Single-molecule Microscopy Research Articles

Molecular crowding in single eukaryotic cells: Using cell environment biosensing and single-molecule optical microscopy to probe dependence on extracellular ionic strength, local glucose conditions, and sensor copy number.

The physical and chemical environment inside cells is of fundamental importance to all life but has traditionally been difficult to determine on a subcellular basis. Here we combine cutting-edge genomically integrated FRET biosensing to readout localized molecular crowding in single live yeast cells. Confocal microscopy allows us to build subcellular crowding heatmaps using ratiometric FRET, while whole-cell analysis demonstrates crowding is reduced when yeast is grown in elevated glucose concentrations. Simulations indicate that the cell membrane is largely inaccessible to these sensors and that cytosolic crowding is broadly uniform across each cell over a timescale of seconds. Millisecond single-molecule optical microscopy was used to track molecules and obtain brightness estimates that enabled calculation of crowding sensor copy numbers. The quantification of diffusing molecule trajectories paves the way for correlating subcellular processes and the physicochemical environment of cells under stress.

Advanced fluorescence microscopy reveals disruption of dynamic CXCR4 dimerization by subpocket-specific inverse agonists

Although class A G protein-coupled receptors (GPCRs) can function as monomers, many of them form dimers and oligomers, but the mechanisms and functional relevance of such oligomerization is ill understood. Here, we investigate this problem for the CXC chemokine receptor 4 (CXCR4), a GPCR that regulates immune and hematopoietic cell trafficking, and a major drug target in cancer therapy. We combine single-molecule microscopy and fluorescence fluctuation spectroscopy to investigate CXCR4 membrane organization in living cells at densities ranging from a few molecules to hundreds of molecules per square micrometer of the plasma membrane. We observe that CXCR4 forms dynamic, transient homodimers, and that the monomer-dimer equilibrium is governed by receptor density. CXCR4 inverse agonists that bind to the receptor minor pocket inhibit CXCR4 constitutive activity and abolish receptor dimerization. A mutation in the minor binding pocket reduced the dimer-disrupting ability of these ligands. In addition, mutating critical residues in the sixth transmembrane helix of CXCR4 markedly diminished both basal activity and dimerization, supporting the notion that CXCR4 basal activity is required for dimer formation. Together, these results link CXCR4 dimerization to its density and to its activity. They further suggest that inverse agonists binding to the minor pocket suppress both dimerization and constitutive activity and may represent a specific strategy to target CXCR4.

Endogenous Labeling for Light Microscopy during HIV-1 Immune Responses

New approaches in single molecule spectroscopy and microscopy are able to resolve the spatial and temporal resolution of T cell receptor signaling in the context of immune responses to HIV-1 infection. These approaches need to be complemented with novel techniques that endogenously tag the protein or proteins of interest, yet avoid overexpression, to image protein dynamics under physiological conditions.

Probing Nanoscale Diffusional Heterogeneities in Cellular Membranes through Multidimensional Single-Molecule and Super-Resolution Microscopy.

Diffusion properties notably determine the behavior of biomembranes. Here we report the concurrent nanoscale fine-mapping of membrane topography, diffusivity, and packing order in live mammalian cells through a synergy of single-molecule and super-resolution methods. By identifying a bright, lipophilic fluorescence turn-on probe that enables sustained single-molecule imaging of cellular membranes under stroboscopic excitation, we accumulate the positions and transient displacements of >106 probe molecules to achieve super-resolution topography and diffusivity mapping. We thus determine a trend that the membrane diffusivity drops with increased lipid packing order when comparing the endoplasmic reticulum (ER) membrane, plasma membrane, and nanodomains induced by cholera toxin B. Utilizing our nanoscale mapping capability, we further unveil reduced diffusivity in the ER membrane at ER-plasma membrane contact sites. By next integrating spectrally resolved single-molecule imaging, we show that this localized diffusion slowdown is not due to altered lipid packing order but may instead be attributed to local protein crowding. Our integrated multidimensional single-molecule approach thus unveils and differentiates between nanoscale diffusional heterogeneities of different origins in live-cell membranes.

Super-resolution Microscopy with Single Molecules in Biology and Beyond-Essentials, Current Trends, and Future Challenges.

Single-molecule super-resolution microscopy has developed from a specialized technique into one of the most versatile and powerful imaging methods of the nanoscale over the past two decades. In this perspective, we provide a brief overview of the historical development of the field, the fundamental concepts, the methodology required to obtain maximum quantitative information, and the current state of the art. Then, we will discuss emerging perspectives and areas where innovation and further improvement are needed. Despite the tremendous progress, the full potential of single-molecule super-resolution microscopy is yet to be realized, which will be enabled by the research ahead of us.

Nanoelectromechanical photothermal polarization microscopy with 3 Å localization precision

Single-molecule microscopy has become an indispensable tool for biochemical analysis. The capability of characterizing distinct properties of individual molecules without averaging has provided us with a different perspective for the existing scientific issues and phenomena. Recently, super-resolution fluorescence microscopy techniques have overcome the optical diffraction limit by the localization of molecule positions. However, the labeling process can potentially modify the intermolecular dynamics. Based on the highly sensitive nanomechanical photothermal microscopy reported previously, we propose optimizations on this label-free microscopy technique toward localization microscopy. A localization precision of 3 Å is achieved with gold nanoparticles, and the detection of polarization-dependent absorption is demonstrated, which opens the door for further improvement with polarization modulation imaging.

EJE AWARD 2020: Signalling by G protein-coupled receptors: why space and time matter.

G protein-coupled receptors (GPCRs) are the largest family of membrane receptors and major drug targets. They play a fundamental role in the endocrine system, where they mediate the effects of several hormones and neurotransmitters. As a result, alterations of GPCR signalling are a major cause of endocrine disorders such as congenital hypothyroidism or Cushing's syndrome. My group develops innovative optical methods such as fluorescence resonance energy transfer (FRET) and single-molecule microscopy, which allow us to investigate GPCR signalling in living cells with unprecedented spatiotemporal resolution. Using this innovative approach, we have contributed to elucidate some long-debated questions about the mechanisms of GPCR signalling and their involvement in human disease. Among other findings, these studies have led to the unexpected discovery that GPCRs are not only signalling at the cell surface, as previously assumed, but also at various intracellular sites. This has important implications to understand how hormones and neurotransmitters produce specific responses in our cells and might pave the way to innovative treatments for common diseases like diabetes or heart failure.

From single particle motion to structure of biological systems

Single molecule and single particle microscopy opened the door to observing dynamical processes in noisy living systems. Recent studies demonstrate how the stochastic motion of tracer particles can also provide us with information about the structure and flow properties of active and complex biological systems.

SLC6 transporter oligomerization

Transporters of the solute carrier 6 (SLC6) family mediate the reuptake of neurotransmitters such as dopamine, norepinephrine, serotonin, GABA, and glycine. SLC6 family members are 12 transmembrane helix‐spanning proteins that operate using the transmembrane sodium gradient for transport. These transporters assume various quaternary arrangements ranging from monomers to complex stoichiometries with multiple subunits. Dopamine and serotonin transporter oligomerization has been implicated in trafficking of newly formed proteins from the endoplasmic reticulum to the plasma membrane with a pre‐fixed assembly. Once at the plasma membrane, oligomers are kept fixed in their quaternary assembly by interaction with phosphoinositides. While it remains unclear how oligomer formation precisely affects physiological transporter function, it has been shown that oligomerization supports the activity of release‐type psychostimulants. Most recently, single molecule microscopy experiments unveiled that the stoichiometry differs between individual members of the SLC6 family. The present overview summarizes our understanding of the influence of plasma membrane constituents on transporter oligomerization, describes the known interfaces between protomers and discusses open questions.

Rad54 and Rdh54 occupy spatially and functionally distinct sites within the Rad51-ssDNA presynaptic complex.

Rad54 and Rdh54 are closely related ATP-dependent motor proteins that participate in homologous recombination (HR). During HR, these enzymes functionally interact with the Rad51 presynaptic complex (PSC). Despite their importance, we know little about how they are organized within the PSC, or how their organization affects PSC function. Here, we use single-molecule optical microscopy and genetic analysis of chimeric protein constructs to evaluate the binding distributions of Rad54 and Rdh54 within the PSC. We find that Rad54 and Rdh54 have distinct binding sites within the PSC, which allow these proteins to act cooperatively as DNA sequences are aligned during homology search. Our data also reveal that Rad54 must bind to a specific location within the PSC, whereas Rdh54 retains its function in the repair of MMS-induced DNA damage even when recruited to the incorrect location. These findings support a model in which the relative binding sites of Rad54 and Rdh54 help to define their functions during mitotic HR.

Multiple Factors Regulate the Spirocyclization Equilibrium of Si-Rhodamines.

Si-rhodamine has been extensively used in super-resolution fluorescence imaging in recent years. Its equilibrium between ring-closed nonfluorescent spirolactones and ring-opened fluorescent zwitterions endows Si-rhodamine with excellent fluorogenicity, membrane permeability, and photostability. In this paper, the equilibrium of Si-rhodamine between lactones and zwitterions was revealed to be greatly affected by various environmental factors, including molecular aggregation, solvent polarity, pH, metal ions, irradiation, and temperature. These environmental sensitivities make Si-rhodamine useful as a hydrochromic material, a fluorescent sensor array for metal ions or solvents, and a photoactivatable switch. Importantly, these results indicate that using Si-rhodamine as a fluorogenic probe or a blinking fluorophore in single-molecule localization super-resolution microscopy requires caution on possible false signals caused by its environmental sensitivity.

Sequential Folding of the Nickel/Cobalt Riboswitch Is Facilitated by a Conformational Intermediate: Insights from Single-Molecule Kinetics and Thermodynamics.

The present work presents the first single-molecule fluorescence resonant energy transfer (smFRET) studies of the nickel/cobalt (NiCo) riboswitch, with temperature-dependent, single-molecule confocal microscopy to provide comprehensive kinetic and thermodynamic information on folding into a biochemically competent structure. The results indicate that the NiCo riboswitch first folds into a more compact "prefolded" conformation, with a preorganized binding pocket partially stabilized under physiological conditions by noncognate monovalent/divalent cations. Such a prefolded intermediate then has opportunity to fold further into a tightly ligand-bound structure, in response to the cognate ligands, Ni2+ or Co2+, with submicromolar affinities. Such stepwise ligand-induced folding represents a particularly clean example of a conformational selection ("fold-then-bind") mechanism, whereby a configuration dynamically accessible by thermal fluctuation is stabilized into the final folded state by ligand association. In addition, we observe a strong positive cooperativity in the ligand-induced folding kinetics with respect to both Ni2+ and Co2+ ligands. This provides maximal sensitivity in the riboswitch conformational response near [Ni2+] or [Co2+] ≈ Kd, which facilitates more accurate biochemical probing of the cell environment and therefore bioregulation of gene expression. Temperature-dependent kinetics at the single-molecule level has also been explored, which permits free energies to be deconstructed into enthalpic and entropic components along the folding coordinate. In the absence of the cognate ligand, a predominantly enthalpic barrier between the unfolded riboswitch (U) and the prefolded intermediate (I) suggests a rearrangement of the hydrogen bonding network, whereas in the presence of the cognate ligand, a large entropic penalty (-TΔS0 > 0) in forming the folded riboswitch conformation (F) is almost perfectly counterbalanced by an equivalent enthalpic gain (ΔH0 < 0) to yield ΔG0 ≈ 0. The thermodynamic results are therefore consistent with a simple physical picture of riboswitch folding, whereby association of the cognate ligand is strongly stabilized by Coulombic attraction while forming an entropically more ordered structure around the binding site.

Single-molecule live-cell imaging reveals RecB-dependent function of DNA polymerase IV indouble strand break repair.

Several functions have been proposed for the Escherichia coli DNA polymerase IV (pol IV). Although much research has focused on a potential role for pol IV in assisting pol III replisomes in the bypass of lesions, pol IV is rarely found at the replication fork in vivo. Pol IV is expressed at increased levels in E. coli cells exposed to exogenous DNA damaging agents, including many commonly used antibiotics. Here we present live-cell single-molecule microscopy measurements indicating that double-strand breaks induced by antibiotics strongly stimulate pol IV activity. Exposure to the antibiotics ciprofloxacin and trimethoprim leads to the formation of double strand breaks in E. coli cells. RecA and pol IV foci increase after treatment and exhibit strong colocalization. The induction of the SOS response, the appearance of RecA foci, the appearance of pol IV foci and RecA-pol IV colocalization are all dependent on RecB function. The positioning of pol IV foci likely reflects a physical interaction with the RecA* nucleoprotein filaments that has been detected previously in vitro. Our observations provide an in vivo substantiation of a direct role for pol IV in double strand break repair in cells treated with double strand break-inducing antibiotics.

CARs need a jump start to get going.

CAR T cells are engineered to recognize tumor-specific antigens and kill tumor cells. A study of CAR T cell activation using single-molecule microscopy reveals that CARs fail to efficiently convert antigen binding into early T cell signaling events.

The transition state and regulation of γ-TuRC-mediated microtubule nucleation revealed by single molecule microscopy.

Determining how microtubules (MTs) are nucleated is essential for understanding how the cytoskeleton assembles. While the MT nucleator, γ-tubulin ring complex (γ-TuRC) has been identified, precisely how γ-TuRC nucleates a MT remains poorly understood. Here, we developed a single molecule assay to directly visualize nucleation of a MT from purified Xenopus laevis γ-TuRC. We reveal a high γ-/αβ-tubulin affinity, which facilitates assembly of a MT from γ-TuRC. Whereas spontaneous nucleation requires assembly of 8 αβ-tubulins, nucleation from γ-TuRC occurs efficiently with a cooperativity of 4 αβ-tubulin dimers. This is distinct from pre-assembled MT seeds, where a single dimer is sufficient to initiate growth. A computational model predicts our kinetic measurements and reveals the rate-limiting transition where laterally associated αβ-tubulins drive γ-TuRC into a closed conformation. NME7, TPX2, and the putative activation domain of CDK5RAP2 h γ-TuRC-mediated nucleation, while XMAP215 drastically increases the nucleation efficiency by strengthening the longitudinal γ-/αβ-tubulin interaction.

Single-molecule analysis reveals agonist-specific dimer formation of µ-opioid receptors

G-protein-coupled receptors (GPCRs) are key signaling proteins that mostly function as monomers, but for several receptors constitutive dimer formation has been described and in some cases is essential for function. Using single-molecule microscopy combined with super-resolution techniques on intact cells, we describe here a dynamic monomer-dimer equilibrium of µ-opioid receptors (µORs), where dimer formation is driven by specific agonists. The agonist DAMGO, but not morphine, induces dimer formation in a process that correlates both temporally and in its agonist- and phosphorylation-dependence with β-arrestin2 binding to the receptors. This dimerization is independent from, but may precede, µOR internalization. These data suggest a new level of GPCR regulation that links dimer formation to specific agonists and their downstream signals.

Dynamic topology of double-stranded telomeric DNA studied by single-molecule manipulation in vitro.

The dynamic topological structure of telomeric DNA is closely related to its biological function; however, no such structural information on full-length telomeric DNA has been reported due to difficulties synthesizing long double-stranded telomeric DNA. Herein, we developed an EM-PCR and TA cloning-based approach to synthesize long-chain double-stranded tandem repeats of telomeric DNA. Using mechanical manipulation assays based on single-molecule atomic force microscopy, we found that mechanical force can trigger the melting of double-stranded telomeric DNA and the formation of higher-order structures (G-quadruplexes or i-motifs). Our results show that only when both the G-strand and C-strand of double-stranded telomeric DNA form higher-order structures (G-quadruplexes or i-motifs) at the same time (e.g. in the presence of 100 mM KCl under pH 4.7), that the higher-order structure(s) can remain after the external force is removed. The presence of monovalent K+, single-wall carbon nanotubes (SWCNTs), acidic conditions, or short G-rich fragments (∼30 nt) can shift the transition from dsDNA to higher-order structures. Our results provide a new way to regulate the topology of telomeric DNA.

Polyelectrolyte Surface Diffusion in a Nanoslit Geometry

The surface diffusion of poly-l-lysine (PLL) in a planar nanoslit was studied using convex lens-induced confinement (CLiC) single-molecule tracking microscopy. Three surface chemistries were employ...

Imaging the fibroblast growth factor receptor network on the plasma membrane with DNA-assisted single-molecule super-resolution microscopy.

Fibroblast growth factor receptors (FGFRs) are a subfamily of receptor tyrosine kinases and central players in health and disease. Following ligand binding and the formation of homo- and heteromeric complexes, FGFRs initiate a cellular response. Challenges in studying FGFR activation are inner-subfamily interactions and a complex heterogeneity of these in the cell membrane, which demand for observation techniques that can resolve individual protein complexes and that are compatible with endogenous protein levels. Here, we established an imaging and analysis pipeline for multiplexed single-molecule localization microscopy (SMLM) of the FGFR network at the plasma membrane. Using DNA-labeled primary antibodies, we visualize all four FGFRs in the same cell with near-molecular spatial resolution. From the super-resolution imaging data, we extract information on FGFR density, spatial distribution, and inner-subfamily colocalization. Our approach is straightforward and easily adaptable to other multiplexed SMLM data of membrane proteins.

Nucleoplasmic signals promote directed transmembrane protein import simultaneously via multiple channels of nuclear pores

Roughly 10% of eukaryotic transmembrane proteins are found on the nuclear membrane, yet how such proteins target and translocate to the nucleus remains in dispute. Most models propose transport through the nuclear pore complexes, but a central outstanding question is whether transit occurs through their central or peripheral channels. Using live-cell high-speed super-resolution single-molecule microscopy we could distinguish protein translocation through the central and peripheral channels, finding that most inner nuclear membrane proteins use only the peripheral channels, but some apparently extend intrinsically disordered domains containing nuclear localization signals into the central channel for directed nuclear transport. These nucleoplasmic signals are critical for central channel transport as their mutation blocks use of the central channels; however, the mutated proteins can still complete their translocation using only the peripheral channels, albeit at a reduced rate. Such proteins can still translocate using only the peripheral channels when central channel is blocked, but blocking the peripheral channels blocks translocation through both channels. This suggests that peripheral channel transport is the default mechanism that was adapted in evolution to include aspects of receptor-mediated central channel transport for directed trafficking of certain membrane proteins.