Single-molecule Imaging Research Articles

Cohesin prevents cross-domain gene coactivation

The contrast between the disruption of genome topology after cohesin loss and the lack of downstream gene expression changes instigates intense debates regarding the structure–function relationship between genome and gene regulation. Here, by analyzing transcriptome and chromatin accessibility at the single-cell level, we discover that, instead of dictating population-wide gene expression levels, cohesin supplies a general function to neutralize stochastic coexpression tendencies of cis-linked genes in single cells. Notably, cohesin loss induces widespread gene coactivation and chromatin co-opening tens of million bases apart in cis. Spatial genome and protein imaging reveals that cohesin prevents gene co-bursting along the chromosome and blocks spatial mixing of transcriptional hubs. Single-molecule imaging shows that cohesin confines the exploration of diverse enhancer and core promoter binding transcriptional regulators. Together, these results support that cohesin arranges nuclear topology to control gene coexpression in single cells.

Single-molecule imaging reveals allosteric stimulation of SARS-CoV-2 spike receptor binding domain by host sialic acid.

Conformational dynamics of the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) spike glycoprotein (S) mediate exposure of the binding site for the cellular receptor, angiotensin-converting enzyme 2 (ACE2). The N-terminal domain (NTD) of S binds terminal sialic acid (SA) moieties on the cell surface, but the functional role of this interaction in virus entry is unknown. Here, we report that NTD-SA interaction enhances both S-mediated virus attachment and ACE2 binding. Through single-molecule Förster resonance energy transfer imaging of individual S trimers, we demonstrate that SA binding to the NTD allosterically shifts the S conformational equilibrium, favoring enhanced exposure of the ACE2-binding site. Antibodies that target the NTD block SA binding, which contributes to their mechanism of neutralization. These findings inform on mechanisms of S activation at the cell surface.

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 Imaging of Integral Membrane Protein Dynamics and Function.

Integral membrane proteins (IMPs) play central roles in cellular physiology and represent the majority of known drug targets. Single-molecule fluorescence and fluorescence resonance energy transfer (FRET) methods have recently emerged as valuable tools for investigating structure-function relationships in IMPs. This review focuses on the practical foundations required for examining polytopic IMP function using single-molecule FRET (smFRET) and provides an overview of the technical and conceptual frameworks emerging from this area of investigation. In this context, we highlight the utility of smFRET methods to reveal transient conformational states critical to IMP function and the use of smFRET data to guide structural and drug mechanism-of-action investigations. We also identify frontiers where progress is likely to be paramount to advancing the field.

Stereoscopic Imaging of Single Molecules at Plasma Membrane of Single Cell Using Photoreduction-Assisted Electrochemistry.

Stereoscopic imaging of single molecules at the plasma membrane of single cell requires spatial resolutions in 3 dimensions (x-y-z) at 10-nm level, which is rarely achieved using most optical super-resolution microscopies. Here, electrochemical stereoscopic microscopy with a detection limit down to a single molecule is achieved using a photoreduction-assisted cycle inside a 20-nm gel electrolyte nanoball at the tip of a nanopipette. On the basis of the electrochemical oxidation of Ru(bpy)3 2+ into Ru(bpy)3 3+ followed by the reduction of Ru(bpy)3 3+ into Ru(bpy)3 2+ by photogenerated isopropanol radicals, a charge of 1.5 fC is obtained from the cycling electron transfers involving one Ru(bpy)3 2+/3+ molecule. By using the nanopipette to scan the cellular membrane modified with Ru(bpy)3 2+-tagged antibody, the morphology of the cell membrane and the distribution of carcinoembryonic antigen (CEA) on the membrane are electrochemically visualized with a spatial resolution of 14 nm. The resultant stereoscopic image reveals more CEA on membrane protrusions, providing direct evidence to support easy access of membrane CEA to intravenous antibodies. The breakthrough in single-molecule electrochemistry at the cellular level leads to the establishment of high-resolution 3-dimensional single-cell electrochemical microscopy, offering an alternative strategy to remedy the imperfection of stereoscopic visualization in optical microscopes.

Highly sensitive volumetric single-molecule imaging.

Volumetric subcellular imaging has long been essential for studying structures and dynamics in cells and tissues. However, due to limited imaging speed and depth of field, it has been challenging to perform live-cell imaging and single-particle tracking. Here we report a 2.5D fluorescence microscopy combined with highly inclined illumination beams, which significantly reduce not only the image acquisition time but also the out-of-focus background by ∼2-fold compared to epi-illumination. Instead of sequential z-scanning, our method projects a certain depth of volumetric information onto a 2D plane in a single shot using multi-layered glass for incoherent wavefront splitting, enabling high photon detection efficiency. We apply our method to multi-color immunofluorescence imaging and volumetric super-resolution imaging, covering ∼3-4 µm thickness of samples without z-scanning. Additionally, we demonstrate that our approach can substantially extend the observation time of single-particle tracking in living cells.

DNA-PK: A synopsis beyond synapsis

Given its central role in life, DNA is remarkably easy to damage. Double strand breaks (DSBs) are the most toxic form of DNA damage, and DSBs pose the greatest danger to genomic integrity. In higher vertebrates, the non-homologous end joining pathway (NHEJ) is the predominate pathway that repairs DSBs. NHEJ has three steps: 1) DNA end recognition by the DNA dependent protein kinase [DNA-PK], 2) DNA end-processing by numerous NHEJ accessory factors, and 3) DNA end ligation by the DNA ligase IV complex (LX4). Although this would appear to be a relatively simple mechanism, it has become increasingly apparent that it is not.Recently, much insight has been derived regarding the mechanism of non-homologous end joining through a proliferation of cryo-EM studies, structure-function mutational experiments informed by these new structural data, and novel single-molecule imaging approaches. An emerging consensus in the field is that NHEJ progresses from initial DSB end recognition by DNA-PK to synapsis of the two DNA ends in a long-range synaptic complex where ends are held too far apart (115 Å) for ligation, and then progress to a short-range synaptic complex where ends are positioned close enough for ligation. What was surprising from these structural studies was the observation of two distinct types of DNA-PK dimers that represent NHEJ long-range complexes. In this review, we summarize current knowledge about the function of the distinct NHEJ synaptic complexes and align this new information with emerging cellular single-molecule microscopy studies as well as with previous studies of DNA-PK’s function in repair.

Direct observation of translational activation by a ribonucleoprotein granule

Biomolecular condensates organize biochemical processes at the subcellular level and can provide spatiotemporal regulation within a cell. Among these, ribonucleoprotein (RNP) granules are storage hubs for translationally repressed mRNA. Whether RNP granules can also activate translation and how this could be achieved remains unclear. Here, using single-molecule imaging, we demonstrate that the germ cell-determining RNP granules in Drosophila embryos are sites for active translation of nanos mRNA. Nanos translation occurs preferentially at the germ granule surface with the 3′ UTR buried within the granule. Smaug, a cytosolic RNA-binding protein, represses nanos translation, which is relieved when Smaug is sequestered to the germ granule by the scaffold protein Oskar. Together, our findings uncover a molecular process by which RNP granules achieve localized protein synthesis through the compartmentalized loss of translational repression.

Laser-induced electron diffraction: Imaging of a single gas-phase molecular structure with one of its own electrons.

Among the many methods to image molecular structure, laser-induced electron diffraction (LIED) can image a single gas-phase molecule by locating all of a molecule's atoms in space and time. The method is based on attosecond electron recollision driven by a laser field and can reach attosecond temporal resolution. Implementation with a mid-IR laser and cold-target recoil ion-momentum spectroscopy, single molecules are measured with picometer resolution due to the keV electron impact energy without ensemble averaging or the need for molecular orientation. Nowadays, the method has evolved to detect single complex and chiral molecular structures in 3D. The review will touch on the various methods to discuss the implementations of LIED toward single-molecule imaging and complement the discussions with noteworthy experimental findings in the field.

Positive Allosteric Modulation of NMDARs Prevents the Altered Surface Dynamics Caused by Patients' Antibodies.

A positive allosteric modulator of the NMDAR, SGE-301, has been shown to reverse the alterations caused by the antibodies of patients with anti-NMDAR encephalitis (NMDARe). However, the mechanisms involved beyond receptor modulation are unclear. In this study, we aimed to investigate how this modulator affects NMDAR membrane dynamics. Cultured hippocampal neurons were treated with SGE-301 or vehicle, alongside with immunoglobulins G (IgG) from patients with NMDARe or healthy controls. NMDAR surface dynamics were assessed with single-molecule imaging by photoactivated localization microscopy. NMDAR trajectories from neurons treated with SGE-301 were less confinement, with increased diffusion coefficients. This effect mainly occurred at synapses because extrasynaptic diffusion and confinement were minimally affected by SGE-301. Treatment with patients' IgG reduced NMDAR surface dynamics and increased their confinement. Remarkably, SGE-301 incubation antagonized patients' IgG effects in both synaptic and extrasynaptic membrane compartments, restoring diffusion and confinement values similar to those from neurons exposed to control IgG. We demonstrate that SGE-301 upregulates NMDAR surface diffusion and antagonizes the pathogenic effects of patients' IgG on NMDAR membrane organization. These findings suggest a potential therapeutic strategy for NMDARe.

Behaviors of nucleosomes with mutant histone H4s in euchromatic domains of living human cells.

Since Robert Feulgen first stained DNA in the cell, visualizing genome chromatin has been a central issue in cell biology to uncover how chromatin is organized and behaves in the cell. To approach this issue, we have developed single-molecule imaging of nucleosomes, a basic unit of chromatin, to unveil local nucleosome behavior in living cells. In this study, we investigated behaviors of nucleosomes with various histone H4 mutants in living HeLa cells to address the role of H4 tail acetylation, including H4K16Ac and others, which are generally associated with more transcriptionally active chromatin regions. We ectopically expressed wild-type (wt) or mutated H4s (H4K16 point; H4K5,8,12,16 quadruple; and H4 tail deletion) fused with HaloTag in HeLa cells. Cells that expressed wtH4-Halo, H4K16-Halo mutants, and multiple H4-Halo mutants had euchromatin-concentrated distribution. Consistently, the genomic regions of the wtH4-Halo nucleosomes corresponded to Hi-C contact domains (or topologically associating domains, TADs)with active chromatin marks (A-compartment). Utilizing single-nucleosome imaging, we found that none of the H4 deacetylation or acetylation mimicked H4 mutants altered the overall local nucleosome motion. This finding suggests that H4 mutant nucleosomes embedded in the condensed euchromatic domains with excess endogenous H4 nucleosomes cannot cause an observable change in the local motion. Interestingly, H4 with four lysine-to-arginine mutations displayed a substantial freely diffusing fraction in the nucleoplasm, whereas H4 with a truncated N-terminal tail was incorporated in heterochromatic regions as well as euchromatin. Our study indicates the power of single-nucleosome imaging to understand individual histone/nucleosome behavior reflecting chromatin environments in living cells.

A predicted model-aided reconstruction algorithm for X-ray free-electron laser single-particle imaging.

Ultra-intense, ultra-fast X-ray free-electron lasers (XFELs) enable the imaging of single protein molecules under ambient temperature and pressure. A crucial aspect of structure reconstruction involves determining the relative orientations of each diffraction pattern and recovering the missing phase information. In this paper, we introduce a predicted model-aided algorithm for orientation determination and phase retrieval, which has been tested on various simulated datasets and has shown significant improvements in the success rate, accuracy and efficiency of XFEL data reconstruction.

The condensation of HP1α/Swi6 imparts nuclear stiffness

Biomolecular condensates have emerged as major drivers of cellular organization. It remains largely unexplored, however, whether these condensates can impart mechanical function(s) to the cell. The heterochromatin protein HP1α (Swi6 in Schizosaccharomyces pombe) crosslinks histone H3K9 methylated nucleosomes and has been proposed to undergo condensation to drive the liquid-like clustering of heterochromatin domains. Here, we leverage the genetically tractable S.pombe model and a separation-of-function allele to elucidate a mechanical function imparted by Swi6 condensation. Using single-molecule imaging, force spectroscopy, and high-resolution live-cell imaging, we show that Swi6 is critical for nuclear resistance to external force. Strikingly, it is the condensed yet dynamic pool of Swi6, rather than the chromatin-bound molecules, that is essential to imparting mechanical stiffness. Our findings suggest that Swi6 condensates embedded in the chromatin meshwork establish the emergent mechanical behavior of the nucleus as a whole, revealing that biomolecular condensation can influence organelle and cell mechanics.

Escherichia coli DNA replication: the old model organism still holds many surprises.

Research on Escherichia coli DNA replication paved the groundwork for many breakthrough discoveries with important implications for our understanding of human molecular biology, due to the high level of conservation of key molecular processes involved. To this day, it attracts a lot of attention, partially by virtue of being an important model organism, but also because the understanding of factors influencing replication fidelity might be important for studies on the emergence of antibiotic resistance. Importantly, the wide access to high-resolution single-molecule and live-cell imaging, whole genome sequencing, and cryo-electron microscopy techniques, which were greatly popularized in the last decade, allows us to revisit certain assumptions about the replisomes and offers very detailed insight into how they work. For many parts of the replisome, step-by-step mechanisms have been reconstituted, and some new players identified. This review summarizes the latest developments in the area, focusing on (a) the structure of the replisome and mechanisms of action of its components, (b) organization of replisome transactions and repair, (c) replisome dynamics, and (d) factors influencing the base and sugar fidelity of DNA synthesis.

Near-Infrared Spontaneously Blinking Fluorophores for Live Cell Super-Resolution Imaging with Minimized Phototoxicity.

Single-molecule localization microscopy (SMLM) requires high-intensity laser irradiation, typically exceeding kW/cm2, to yield a sufficient photon count. However, this intense visible light exposure incurs substantial cellular toxicity, hindering its use in living cells. Here, we developed a class of near-infrared (NIR) spontaneously blinking fluorophores for SMLM. These NIR fluorophores are a combination of rhodamine spirolactams and merocyanine derivatives, where the rhodamine spirolactam component converts between a bright and dark state based on pH-dependent spirocyclization and merocyanine derivatives shift the excitation wavelength into the infrared. Single-molecule characterizations demonstrated their potential for SMLM. At a moderate power density of 3.93 kW/cm2, these probes exhibit duty cycle as low as 0.18% and an emission rate as high as 26,700 photons/s. Phototoxicity assessment under single-molecule imaging conditions reveals that NIR illumination (721 nm) minimizes harm to living cells. Employing these NIR fluorophores, we successfully captured time-lapse super-resolution tracking of mitochondria at a Fourier ring correlation (FRC) resolution of 69.4 nm and reconstructed the ultrastructures of endoplasmic reticulum (ER) in living cells.

Insight into Single-Molecule Imaging Techniques for the Study of Prokaryotic Genome Maintenance.

Genome maintenance comprises a group of complex and interrelated processes crucial for preserving and safeguarding genetic information within all organisms. Key aspects of genome maintenance involve DNA replication, transcription, recombination, and repair. Improper regulation of these processes could cause genetic changes, potentially leading to antibiotic resistance in bacterial populations. Due to the complexity of these processes, ensemble averaging studies may not provide the level of detail required to capture the full spectrum of molecular behaviors and dynamics of each individual biomolecule. Therefore, researchers have increasingly turned to single-molecule approaches, as these techniques allow for the direct observation and manipulation of individual biomolecules, and offer a level of detail that is unattainable with traditional ensemble methods. In this review, we provide an overview of recent in vitro and in vivo single-molecule imaging approaches employed to study the complex processes involved in prokaryotic genome maintenance. We will first highlight the principles of imaging techniques such as total internal reflection fluorescence microscopy and atomic force microscopy, primarily used for in vitro studies, and highly inclined and laminated optical sheet and super-resolution microscopy, mainly employed in in vivo studies. We then demonstrate how applying these single-molecule techniques has enabled the direct visualization of biological processes such as replication, transcription, DNA repair, and recombination in real time. Finally, we will showcase the results obtained from super-resolution microscopy approaches, which have provided unprecedented insights into the spatial organization of different biomolecules within bacterial organisms.

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.

Single-molecule imaging reveals the mechanism of bidirectional replication initiation in metazoa

Metazoan genomes are copied bidirectionally from thousands of replication origins. Replication initiation entails the assembly and activation of two CMG helicases (Cdc45⋅Mcm2-7⋅GINS) at each origin. This requires several replication firing factors (including TopBP1, RecQL4, and DONSON) whose exact roles are still under debate. How two helicases are correctly assembled and activated at each origin is a long-standing question. By visualizing the recruitment of GINS, Cdc45, TopBP1, RecQL4, and DONSON in real time, we uncovered that replication initiation is surprisingly dynamic. First, TopBP1 transiently binds to the origin and dissociates before the start of DNA synthesis. Second, two Cdc45 are recruited together, even though Cdc45 alone cannot dimerize. Next, two copies of DONSON and two GINS simultaneously arrive at the origin, completing the assembly of two CMG helicases. Finally, RecQL4 is recruited to the CMG⋅DONSON⋅DONSON⋅CMG complex and promotes DONSON dissociation and CMG activation via its ATPase activity.

Robust Quantification of Live-Cell Single-Molecule Tracking Data for Fluorophores with Different Photophysical Properties.

High-speed single-molecule tracking in live cells is becoming an increasingly popular method for quantifying the spatiotemporal behavior of proteins in vivo. The method provides a wealth of quantitative information, but users need to be aware of biases that can skew estimates of molecular mobilities. The range of suitable fluorophores for live-cell single-molecule imaging has grown substantially over the past few years, but it remains unclear to what extent differences in photophysical properties introduce biases. Here, we tested two fluorophores with entirely different photophysical properties, one that photoswitches frequently between bright and dark states (TMR) and one that shows exceptional photostability without photoswitching (JFX650). We used a fusion of the Escherichia coli DNA repair enzyme MutS to the HaloTag and optimized sample preparation and imaging conditions for both types of fluorophore. We then assessed the reliability of two common data analysis algorithms, mean-square displacement (MSD) analysis and Hidden Markov Modeling (HMM), to estimate the diffusion coefficients and fractions of MutS molecules in different states of motion. We introduce a simple approach that removes discrepancies in the data analyses and show that both algorithms yield consistent results, regardless of the fluorophore used. Nevertheless, each dye has its own strengths and weaknesses, with TMR being more suitable for sampling the diffusive behavior of many molecules, while JFX650 enables prolonged observation of only a few molecules per cell. These characterizations and recommendations should help to standardize measurements for increased reproducibility and comparability across studies.

Multicolor, Cell-Impermeable, and High Affinity BACE1 Inhibitor Probes Enable Superior Endogenous Staining and Imaging of Single Molecules.

The prevailing but not undisputed amyloid cascade hypothesis places the β-site of APP cleaving enzyme 1 (BACE1) center stage in Alzheimer's Disease pathogenesis. Here, we investigated functional properties of BACE1 with novel tag- and antibody-free labeling tools, which are conjugates of the BACE1-inhibitor IV (also referred to as C3) linked to different impermeable Alexa Fluor dyes. We show that these fluorescent small molecules bind specifically to BACE1, with a 1:1 labeling stoichiometry at their orthosteric site. This is a crucial property especially for single-molecule and super-resolution microscopy approaches, allowing characterization of the dyes' labeling capabilities in overexpressing cell systems and in native neuronal tissue. With multiple colors at hand, we evaluated BACE1-multimerization by Förster resonance energy transfer (FRET) acceptor-photobleaching and single-particle imaging of native BACE1. In summary, our novel fluorescent inhibitors, termed Alexa-C3, offer unprecedented insights into protein-protein interactions and diffusion behavior of BACE1 down to the single molecule level.