Single-molecule Tracking Methods Research Articles

Single molecule analysis of CENP-A chromatin by high-speed atomic force microscopy.

Chromatin accessibility is modulated in a variety of ways to create open and closed chromatin states, both of which are critical for eukaryotic gene regulation. At the single molecule level, how accessibility is regulated of the chromatin fiber composed of canonical or variant nucleosomes is a fundamental question in the field. Here, we developed a single-molecule tracking method where we could analyze thousands of canonical H3 and centromeric variant nucleosomes imaged by high-speed atomic force microscopy. This approach allowed us to investigate how changes in nucleosome dynamics in vitro inform us about transcriptional potential in vivo. By high-speed atomic force microscopy, we tracked chromatin dynamics in real time and determined the mean square displacement and diffusion constant for the variant centromeric CENP-A nucleosome. Furthermore, we found that an essential kinetochore protein CENP-C reduces the diffusion constant and mobility of centromeric nucleosomes along the chromatin fiber. We subsequently interrogated how CENP-C modulates CENP-A chromatin dynamics in vivo. Overexpressing CENP-C resulted in reduced centromeric transcription and impaired loading of new CENP-A molecules. From these data, we speculate that factors altering nucleosome mobility in vitro, also correspondingly alter transcription in vivo. Subsequently, we propose a model in which variant nucleosomes encode their own diffusion kinetics and mobility, and where binding partners can suppress or enhance nucleosome mobility.

Single-molecule tracking of dye diffusion in synthetic polymers: A tutorial review

Single-molecule tracking (SMT) methods have been widely employed to offer a high-resolution characterization of synthetic polymers under ambient conditions and, thus, have advanced our understanding of their unique material properties. SMT is based on the systematic monitoring of the diffusive motions of individual fluorescent dye molecules in the as-prepared polymer thin films or thicker monoliths. Quantitative assessment of the recorded SMT video data involves the systematic analysis of the generated diffusion trajectories of a single molecule using well-established and reported methods. The results have offered a wealth of new information on the structural alignment, orientational order, and long-range continuity of the polymer microdomains; the nanoscale material heterogeneities governed by defects, misalignment, and ill-controlled preparation conditions; as well as the various forms of probe–host interactions on the single-molecule level. In the first part of this Tutorial review, we describe the fundamental principles and instrumentation of SMT, before offering interested readers and potential future SMT users a practical guidance on the selection of fluorescent probe molecules, preparation of suitable samples, and optimization of experimental conditions and imaging parameters. Then, we highlight several representative SMT studies in microphase-separated block copolymers, and semicrystalline and amorphous homopolymers to further emphasize the usefulness of SMT methods for polymer characterization without neglecting some of their shortcomings. This Tutorial review is written with the motivation to attract new researchers to the fast-growing field and assist them in starting their own SMT research of synthetic polymers and other technologically useful soft matter systems.

The glucocorticoid receptor associates with the cohesin loader NIPBL to promote long-range gene regulation.

The cohesin complex is central to chromatin looping, but mechanisms by which these long-range chromatin interactions are formed and persist remain unclear. We demonstrate that interactions between a transcription factor (TF) and the cohesin loader NIPBL regulate enhancer-dependent gene activity. Using mass spectrometry, genome mapping, and single-molecule tracking methods, we demonstrate that the glucocorticoid (GC) receptor (GR) interacts with NIPBL and the cohesin complex at the chromatin level, promoting loop extrusion and long-range gene regulation. Real-time single-molecule experiments show that loss of cohesin markedly diminishes the concentration of TF molecules at specific nuclear confinement sites, increasing TF local concentration and promoting gene regulation. Last, patient-derived acute myeloid leukemia cells harboring cohesin mutations exhibit a reduced response to GCs, suggesting that the GR-NIPBL-cohesin interaction is defective in these patients, resulting in poor response to GC treatment.

Delineating the design features underlying the stepping mechanism of class X myosin

Motor proteins, including myosin, kinesin and dynein, play an important role in transport in cells. Class X myosin (Myo10) is the first reported myosin class to form an anti-parallel dimer and is involved in the formation of and trafficking in filopodia. Delineating the design features underlying the stepping mechanism of Myo10 is necessary for understanding these processes, but remains elusive. Here, we investigate the stepping mechanism of Myo10 by creating several chimeras between a vertebrate (chicken) class V myosin (Myo5) and the human Myo10 and by implementing the single-molecule tracking method called FIONA.

Single-Molecule Fluorescence Imaging Reveals GABAB Receptor Aggregation State Changes.

The GABAB receptor is a typical G protein–coupled receptor, and its functional impairment is related to a variety of diseases. While the premise of GABAB receptor activation is the formation of heterodimers, the receptor also forms a tetramer on the cell membrane. Thus, it is important to study the effect of the GABAB receptor aggregation state on its activation and signaling. In this study, we have applied single-molecule photobleaching step counting and single-molecule tracking methods to investigate the formation and change of GABAB dimers and tetramers. A single-molecule stoichiometry assay of the wild-type and mutant receptors revealed the key sites on the interface of ligand-binding domains of the receptor for its dimerization. Moreover, we found that the receptor showed different aggregation behaviors at different conditions. Our results offered new evidence for a better understanding of the molecular basis for GABAB receptor aggregation and activation.

Single-molecule tracking measurement of PDMS layer during curing process

The curing process of poly(dimethylsiloxane) (PDMS) was microscopically investigated by the single-molecule tracking method based on the diffusion motion of fluorescent dye molecules adding to the PDMS layer stored at a temperature of 308K. The PDMS layer was completely cured at 900 min after adding a curing agent. We compared the time- and ensemble-averaged mean square displacements (MSDs) of the tracked molecules at 90, 510, and 900 min after adding the curing agent into the PDMS layer. The discrepancies were observed between the time- and ensemble-averaged MSDs, indicating weak ergodicity breaking. The spatially averaged diffusion coefficient exhibited a two-step decrease: first step was rapid decrease suggesting the extent of crosslinking, and second step was slow one suggesting the increase of crosslink density. The single-molecule trajectory scale analysis revealed the heterogeneous distribution of the diffusion coefficient. By calculating the heat map from the slope of moment scaling spectrum (MSS) of each single-molecule trajectory, cluster structures were recognized. The spatial correlation of the slope of MSS decreased with the time elapsed. These results suggested the existence of the heterogeneous structure in the PDMS layer during the curing process.

Abstract IA07: KRAS4b’s unique diffusion behavior is defined by plasma membrane and effector interactions

Abstract RAS proteins are GTP-dependent switches that control and regulate signaling pathways involved in cell fate and are frequently mutated in cancer. RAS association with the plasma membrane, or with certain endomembrane compartments, is a required step for its activity. Why precisely this is so remains an open question. One possibility is that RAS is merely required for recruitment of RAF and other effectors to the membrane where it can associate with key regulatory molecules that lead to signaling activation. However, four isoforms exist in humans (HRAS, NRAS, and two splice variants, KRAS4b and KRAS4a) and their differences lie within 22 amino acids in the C-terminal hypervariable region (HVR, aa 167-189). These differences in the domain responsible for membrane association result in the recruitment and organization of RAS into distinct membrane nanodomains, and it is thought that this results in differential signaling behavior from RAS isoforms. Isoform-specific RAS nanodomains are thought to both be highly dynamic, differentially composed of a variety of lipids and proteins, and supported by interactions with cytoskeletal structures. How these domains are regulated, and how they influence isoform-specific RAS signaling, remains unclear. Here, we report the molecular mobility of RAS variants in the plasma membrane of living cancer cells using single-molecule tracking methods. Detailed analysis of tracks revealed that KRAS4b molecules exhibit confined mobility with three diffusive states in the active plasma membrane of living cells. This diffusion characteristic was unique to KRAS4b and influenced by both the hypervariable region and globular domain of the protein, compared to all the other Ras isoforms. Importantly, the occupancy of each diffusive states was altered for the oncogenic mutant of KRAS4b, suggesting that the diffusive states we observe are related to signaling events. Our working hypothesis is that the HVR of KRAS4b may be directly involved in assembling the membrane lipid and protein nanodomain necessary for KRAS4b signaling activity. In addition, using two-color single-molecule tracking studies, we are beginning to characterize the interactions of KRAS4b with its major effector RAF. From these studies, we are learning about the kinetics of RAS/RAF interactions in the membrane of living cells. Understanding the underlying principle of KRAS4b functionality on cell membranes is useful for developing novel therapeutic strategies for targeting oncogenic KRAS4b. Citation Format: Debanjan Goswami, De Chen, John Columbus, Yue Yang, Felice Lightstone, Thomas Turbyville. KRAS4b’s unique diffusion behavior is defined by plasma membrane and effector interactions [abstract]. In: Proceedings of the AACR Special Conference on Targeting RAS-Driven Cancers; 2018 Dec 9-12; San Diego, CA. Philadelphia (PA): AACR; Mol Cancer Res 2020;18(5_Suppl):Abstract nr IA07.

Entangled polymer dynamics beyond reptation

Macroscopic properties of polymers arise from microscopic entanglement of polymer chains. Entangled polymer dynamics have been described theoretically by time- and space-averaged relaxation modes of single chains occurring at different time and length scales. However, theoretical and experimental studies along this framework provide oversimplified picture of spatiotemporally heterogeneous polymer dynamics. Characterization of entangled polymer dynamics beyond this paradigm requires a method that enables to capture motion and relaxation occurring in real space at different length and time scales. Here we develop new single-molecule characterization platform by combining super-resolution fluorescence imaging and recently developed single-molecule tracking method, cumulative-area tracking, which enables to quantify the chain motion in the length and time scale of nanometres to micrometres and milliseconds to minutes. Using linear and cyclic dsDNA molecules as model systems, our new method reveals chain-position-dependent motion of the entangled linear chains, which is beyond the scope of current theoretical framework.

Visualizing the molecular mode of motion from a correlative analysis of localization microscopy datasets

The determination of the mode and rapidity of motion of individual molecules within a biological sample is becoming a more and more common analysis in biophysical investigations. Single molecule tracking (SMT) techniques allow reconstructing the trajectories of individual molecules within a movie, provided that the position from one frame to the other can be correctly linked. The outcomes, however, appear to depend on the specific method used, and most techniques display a limitation to capture fast modes of motion in a crowded environment. We demonstrate here that the limitations encountered by conventional SMT can be significantly overcome by employing alternative approaches based on image spatial-temporal correlations, enabling to visually extract quantitative insights on the ensemble mode of motion of fluorescently labeled biomolecules that would otherwise be inaccessible.

Dense Poly(ethylene glycol) Brushes Reduce Adsorption and Stabilize the Unfolded Conformation of Fibronectin.

Polymer brushes, in which polymers are end-tethered densely to a grafting surface, are commonly proposed for use as stealth coatings for various biomaterials. However, although their use has received considerable attention, a mechanistic understanding of the impact of brush properties on protein adsorption and unfolding remains elusive. We investigated the effect of the grafting density of poly(ethylene glycol) (PEG) brushes on the interactions of the brush with fibronectin (FN) using high-throughput single-molecule tracking methods, which directly measure protein adsorption and unfolding within the brush. We observed that, as grafting density increased, the rate of FN adsorption decreased; however, surface-adsorbed FN unfolded more readily, and unfolded molecules were retained on the surface for longer residence times relative to those of folded molecules. These results, which are critical for the rational design of PEG brushes, suggest that there is a critical balance between protein adsorption and conformation that underlies the utility of such brushes in physiological environments.

Tracking single molecules at work in living cells.

Methods for imaging and tracking single molecules conjugated with fluorescent probes, called single-molecule tracking (SMT), are now providing researchers with the unprecedented ability to directly observe molecular behaviors and interactions in living cells. Current SMT methods are achieving almost the ultimate spatial precision and time resolution for tracking single molecules, determined by the currently available dyes. In cells, various molecular interactions and reactions occur as stochastic and probabilistic processes. SMT provides an ideal way to directly track these processes by observing individual molecules at work in living cells, leading to totally new views of the biochemical and molecular processes used by cells whether in signal transduction, gene regulation or formation and disintegration of macromolecular complexes. Here we review SMT methods, summarize the recent results obtained by SMT, including related superresolution microscopy data, and describe the special concerns when SMT applications are shifted from the in vitro paradigms to living cells.

Following Single Molecules to a Better Understanding of Self-Assembled One-Dimensional Nanostructures

Single-molecule tracking (SMT) methods are now being employed to probe the morphologies and mass-transport characteristics of self-assembled one-dimensional (1D) nanostructures. Such nanostructures are found in surfactant-templated mesoporous metal oxides, phase-separated block copolymers, and lyotropic liquid-crystal mesophases. This Perspective begins with a review of investigations in which SMT methods have been employed for in situ visualization of 1D nanostructures and their ability to guide and support 1D diffusion of fluorescent probe molecules. New orthogonal regression methods for the quantitative characterization of 1D nanostructure alignment and order are subsequently discussed. Recent investigations in which the confined orientational motions of single molecules are probed by single-molecule emission polarization measurements are highlighted. These data are used to access high-precision estimates of the effective lateral nanostructure dimensions. The results reveal the important role played by...

Single-Molecule Tracking Studies of Millimeter-Scale Cylindrical Domain Alignment in Polystyrene–Poly(ethylene oxide) Diblock Copolymer Films Induced by Solvent Vapor Penetration

Single-molecule tracking (SMT) was used to probe the alignment of cylindrical microdomains in polystyrene–poly(ethylene oxide) diblock copolymer (PS-b-PEO) films induced by directional solvent penetration. PS-b-PEO films were supported between two glass plates. Aligned PEO domains were obtained upon horizontal penetration of 1,4-dioxane vapor through the films, while no observable alignment occurred when benzene or toluene was used. Domain alignment was assessed by SMT, using a dye that partitions into PEO. More than 70% of the dye molecules exhibited one-dimensional (1D) diffusion along the dioxane penetration direction. Quantitative assessment of 1D trajectory alignment yielded an order parameter of ca. 0.9. A high degree of domain order was observed across millimeter distances throughout the 4 μm thick films. PEO domain size (ca. 11 nm radius) was determined from the probe positioning error. These results demonstrate the utility of SMT methods for characterization of nanoscale domains in cylinder-forming block copolymer films.

Acetylcholine Receptor Organization in Membrane Domains in Muscle Cells: EVIDENCE FOR RAPSYN-INDEPENDENT AND RAPSYN-DEPENDENT MECHANISMS

Nicotinic acetylcholine receptors (nAChR) in muscle fibers are densely packed in the postsynaptic region at the neuromuscular junction. Rapsyn plays a central role in directing and clustering nAChR during cellular differentiation and neuromuscular junction formation; however, it has not been demonstrated whether rapsyn is the only cause of receptor immobilization. Here, we used single-molecule tracking methods to investigate nAChR mobility in plasma membranes of myoblast cells during their differentiation to myotubes in the presence and absence of rapsyn. We found that in myoblasts the majority of nAChR were immobile and that ∼20% of the receptors showed restricted diffusion in small domains of ∼50 nm. In myoblasts devoid of rapsyn, the fraction of mobile nAChR was considerably increased, accompanied by a 3-fold decrease in the immobile population of nAChR with respect to rapsyn-expressing cells. Half of the mobile receptors were confined to domains of ∼120 nm. Measurements performed in heterologously transfected HEK cells confirmed the direct immobilization of nAChR by rapsyn. However, irrespective of the presence of rapsyn, about one-third of nAChR were confined in 300-nm domains. Our results show (i) that rapsyn efficiently immobilizes nAChR independently of other postsynaptic scaffold components; (ii) nAChR is constrained in confined membrane domains independently of rapsyn; and (iii) in the presence of rapsyn, the size of these domains is strongly reduced.

Direct Observation and Analysis of 3d Diffusing Fluorescent Proteins in Solution using Single Image Measurements

The low temporal resolution of current single-molecule tracking methods on the order of 100 ms limits the methods' capability to investigate fast diffusion processes, such as a protein's 3D diffusion in solution. Using TIRF microscopy at sub-millisecond exposure times, we have directly imaged 3D-diffusing streptavidin-Alexa533 molecules in solution and recorded the intensity profiles of the diffusion proteins. The intensity profile of a 3D-diffusion protein represents a convolution of the native point spread function (PSF) of the molecule with the protein's pathway distribution function for the given exposure time, and can be approximated by a two-dimensional Gaussian function. The standard deviation (SD) of the Gaussian intensity profile of the 3D diffusing proteins allows us to obtain the molecule's diffusion coefficient to known precision [1], all by using a single image of sub-millisecond exposure time and our theoretical formulation relating SD of the intensity profile of a 3D-diffusing protein to the diffusion coefficient. Our theoretical formulation agrees with results using simulations and experimental studies on proteins of a known diffusion coefficient.[1]. DeSantis, M. C., DeCenzo, S. H., Li, J. -L. & Wang, Y.M. Physical Review E. In Review.

Paradigm Shift of the Plasma Membrane Concept from the Two-Dimensional Continuum Fluid to the Partitioned Fluid: High-Speed Single-Molecule Tracking of Membrane Molecules

Recent advancements in single-molecule tracking methods with nanometer-level precision now allow researchers to observe the movement, recruitment, and activation of single molecules in the plasma membrane in living cells. In particular, on the basis of the observations by high-speed single-particle tracking at a frame rate of 40,000 frames s(1), the partitioning of the fluid plasma membrane into submicron compartments throughout the cell membrane and the hop diffusion of virtually all the molecules have been proposed. This could explain why the diffusion coefficients in the plasma membrane are considerably smaller than those in artificial membranes, and why the diffusion coefficient is reduced upon molecular complex formation (oligomerization-induced trapping). In this review, we first describe the high-speed single-molecule tracking methods, and then we critically review a new model of a partitioned fluid plasma membrane and the involvement of the actin-based membrane-skeleton "fences" and anchored-transmembrane protein "pickets" in the formation of compartment boundaries.