Live-cell Single-molecule Imaging Research Articles

Peptidoglycan synthesis drives a single population of septal cell wall synthases during division in Bacillus subtilis

Bacterial cell division requires septal peptidoglycan (sPG) synthesis by the divisome complex. Treadmilling of the essential tubulin homologue FtsZ has been implicated in septal constriction, though its precise role remains unclear. Here we used live-cell single-molecule imaging of the divisome transpeptidase PBP2B to investigate sPG synthesis dynamics in Bacillus subtilis. In contrast to previous models, we observed a single population of processively moving PBP2B molecules whose motion is driven by peptidoglycan synthesis and is not associated with FtsZ treadmilling. However, despite the asynchronous motions of PBP2B and FtsZ, a partial dependence of PBP2B processivity on FtsZ treadmilling was observed. Additionally, through single-molecule counting experiments we provide evidence that the divisome synthesis complex is multimeric. Our results support a model for B. subtilis division where a multimeric synthesis complex follows a single track dependent on sPG synthesis whose activity and dynamics are asynchronous with FtsZ treadmilling.

Dissecting ER stress signaling with live-cell single-molecule imaging and optogenetics

Dissecting ER stress signaling with live-cell single-molecule imaging and optogenetics

Live-cell single-molecule imaging and mapping of human SWI/SNF chromatin remodelers reveal multi-modal regulatory landscape and cancer-mutant-specific signatures in DNA-binding dynamics

Live-cell single-molecule imaging and mapping of human SWI/SNF chromatin remodelers reveal multi-modal regulatory landscape and cancer-mutant-specific signatures in DNA-binding dynamics

Single-Molecule Measurement of Protein Interaction Dynamics within Biomolecular Condensates.

Biomolecular condensates formed via liquid-liquid phase separation (LLPS) have been considered critical in cellular organization and an increasing number of cellular functions. Characterizing LLPS in live cells is also important because aberrant condensation has been linked to numerous diseases, including cancers and neurodegenerative disorders. LLPS is often driven by selective, transient, and multivalent interactions between intrinsically disordered proteins. Of great interest are the interaction dynamics of proteins participating in LLPS, which are well-summarized by measurements of their binding residence time (RT), that is, the amount of time they spend bound within condensates. Here, we present a method based on live-cell single-molecule imaging that allows us to measure the mean RT of a specific protein within condensates. We simultaneously visualize individual protein molecules and the condensates with which they associate, use single-particle tracking (SPT) to plot single-molecule trajectories, and then fit the trajectories to a model of protein-droplet binding to extract the mean RT of the protein. Finally, we show representative results where this single-molecule imaging method was applied to compare the mean RTs of a protein at its LLPS condensates when fused and unfused to an oligomerizing domain. This protocol is broadly applicable to measuring the interaction dynamics of any protein that participates in LLPS.

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.

Antiviral responses are shaped by heterogeneity in viral replication dynamics.

Antiviral signalling, which can be activated in host cells upon virus infection, restricts virus replication and communicates infection status to neighbouring cells. The antiviral response is heterogeneous, both quantitatively (efficiency of response activation) and qualitatively (transcribed antiviral gene set). To investigate the basis of this heterogeneity, we combined Virus Infection Real-time IMaging (VIRIM), a live-cell single-molecule imaging method, with real-time readouts of the dsRNA sensing pathway to analyse the response of human cells to encephalomyocarditis virus (EMCV) infection. We find that cell-to-cell heterogeneity in viral replication rates early in infection affect the efficiency of antiviral response activation, with lower replication rates leading to more antiviral response activation. Furthermore, we show that qualitatively distinct antiviral responses can be linked to the strength of the antiviral signalling pathway. Our analyses identify variation in early viral replication rates as an important parameter contributing to heterogeneity in antiviral response activation.

Systematic analysis of the molecular and biophysical properties of key DNA damage response factors.

Repair of DNA double strand breaks (DSBs) is integral to preserving genomic integrity. Therefore, defining the mechanisms underlying DSB repair will enhance our understanding of how defects in these pathways contribute to human disease and could lead to the discovery of new approaches for therapeutic intervention. Here, we established a panel of HaloTagged DNA damage response factors in U2OS cells which enables concentration-dependent protein labeling by fluorescent HaloTag ligands. Genomic insertion of HaloTag at the endogenous loci of these repair factors preserves expression levels and proteins retain proper subcellular localization, foci-forming ability, and functionally support DSB repair. We systematically analyzed total cellular protein abundance, measured recruitment kinetics to laser-induced DNA damage sites, and defined the diffusion dynamics and chromatin binding characteristics by live-cell single-molecule imaging. Our work demonstrates that the Shieldin complex, a critical factor in end-joining, does not exist in a preassembled state and that relative accumulation of these factors at DSBs occurs with different kinetics. Additionally, live-cell single-molecule imaging revealed the constitutive interaction between MDC1 and chromatin mediated by its PST repeat domain. Altogether, our studies demonstrate the utility of single-molecule imaging to provide mechanistic insights into DNA repair, which will serve as a powerful resource for characterizing the biophysical properties of DNA repair factors in living cells.

Dynamic epistasis analysis reveals how chromatin remodeling regulates transcriptional bursting

Transcriptional bursting has been linked to the stochastic positioning of nucleosomes. However, how bursting is regulated by the remodeling of promoter nucleosomes is unknown. Here, we use single-molecule live-cell imaging of GAL10 transcription in Saccharomyces cerevisiae to measure how bursting changes upon combined perturbations of chromatin remodelers, the transcription factor Gal4 and preinitiation complex components. Using dynamic epistasis analysis, we reveal how the remodeling of different nucleosomes regulates transcriptional bursting parameters. At the nucleosome covering the Gal4 binding sites, RSC and Gal4 binding synergistically facilitate each burst. Conversely, nucleosome remodeling at the TATA box controls only the first burst upon galactose induction. At canonical TATA boxes, the nucleosomes are displaced by TBP binding to allow for transcription activation even in the absence of remodelers. Overall, our results reveal how promoter nucleosome remodeling together with Gal4 and preinitiation complex binding regulates transcriptional bursting.

Structure and dynamics of the EGFR/HER2 heterodimer

HER2 belongs to the human epidermal growth factor receptor tyrosine kinase family. Its overexpression or hyperactivation is a leading cause for multiple types of cancers. HER2 functions mainly through dimerization with other family members, such as EGFR. However, the molecular details for heterodimer assembly have not been completely understood. Here, we report cryo-EM structures of the EGF- and epiregulin-bound EGFR/HER2 ectodomain complexes at resolutions of 3.3 Å and 4.5 Å, respectively. Together with the functional analyses, we demonstrate that only the dimerization arm of HER2, but not that of EGFR, is essential for their heterodimer formation and signal transduction. Moreover, we analyze the differential membrane dynamics and transient interactions of endogenous EGFR and HER2 molecules in genome-edited cells using single-molecule live-cell imaging. Furthermore, we show that the interaction with HER2 could allow EGFR to resist endocytosis. Together, this work deepens our understanding of the unique structural properties and dynamics of the EGFR/HER2 complex.

Live cell single molecule imaging reveals concentration dependent changes to dynamic behavior of nuclear receptors RAR and RXR.

Type 2 Nuclear Receptors (T2NRs) like Retinoic Acid Receptor (RAR) are believed to require heterodimerization with a common factor, the Retinoid X Receptor (RXR), to bind chromatin and regulate its target genes. Here, we deploy stroboscopic photo-activation SPT (spaSPT) to understand the molecular dynamics of the RAR/RXR system and infer different diffusion states acquired by RAR and RXR in the nucleoplasm. These states occupied by RAR and RXR presumably exist in dynamic equilibrium. Next, we ask in a complex heterodimer driven transcriptional regulatory system such as this, how is the dynamic equilibrium shifted when either of the heterodimeric partner is in excess. We find that in live cells, increasing the number of RAR molecules and not RXR molecules leads to altered chromatin binding of RAR. While increasing the number of molecules of RAR and RXR both alter the chromatin binding of RXR. Since pharmacological modulation of the RAR/RXR system is an important avenue for cancer treatments, this study constitutes a step toward understanding how perturbations of this system affect chromatin binding and possibly downstream transcriptional regulatory function.

Single-molecule localization-integrated trajectory analysis for the dynamic interaction of chromatin structure and transcription machinery.

The localization and dynamics of individual proteins in a living cell are essential information for investigating protein functions. Live-cell single-molecule imaging has provided direct information on dynamics involved in biological processes. We have identified the characteristic movement of transcription-related factors in elongation sites and directly measured the viscoelastic properties of phase-separated nucleolus proteins using single-molecule trajectory analysis in living cells. However, the functional dynamics are difficult to identify because of the highly heterogeneous distribution and mobility of individual molecules. Here, we developed localization-integrated single-molecule trajectory analysis with machine-learning-based trajectory classification for simultaneous single-molecule imaging. We applied this method to the dynamics of the largest subunit of RNA polymerase II (Pol II) in a crowded chromatin structure visualized by histone H3.1 labeled with spontaneously blinking protein in a living cell nucleus. We identified distinct Pol II mobility features by calculating various mobility indicators, including diffusion coefficient, spring coefficient, and confinement radius, then applied machine-learning methods, including dimensionality reduction and clustering. We also developed a series of analysis methods to quantify the spatial characteristics of the point distribution adjacent to the trajectory. These methods reveal that the spatial probability of surrounding histone distribution depends on the mobility state of Pol II. The simultaneous imaging technique and the new analysis method developed in this study directly link the physical properties of the transcription machinery with the biological function of 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.

Dynamics of endogenous PARP1 and PARP2 during DNA damage revealed by live-cell single-molecule imaging

PARP1 contributes to genome architecture and DNA damage repair through its dynamic association with chromatin. PARP1 and PARP2 (PARP1/2) recognize damaged DNA and recruit the DNA repair machinery. Using single-molecule microscopy in live cells, we monitored the movement of PARP1/2 on undamaged and damaged chromatin. We identify two classes of freely diffusing PARP1/2 and two classes of bound PARP1/2. The majority (>60%) of PARP1/2 diffuse freely in both undamaged and damaged nuclei and in the presence of inhibitors of PARP1/2 used for cancer therapy (PARPi). Laser-induced DNA damage results in a small fraction of slowly diffusing PARP1 and PARP2 to become transiently bound. Treatment of cells with PARPi in the presence of DNA damage causes subtle changes in the dynamics of bound PARP1/2, but not the high levels of PARP1/2 trapping seen previously. Our results imply that next-generation PARPi could specifically target the small fraction of DNA-bound PARP1/2.

Abstract IA019: Ectopic levels of low-complexity domain interactions repress endogenous oncogenic transcription

Abstract Mammalian transcription factors (TFs) ubiquitously contain intrinsically disordered low-complexity domains (LCDs) that perform transactivation functions. Recent studies have shown that dynamic, multivalent, and selective interactions between TF LCDs play an essential role in transactivation, but how such interactions mediate gene transcription remains unclear. It has been proposed that liquid-liquid phase separation (LLPS) of TFs driven by extensive LCD-LCD interactions is the mechanism underlying transactivation. Here, we investigate how tuning the level and localization of LCD-LCD interactions in vivo affects transcription of endogenous human genes. By quantitative single-cell imaging, we reveal that efficient gene activation by the oncogenic TF EWS/FLI1 requires narrow optimum of LCD-LCD interactions at its target genes associated with GGAA microsatellites. Increasing LCD-LCD interactions toward putative LLPS represses transcription of these genes in Ewing sarcoma cells. Likewise, ectopically creating LCD-LCD interactions to sequester EWS/FLI1 into a well-documented LLPS compartment, the nucleolus, represses EWS/FLI1 -driven transcription and inhibits malignant transformation of Ewing sarcoma. In addition, using live-cell single-molecule imaging, we show that a TF diffuses significantly more slowly in the nucleolus than in the nucleoplasm, demonstrating an important impact of LLPS on TF dynamic behaviors and single-molecule diffusion measurement as an effective approach to diagnose LLPS in vivo. Our findings show how altering the balance of LCD-LCD interactions can influence transcriptional regulation and suggest a potential therapeutic strategy for targeting disease-causing TFs. Citation Format: Shasha Chong. Ectopic levels of low-complexity domain interactions repress endogenous oncogenic transcription. [abstract]. In: Proceedings of the AACR Special Conference: Cancer Epigenomics; 2022 Oct 6-8; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2022;82(23 Suppl_2):Abstract nr IA019.

Fixation can change the appearance of phase separation in living cells.

Fixing cells with paraformaldehyde (PFA) is an essential step in numerous biological techniques as it is thought to preserve a snapshot of biomolecular transactions in living cells. Fixed-cell imaging techniques such as immunofluorescence have been widely used to detect liquid-liquid phase separation (LLPS) in vivo. Here, we compared images, before and after fixation, of cells expressing intrinsically disordered proteins that are able to undergo LLPS. Surprisingly, we found that PFA fixation can both enhance and diminish putative LLPS behaviors. For specific proteins, fixation can even cause their droplet-like puncta to artificially appear in cells that do not have any detectable puncta in the live condition. Fixing cells in the presence of glycine, a molecule that modulates fixation rates, can reverse the fixation effect from enhancing to diminishing LLPS appearance. We further established a kinetic model of fixation in the context of dynamic protein-protein interactions. Simulations based on the model suggest that protein localization in fixed cells depends on an intricate balance of protein-protein interaction dynamics, the overall rate of fixation, and notably, the difference between fixation rates of different proteins. Consistent with simulations, live-cell single-molecule imaging experiments showed that a fast overall rate of fixation relative to protein-protein interaction dynamics can minimize fixation artifacts. Our work reveals that PFA fixation changes the appearance of LLPS from living cells, presents a caveat in studying LLPS using fixation-based methods, and suggests a mechanism underlying the fixation artifact.

Detecting molecular interactions in live-cell single-molecule imaging with proximity-assisted photoactivation (PAPA).

Single-molecule imaging provides a powerful way to study biochemical processes in live cells, yet it remains challenging to track single molecules while simultaneously detecting their interactions. Here, we describe a novel property of rhodamine dyes, proximity-assisted photoactivation (PAPA), in which one fluorophore (the 'sender') can reactivate a second fluorophore (the 'receiver') from a dark state. PAPA requires proximity between the two fluorophores, yet it operates at a longer average intermolecular distance than Förster resonance energy transfer (FRET). We show that PAPA can be used in live cells both to detect protein-protein interactions and to highlight a subpopulation of labeled protein complexes in which two different labels are in proximity. In proof-of-concept experiments, PAPA detected the expected correlation between androgen receptor self-association and chromatin binding at the single-cell level. These results establish a new way in which a photophysical property of fluorophores can be harnessed to study molecular interactions in single-molecule imaging of live cells.

Direct Observation of Compartment-Specific Localization and Dynamics of Voltage-Gated Sodium Channels.

Brain enriched voltage-gated sodium channel (VGSC) Nav1.2 and Nav1.6 are critical for electrical signaling in the CNS. Previous studies have extensively characterized cell-type-specific expression and electrophysiological properties of these two VGSCs and how their differences contribute to fine-tuning of neuronal excitability. However, because of a lack of reliable labeling and imaging methods, the subcellular localization and dynamics of these homologous Nav1.2 and Nav1.6 channels remain understudied. To overcome this challenge, we combined genome editing, super-resolution, and live-cell single-molecule imaging to probe subcellular composition, relative abundances, and trafficking dynamics of Nav1.2 and Nav1.6 in cultured mouse and rat neurons and in male and female mouse brain. We discovered a previously uncharacterized trafficking pathway that targets Nav1.2 to the distal axon of unmyelinated neurons. This pathway uses distinct signals residing in the intracellular loop 1 between transmembrane domain I and II to suppress the retention of Nav1.2 in the axon initial segment and facilitate its membrane loading at the distal axon. As mouse pyramidal neurons undergo myelination, Nav1.2 is gradually excluded from the distal axon as Nav1.6 becomes the dominant VGSC in the axon initial segment and nodes of Ranvier. In addition, we revealed exquisite developmental regulation of Nav1.2 and Nav1.6 localizations in the axon initial segment and dendrites, clarifying the molecular identity of sodium channels in these subcellular compartments. Together, these results unveiled compartment-specific localizations and trafficking mechanisms for VGSCs, which could be regulated separately to modulate membrane excitability in the brain.SIGNIFICANCE STATEMENT Direct observation of endogenous voltage-gated sodium channels reveals a previously uncharacterized distal axon targeting mechanism and the molecular identity of sodium channels in distinct subcellular compartments.

Single-molecule study of VEGFR-2 spatiotemporal organization and activation on the surface of live endothelial cells

The dynamic organization of cell surface receptors plays an important role in signaling. Here we investigated the spatiotemporal organization of VEGF receptor-2 (VEGFR-2), a critical pro-angiogenic receptor tyrosine kinase in live, microvascular endothelial cells. Our study strategy combined live-cell single-molecule imaging of endogenous VEGFR-2 with computational image analysis and multiscale data analysis. We found that VEGFR-2, even in its basal, unstimulated state, possesses diffusion heterogeneity, with a mobile subpopulation and a subpopulation of restricted mobility, and assembly state heterogeneity, where receptors can be organized as monomeric or non-monomeric.

Dynamics of Endogenous PARP1 and PARP2 During DNA Damage Revealed by Live-Cell Single-Molecule Imaging

Dynamics of Endogenous PARP1 and PARP2 During DNA Damage Revealed by Live-Cell Single-Molecule Imaging

Live-Cell Imaging of Single Neurotrophin Receptor Molecules on Human Neurons in Alzheimer's Disease.

Neurotrophin receptors such as the tropomyosin receptor kinase A receptor (TrkA) and the low-affinity binding p75 neurotrophin receptor p75NTR play a critical role in neuronal survival and their functions are altered in Alzheimer’s disease (AD). Changes in the dynamics of receptors on the plasma membrane are essential to receptor function. However, whether receptor dynamics are affected in different pathophysiological conditions is unexplored. Using live-cell single-molecule imaging, we examined the surface trafficking of TrkA and p75NTR molecules on live neurons that were derived from human-induced pluripotent stem cells (hiPSCs) of presenilin 1 (PSEN1) mutant familial AD (fAD) patients and non-demented control subjects. Our results show that the surface movement of TrkA and p75NTR and the activation of TrkA- and p75NTR-related phosphoinositide-3-kinase (PI3K)/serine/threonine-protein kinase (AKT) signaling pathways are altered in neurons that are derived from patients suffering from fAD compared to controls. These results provide evidence for altered surface movement of receptors in AD and highlight the importance of investigating receptor dynamics in disease conditions. Uncovering these mechanisms might enable novel therapies for AD.