation and dissect this intricate protein-assembly process. In a similar manner, single-molecule visualization of fluorescent replicative helicases acting in replication identified that, in contrast to the replicative polymerases, the helicase does not exchange. Instead, the replicative helicase acts as the stable component that serves to anchor the other replication factors to the replisome.

Effect of Spherical Elements of Biosensors and Bioreactors on the Physicochemical Properties of a Peroxidase Protein.

External electromagnetic fields are known to be able to concentrate inside the construction elements of biosensors and bioreactors owing to reflection from their surface. This can lead to changes in the structure of biopolymers (such as proteins), incubated inside these elements, thus influencing their functional properties. Our present study concerned the revelation of the effect of spherical elements, commonly employed in biosensors and bioreactors, on the physicochemical properties of proteins with the example of the horseradish peroxidase (HRP) enzyme. In our experiments, a solution of HRP was incubated within a 30 cm-diameter titanium half-sphere, which was used as a model construction element. Atomic force microscopy (AFM) was employed for the single-molecule visualization of the HRP macromolecules, adsorbed from the test solution onto mica substrates in order to find out whether the incubation of the test HRP solution within the half-sphere influenced the HRP aggregation state. Attenuated total reflection Fourier transform infrared spectroscopy (ATR-FTIR) was employed in order to reveal whether the incubation of HRP solution within the half-sphere led to any changes in its secondary structure. In parallel, spectrophotometry-based estimation of the HRP enzymatic activity was performed in order to find out if the HRP active site was affected by the electromagnetic field under the conditions of our experiments. We revealed an increased aggregation of HRP after the incubation of its solution within the half-sphere in comparison with the control sample incubated far outside the half-sphere. ATR-FTIR allowed us to reveal alterations in HRP’s secondary structure. Such changes in the protein structure did not affect its active site, as was confirmed by spectrophotometry. The effect of spherical elements on a protein solution should be taken into account in the development of the optimized design of biosensors and bioreactors, intended for performing processes involving proteins in biomedicine and biotechnology, including highly sensitive biosensors intended for the diagnosis of socially significant diseases in humans (including oncology, cardiovascular diseases, etc.) at early stages.