Single-molecule Imaging Techniques Research Articles

Single molecule characterisation of salivary protein aggregates from Parkinson's disease patients – a pilot study

Abstract Saliva is a convenient and accessible biofluid that has potential as a future diagnostic tool for Parkinson’s disease. Candidate diagnostic tests for Parkinson’s disease to date have predominantly focused on measurements of alpha-synuclein in cerebrospinal fluid, but there is a need for accurate tests utilising more easily accessible sample types. Prior studies utilising saliva have used bulk measurements of salivary α-synuclein to provide diagnostic insight. Aggregate structure may influence the contribution of α-synuclein to disease pathology. Single molecule approaches can characterise the structure of individual aggregates present in the biofluid and may therefore provide greater insight than bulk measurements. We have employed an antibody-based single-molecule pulldown assay to quantify salivary α-synuclein and amyloid-β peptide aggregate number and subsequently super-resolved captured aggregates using direct Stochastic Optical Reconstruction Microscopy to describe their morphological features. We show that the salivary α-synuclein aggregate/amyloid-β aggregate ratio is increased almost two-fold in Parkinson’s disease patients (n = 20) compared to controls (n = 20, p <0.05). Morphological information also provides insight with saliva from Parkinson’s disease patients containing a greater proportion of larger and more fibrillar amyloid-β aggregates than control saliva (p <0.05). Furthermore, the combination of count and morphology data provides greater diagnostic value than either measure alone distinguishing between the people with Parkinson’s disease (n = 17) and controls (n = 18) with a high degree of accuracy (AUC = 0.87, p <0.001) and a larger dynamic range. We therefore demonstrate for the first time the application of highly sensitive single molecule imaging techniques to saliva. In addition, we show that aggregates present within saliva retain relevant structural information further expanding the potential utility of saliva-based diagnostic methods.

Atomic single-molecule imaging by the confinement methods in advanced microscopy

Atomic single-molecule imaging has drawn extensive attention for exploring different behaviors and properties of small molecules. However, current techniques still face challenges, due to the thermal activity and beam sensitivity of small molecules. Recently, ångström-level spatial resolution for single molecules was successfully demonstrated using different imaging methods, including scanning tunneling microscopy, atomic force microscopy, cryogenic and transmission electron microscopy. In this perspective, we focus on different confinement strategies in these single-molecule imaging techniques, and summarize the recent studies of the structures and behaviors of single molecules. Especially, a new concept of spatial confinement at room temperature has been achieved by using microporous materials, such as zeolites, which has made it possible to fix and visualize the configurations of single molecule inside channels. In outlook, we describe what progress we can make along such a confinement strategy, and what new perspectives and discoveries we may find beyond our imagination.

Engineering of Multivalent Membrane-Anchored DNA Frameworks for Precise Profiling of Variable Membrane Permeability During Reversible Electroporation.

Electroporation techniques have emerged as attractive tools for intracellular delivery, rendering promising prospects towards clinical therapies. Transient disruption of membrane permeability is the critical process for efficient electroporation-based cargo delivery. However, smart nanotools for precise characterization of transient membrane changes induced by strong electric pulses are extremely limited. Herein, multivalent membrane-anchored fluorescent nanoprobes (MMFNPs) that take advantages of flexible functionalization and spatial arrangement of DNA frameworks are developed for in situ evaluation of electric field-induced membrane permeability during reversible electroporation . Single-molecule fluorescence imaging techniques are adopted to precisely verify the excellent analytical performance of the engineered MMFNPs. Benefited from tight membrane anchoring and sensitive adenosine triphosphate (ATP)profiling, varying degrees of membrane disturbances are visually exhibited under different intensities of the microsecond pulse electric field (µsPEF). Significantly, the dynamic process of membrane repair during reversible electroporation is well demonstrated via ATP fluctuations monitored by the designed MMFNPs. Furthermore, molecular dynamics (MD) simulations are performed for accurate verification of electroporation-driven dynamic cargo entry via membrane nanopores. This work provides an avenue for effectively capturing transient fluctuations of membrane permeability under external stimuli, offering valuable guidance for developing efficient and safe electroporation-driven delivery strategies for clinical diagnosis and therapeutics.

Single-Molecule Imaging Reveals Differential AT1R Stoichiometry Change in Biased Signaling.

G protein-coupled receptors (GPCRs) represent promising therapeutic targets due to their involvement in numerous physiological processes mediated by downstream G protein- and β-arrestin-mediated signal transduction cascades. Although the precise control of GPCR signaling pathways is therapeutically valuable, the molecular details for governing biased GPCR signaling remain elusive. The Angiotensin II type 1 receptor (AT1R), a prototypical class A GPCR with profound implications for cardiovascular functions, has become a focal point for biased ligand-based clinical interventions. Herein, we used single-molecule live-cell imaging techniques to evaluate the changes in stoichiometry and dynamics of AT1R with distinct biased ligand stimulations in real time. It was revealed that AT1R existed predominantly in monomers and dimers and underwent oligomerization upon ligand stimulation. Notably, β-arrestin-biased ligands induced the formation of higher-order aggregates, resulting in a slower diffusion profile for AT1R compared to G protein-biased ligands. Furthermore, we demonstrated that the augmented aggregation of AT1R, triggered by activation from each biased ligand, was completely abrogated in β-arrestin knockout cells. These findings furnish novel insights into the intricate relationship between GPCR aggregation states and biased signaling, underscoring the pivotal role of molecular behaviors in guiding the development of selective therapeutic agents.

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.

Quantitatively Lighting up the Spatial Organization of CD47/SIRPα Immune Checkpoints on the Cellular Membrane with Single-Molecule Localization Microscopy.

Immunotherapy including immune checkpoint inhibition has reinvigorated the current cancer treatment field. The development of efficient cancer immunotherapies depends on a thorough understanding of the status of immune checkpoints and how they interact. However, the distribution and spatial organization changes of immune checkpoints during their interactions at the single-molecule level remain difficult to directly visualize due to the lack of in situ imaging techniques with appropriate spatial and stoichiometric resolution. Herein, we report the direct visualization and quantification of the spatial distribution and organization of CD47 on the bladder tumor cell membrane and SIRPα on the macrophage membrane by using a single-molecule localization imaging technique called quantitative direct stochastic optical reconstruction microscopy (QdSTORM). Results showed that a portion of CD47 and SIRPα was present on cell membranes as heterogeneous clusters of varying sizes and densities prior to activation. Quantitative analyses of the reconstructed super-resolution images and theoretical simulation revealed that CD47 and SIRPα were reorganized into larger clusters upon binding to each other. Furthermore, we found that blocking the immune checkpoint interaction with small-molecule inhibitors or antibodies significantly impacted the spatial clustering behavior of CD47 on bladder tumor cells, demonstrating the promise of our QdSTORM strategy in elucidating the molecular mechanisms underlying immunotherapy. This work offers a promising strategy to advance our understanding of immune checkpoint state and interactions while also contributing to the fields including signal regulation and cancer therapy.

Single-molecule imaging reveals distinct elongation and frameshifting dynamics between frames of expanded RNA repeats in C9ORF72-ALS/FTD

C9ORF72 hexanucleotide repeat expansion is the most common genetic cause of both amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD). One pathogenic mechanism is the accumulation of toxic dipeptide repeat (DPR) proteins like poly-GA, GP and GR, produced by the noncanonical translation of the expanded RNA repeats. However, how different DPRs are synthesized remains elusive. Here, we use single-molecule imaging techniques to directly measure the translation dynamics of different DPRs. Besides initiation, translation elongation rates vary drastically between different frames, with GP slower than GA and GR the slowest. We directly visualize frameshift events using a two-color single-molecule translation assay. The repeat expansion enhances frameshifting, but the overall frequency is low. There is a higher chance of GR-to-GA shift than in the reversed direction. Finally, the ribosome-associated protein quality control (RQC) factors ZNF598 and Pelota modulate the translation dynamics, and the repeat RNA sequence is important for invoking the RQC pathway. This study reveals that multiple translation steps modulate the final DPR production. Understanding repeat RNA translation is critically important to decipher the DPR-mediated pathogenesis and identify potential therapeutic targets in C9ORF72-ALS/FTD.

Trends in Single-Molecule Total Internal Reflection Fluorescence Imaging and Their Biological Applications with Lab-on-a-Chip Technology.

Single-molecule imaging technologies, especially those based on fluorescence, have been developed to probe both the equilibrium and dynamic properties of biomolecules at the single-molecular and quantitative levels. In this review, we provide an overview of the state-of-the-art advancements in single-molecule fluorescence imaging techniques. We systematically explore the advanced implementations of in vitro single-molecule imaging techniques using total internal reflection fluorescence (TIRF) microscopy, which is widely accessible. This includes discussions on sample preparation, passivation techniques, data collection and analysis, and biological applications. Furthermore, we delve into the compatibility of microfluidic technology for single-molecule fluorescence imaging, highlighting its potential benefits and challenges. Finally, we summarize the current challenges and prospects of fluorescence-based single-molecule imaging techniques, paving the way for further advancements in this rapidly evolving field.

Single-molecule imaging of genome maintenance proteins encountering specific DNA sequences and structures

DNA repair pathways are tightly regulated processes that recognize specific hallmarks of DNA damage and coordinate lesion repair through discrete mechanisms, all within the context of a three-dimensional chromatin landscape. Dysregulation or malfunction of any one of the protein constituents in these pathways can contribute to aging and a variety of diseases. While the collective action of these many proteins is what drives DNA repair on the organismal scale, it is the interactions between individual proteins and DNA that facilitate each step of these pathways. In much the same way that ensemble biochemical techniques have characterized the various steps of DNA repair pathways, single-molecule imaging (SMI) approaches zoom in further, characterizing the individual protein-DNA interactions that compose each pathway step. SMI techniques offer the high resolving power needed to characterize the molecular structure and functional dynamics of individual biological interactions on the nanoscale. In this review, we highlight how our lab has used SMI techniques – traditional atomic force microscopy (AFM) imaging in air, high-speed AFM (HS-AFM) in liquids, and the DNA tightrope assay – over the past decade to study protein-nucleic acid interactions involved in DNA repair, mitochondrial DNA replication, and telomere maintenance. We discuss how DNA substrates containing specific DNA sequences or structures that emulate DNA repair intermediates or telomeres were generated and validated. For each highlighted project, we discuss novel findings made possible by the spatial and temporal resolution offered by these SMI techniques and unique DNA substrates.

Deciphering molecular mechanisms of phase separation in RNA biology by single-molecule biophysical technologies.

Ribonucleic acid (RNA) biology has emerged as one of the most important areas in modern biology and biomedicine. RNA and RNA-binding proteins (RBPs) are involved in forming biomolecular condensates, which are crucial for RNA metabolism. To quantitively decipher the molecular mechanisms of RNP granules, researchers have turned to single-molecule biophysical techniques, such as single-molecule Förster resonance energy transfer (smFRET), in vivo single-molecule imaging technique with single particle tracking (SPT), DNA Curtains, optical tweezers, and atomic force microscopy (AFM). These methods are used to investigate the molecular biophysical properties within RNP granules, as well as the molecular interactions between RNA and RBPs and RBPs themselves, which are challenging to study using traditional experimental methods of the liquid-liquid phase separation (LLPS) field, such as fluorescence recovery after photobleaching (FRAP). In this work, we summarize the applications of single-molecule biophysical techniques in RNP granule studies and highlight how these methods can be used to reveal the molecular mechanisms of RNP granules.

Recent Advances in Single-Molecule Tracking and Imaging Techniques.

Since the early 1990s, single-molecule detection in solution at room temperature has enabled direct observation of single biomolecules at work in real time and under physiological conditions, providing insights into complex biological systems that the traditional ensemble methods cannot offer. In particular, recent advances in single-molecule tracking techniques allow researchers to follow individual biomolecules in their native environments for a timescale of seconds to minutes, revealing not only the distinct pathways these biomolecules take for downstream signaling but also their roles in supporting life. In this review, we discuss various single-molecule tracking and imaging techniques developed to date, with an emphasis on advanced three-dimensional (3D) tracking systems that not only achieve ultrahigh spatiotemporal resolution but also provide sufficient working depths suitable for tracking single molecules in 3D tissue models. We then summarize the observables that can be extracted from the trajectory data. Methods to perform single-molecule clustering analysis and future directions are also discussed.

The K2: Open-source simultaneous triple-color TIRF microscope for live-cell and single-molecule imaging

Imaging the dynamics and interactions of biomolecules at the single-molecule level in live cells and reconstituted systems has generated unprecedented knowledge about the biomolecular processes underlying many cellular functions. To achieve the speed and sensitivity needed to detect and follow individual molecules, these experiments typically require custom-built microscopes or custom modifications of commercial systems. The costs of such single-molecule microscopes, their technical complexity and the lack of open-source documentation on how to build custom setups therefore limit the accessibility of single-molecule imaging techniques. To advance the adaptation of dynamic single-molecule imaging by a wider community, we present the “K2”: an open-source, simultaneous triple-color total internal reflection fluorescence (TIRF) microscope specifically designed for live-cell and single-molecule imaging. We explain our design considerations and provide step-by-step building instructions, parts list and full CAD models. The modular design of this TIRF microscope allows users to customize it to their scientific and financial needs, or to re-use parts of our design to improve the capabilities of their existing setups without necessarily having to build a full copy of the K2 microscope.

Pore-Forming Proteins: From Pore Assembly to Structure by Quantitative Single-Molecule Imaging.

Pore-forming proteins (PFPs) play a central role in many biological processes related to infection, immunity, cancer, and neurodegeneration. A common feature of PFPs is their ability to form pores that disrupt the membrane permeability barrier and ion homeostasis and generally induce cell death. Some PFPs are part of the genetically encoded machinery of eukaryotic cells that are activated against infection by pathogens or in physiological programs to carry out regulated cell death. PFPs organize into supramolecular transmembrane complexes that perforate membranes through a multistep process involving membrane insertion, protein oligomerization, and finally pore formation. However, the exact mechanism of pore formation varies from PFP to PFP, resulting in different pore structures with different functionalities. Here, we review recent insights into the molecular mechanisms by which PFPs permeabilize membranes and recent methodological advances in their characterization in artificial and cellular membranes. In particular, we focus on single-molecule imaging techniques as powerful tools to unravel the molecular mechanistic details of pore assembly that are often obscured by ensemble measurements, and to determine pore structure and functionality. Uncovering the mechanistic elements of pore formation is critical for understanding the physiological role of PFPs and developing therapeutic approaches.

Target search dynamics of Sox transcription factors.

Transcription factors (TFs) are central players in regulating gene expression through their ability to bind sequence-specific sites. Nonetheless, how TFs search the genome to locate their binding sites is unclear. The facilitated diffusion theory proposes that TFs combine 1D-sliding on DNA and 3D-diffusion in the nucleus, and that local nonspecific interactions mediated by electrostatic forces drive the search efficiency beyond the one that would be reached by free diffusion alone. While such models are descriptive of the situation in prokaryotes, how eukaryotic TFs search the chromatinized genome is poorly understood. Furthermore, the relevance of regions outside DNA-binding domains (DBDs) for the search process remains unknown. Herein, we hypothesize that positive charges in DBD flanking regions accelerate TF target search through nonspecific interactions with DNA. A comparative study between Sox2 and Sox17 can demonstrate how electric charges within intrinsically disordered regions (IDRs) affect the target search process. These Sox-TF recognize the same motifs but have opposite charge distributions at their DBD C-terminus. Single-molecule imaging techniques allow for investigating the search efficiency. our results show that Sox2 exhibited a two-fold higher pseudo-on-rate than Sox17 and a Sox2 variant having an acidic stretch of Sox17. This is consistent with in vitro measurements, directly reporting a two-fold higher on-rate for wild-type Sox2 compared to the negatively-charged Sox2 variant. Moreover, the Sox2 variant showed a decreased ability to bind target sites within compact chromatin. Since Sox2 is known as a pioneer factor, quantifying their interactions with synthetically-chromatinized DNA will elucidate search mechanisms of pioneer TFs. In this regard, we foresee that more positively-charged IDRs stabilize the specific binding of TFs to nucleosomal DNA and help them to invade chromatin. Altogether, this work will dissect how the charge distribution of DBD flanking regions modulates TF target search on chromatin.

Single-molecule dynamics of ribonuclease H enzymes in Bacillus subtilis.

Transcription and replication are fundamental processes for gene expression during which RNA-DNA hybrids (such as R-loops, single-ribonucleotide errors, RNA primers, ribopatches, and complementary RNA:DNA) may form. When RNA-DNA hybrids are not resolved, replication fork stress can occur leading to compromised genome integrity and an increase in cell mutation rates. Ribonuclease (RNase) H family enzymes contribute to genome maintenance by cleaving the RNA portion of RNA-DNA hybrid structures. Recent work has deepened our understanding of how RNA-DNA hybrids form and what could occur if they are not resolved. However, how these enzymes are recruited to the sites of RNA-DNA hybrids and the individual contributions of each enzyme remain unclear. Here, we use live-cell single-molecule imaging and super-resolution microscopy techniques to study the dynamics of the two RNase H enzymes in Bacillus subtilis. We observe changes in the single-molecule dynamics and localization patterns of each RNase H enzyme upon arresting transcription and inhibiting replication. Our results show that inhibition of transcription and replication both affect the fraction of enzymes existing in a functional binding state, as well as the localization of the enzymes at the replication fork. Our work provides insight into the specific functions of RNase H enzymes for different types of RNA-DNA hybrids in B. subtilis.

Study on biophysical properties of Phi29 DNA polymerase using a novel single-molecule imaging technique DNA curtain.

Phi29 DNA polymerase (Phi29 DNAp) is a bacteriophage polymerase that has high fidelity and processivity due to its structural properties. Based on such unique properties, Phi29 DNAp has been used in various biological techniques such as rolling circle amplification and DNA sequencing. However, the detailed mechanism underlying DNA replication by Phi29 DNAp remains elusive. We study the biophysical properties of phi29 DNAp using a high-throughput single-molecule imaging technique DNA curtain. Using DNA curtain, we visualized single replication events in real-time. The speed of Phi29 DNAp is about 130 bp/sec and the processivity is about 23 kbp. Even though the processivity is smaller than that in the previous ensemble measurement, the processivity shows that Phi29 DNAp can synthesize the entire genome of Phi29 bacteriophage in one replication. We also investigate the collision of Phi29 DNAp and protein roadblocks.

Single-Molecule Imaging of Membrane Proteins on Vascular Endothelial Cells.

Transporting substances such as gases, nutrients, waste, and cells is the primary function of blood vessels. Vascular cells use membrane proteins to perform crucial endothelial functions, including molecular transport, immune cell infiltration, and angiogenesis. A thorough understanding of these membrane receptors from a clinical perspective is warranted to gain insights into the pathogenesis of vascular diseases and to develop effective methods for drug delivery through the vascular endothelium. This review summarizes state-of-the-art single-molecule imaging techniques, such as super-resolution microscopy, single-molecule tracking, and protein-protein interaction analysis, for observing and studying membrane proteins. Furthermore, recent single-molecule studies of membrane proteins such as cadherins, integrins, caveolins, transferrin receptors, vesicle-associated protein-1, and vascular endothelial growth factor receptor are discussed.

Atomic Force Microscopy Reveals Complexity Underlying General Secretory System Activity.

The translocation of specific polypeptide chains across membranes is an essential activity for all life forms. The main components of the general secretory (Sec) system of E. coli include integral membrane translocon SecYEG, peripheral ATPase SecA, and SecDF, an ancillary complex that enhances polypeptide secretion by coupling translocation to proton motive force. Atomic force microscopy (AFM), a single-molecule imaging technique, is well suited to unmask complex, asynchronous molecular activities of membrane-associated proteins including those comprising the Sec apparatus. Using AFM, the dynamic structure of membrane-external protein topography of Sec system components can be directly visualized with high spatial-temporal precision. This mini-review is focused on AFM imaging of the Sec system in near-native fluid conditions where activity can be maintained and biochemically verified. Angstrom-scale conformational changes of SecYEG are reported on 100 ms timescales in fluid lipid bilayers. The association of SecA with SecYEG, forming membrane-bound SecYEG/SecA translocases, is directly visualized. Recent work showing topographical aspects of the translocation process that vary with precursor species is also discussed. The data suggests that the Sec system does not employ a single translocation mechanism. We posit that differences in the spatial frequency distribution of hydrophobic content within precursor sequences may be a determining factor in mechanism selection. Precise AFM investigations of active translocases are poised to advance our currently vague understanding of the complicated macromolecular movements underlying protein export across membranes.

Discrete LAT condensates encode antigen information from single pMHC:TCR binding events

LAT assembly into a two-dimensional protein condensate is a prominent feature of antigen discrimination by T cells. Here, we use single-molecule imaging techniques to resolve the spatial position and temporal duration of each pMHC:TCR molecular binding event while simultaneously monitoring LAT condensation at the membrane. An individual binding event is sufficient to trigger a LAT condensate, which is self-limiting, and neither its size nor lifetime is correlated with the duration of the originating pMHC:TCR binding event. Only the probability of the LAT condensate forming is related to the pMHC:TCR binding dwell time. LAT condenses abruptly, but after an extended delay from the originating binding event. A LAT mutation that facilitates phosphorylation at the PLC-γ1 recruitment site shortens the delay time to LAT condensation and alters T cell antigen specificity. These results identify a function for the LAT protein condensation phase transition in setting antigen discrimination thresholds in T cells.

Regulatory Mechanisms of SNAP-25-Associated Insulin Release Revealed by Live-Cell Confocal and Single-Molecule Localization Imaging.

Impaired insulin release is the key feature of type 2 diabetes. Insulin secretion, mainly mediated by SNARE proteins, is closely related to the blood glucose level. However, the mechanism underlying how glucose controls SNARE proteins to regulate insulin release is largely unexplained. Herein, we investigated the effects of glucose on the subcellular localization and spatial distribution on the plasma membrane (PM) of t-SNAREs (SNAP-25 and STX-1A) using a live-cell confocal microscope and the single-molecule localization imaging technique. Live-cell confocal and dSTORM imaging first revealed that SNAP-25 was mostly localized to the PM as clusters under the basal glucose concentration condition and demonstrated significant colocalization with STX-1A clusters. Furthermore, our data showed that the elevated glucose concentration increased the expression of SNAP-25 and induced more and larger SNAP-25 clusters on the PM, whereas glucotoxicity severely inhibited SNAP-25 transport to the PM and caused fewer and smaller SNAP-25 clusters on the PM. Additionally, we found that glucotoxicity also had an inhibitory effect on the colocalization between SNAP-25 and STX-1A, indicating a decrease of their interactions. Our study sheds light on the regulatory effects of glucose on the functional organization of t-SNAREs at a subcellular and molecular level, thus providing new insights into the mechanisms by which SNAREs regulate insulin release.