Single-molecule Behavior Research Articles

RNA imaging: seeing is believing.

RNA imaging: seeing is believing.

Dissecting the behavior and function of MBD3 in DNA methylation homeostasis by single-molecule spectroscopy and microscopy.

The detailed mechanism for DNA methylation homeostasis relies on an intricate regulatory network with a possible contribution from methyl-CpG-binding domain protein 3 (MBD3). In this study we examine the single-molecule behavior of MBD3 and its functional implication in balancing the activity of DNA methyltransferases (DNMTs). Besides a localization tendency to DNA demethylating sites, MBD3 experiences a concurrent transcription with DNMTs in cell cycle. Fluorescence lifetime correlation spectroscopy (FLCS) and photon counting histogram (PCH) were applied to characterize the chromatin binding kinetics and stoichiometry of MBD3 in different cell phases. In the G1-phase, MBD3, in the context of the Mi-2/NuRD (nucleosome remodeling deacetylase) complex, could adopt a salt-dependent homodimeric association with its target epigenomic loci. Along with cell cycle progression, utilizing fluorescence lifetime imaging microscopy-based Förster resonance energy transfer (FLIM-FRET) we revealed that a proportion of MBD3 and MBD2 would co-localize with DNMT1 during DNA maintenance methylation, providing a proofreading and protective mechanism against a possible excessive methylation by DNMT1. In accordance with our hypothesis, insufficient MBD3 induced by small interfering RNA (siRNA) was found to result in a global DNA hypermethylation as well as increased methylation in the promoter CpG islands (CGIs) of a number of cell cycle related genes.

A Study of Adsorption Behavior of Single Water Molecule on the Surface of Polyhedral Oligomeric Silsesquioxanes

The adsorption of a water molecule on the surface of polyhedral oligomeric silsesquioxanes (POSS, (HSiO3/2) n , n = 4, 6, 8, 10 and 12) with cage structures was studied using the density functional tight-binding method with the inclusion of an empirical dispersion term in total energy. The results revealed that water molecule is favor to adsorb on the ring surfaces which are formed by Si–O–Si bonds of various cage structures. With the increase of the ring size in the same cage, both the adsorption energy and charge transfer increase, while the hydrogen bond length decreases. For all the cage structures, the presence of a single water molecule causes only negligible charge transfer (no more than 0.01 e) from the cages to the water molecule, indicating that the adsorption of a single water molecule has no significant effects on the electronic properties of POSS. Comparing to the large energy gaps (about 8.27–8.63 eV) between the highest occupied molecular orbital and the lowest unoccupied molecular orbital, the water adsorption can only cause slight reductions (within 0.15 eV) in the energy gap. Hence, the energy gap of the systems are primarily determined by the characteristics of POSS, instead of the energy transfer.

Why are Kinesin-2 KIF3AB and KIF3AC so Processive?

Mammalian KIF3AC is a heterotrimeric kinesin-2 that is best known for roles in organelle transport and remodeling of the microtubule cytoskeleton in neuronal tissue, yet in vitro studies to characterize its single molecule behavior are lacking. We report that a bacterially expressed native sequence of KIF3AC is highly processive with run lengths matching those exhibited by conventional kinesin-1 K560 in comparative experiments. This result was unexpected because KIF3AC exhibits the canonical kinesin-2 neck linker sequence that has been reported to be responsible for shorter run lengths observed for kinesin-2 KIF3AB. However, a comparative KIF3AB engineered with native sequence also exhibited long run lengths suggesting that neck linker length is not the sole determinant of run length for native kinesin-2 motors. KIF3C contains a long extension in surface loop L11 not present in KIF3A or KIF3B, or other processive kinesins. L11 is a component of the kinesin microtubule interface and has been implicated in activation of ADP release upon microtubule collision. KIF3AC encoding a truncation in KIF3C L11 (KIF3AC∆L11) is even more processive than wildtype KIF3AC and more similar to KIF3AB, suggesting that L11 also plays a role in determining run length. Steady-state ATPase experiments show that shortening L11 does not alter the kcat, consistent with the observation that single molecule velocities are not affected by this truncation. However, shortening L11 does significantly increase microtubule affinity in these dimers. Analysis of homodimeric KIF3CC and KIF3CC∆L11 reveal that both are processive but exceedingly slow. These results reveal that processivity can be regulated in part by L11, consistent with the interpretation that L11 becomes ordered upon microtubule collision to activate ADP release. These studies also expand our understanding of motor processivity and point to alternative structural and mechanistic modulators of processivity. Supported by NIH R37-GM54141.

Single-Molecule Study of RelA Diffusion in Live E. coli Cells during the Stringent Response

The stringent response is a physiological response that occurs when bacterial cells encounter nutritional stresses such as amino acid starvation or fatty acid starvation. The most marked outcome of this response is an immediate accumulation of the effector nucleotides, guanosine tetra- and pentaphosphate (ppGpp and pppGpp).The RelA protein of Escherichia coli is a (p)ppGpp synthetase that is activated by amino acid starvation. Here, we use single-molecule tracking to investigate the RelA protein diffusive behavior and association with ribosomes both before and after the stringent response. In earlier work from the Elf lab, a RelA-Dendra2 construct diffused like ribosomes (slowly) in normal growth conditions and diffused freely (rapidly) following the stringent response. In sharp contrast, we find that diffusion of a RelA-eYFP construct is heterogeneous in both conditions. Diffusion is much slower under stringent conditions. The single-molecule diffusive behavior is well fit by a hidden Markov model including two diffusive states: free and ribosome-bound. Amino acid starvation increases the dwell time of RelA in the state with lower mobility (ribosome-like diffusion) from 15 ms to 95 ms, while the dwell time of RelA spent in the free state remains unchanged at 20 ms. Correspondingly, the fraction of ribosome-bound RelA increases from 43% to 83% upon amino acid starvation. These observations suggest that RelA binds to ribosomes during synthesis of (p)ppGpp.

Multi-Scale Linkages between Single-Molecule Integrin Dynamics and Cell Protrusion

Recent advances in light microscopy permit the development of cyto-architectural blueprints with single-molecule resolution. It is now conceptually possible to relate the organization of the molecular machinery to cellular function. However, the disparity between the spatial and temporal scales of molecular and cellular behaviors poses substantial challenges in deriving these relationships. New approaches are required to integrate discrete single-molecule behavior with continuous cellular-level processes. Here, we combined intercalated molecular and cellular imaging with an analytical framework to reveal the roles that individual integrin molecules play in the process of initiating cell adhesion and migration. Despite the stochasticity of molecular and cellular behaviors, our data uncovered ‘rules’ of molecular organization for the components of a functional nascent adhesion, namely αvβ3-integrin, talin and actin, as well as the relationship between the local molecular organization of integrins and cell edge movement. We found that integrins exhibit a basal spatial gradient, with highest density and slowest mobility at the cell edge. In preparation for protrusion, this gradient transiently and locally increases, indicative of the initiation of nascent adhesions prior to cell edge protrusion. Moreover, the molecular behavior of talin, actin and β3 integrin mutants revealed that talin binding regulates integrin mobility, while integrin density modulation is primarily dependent on actin. These data establish a multi-scale linkage between characteristic variations in discrete stochastic molecular behaviors and continuum cellular behaviors.

Principles of allosteric interactions in cell signaling.

Linking cell signaling events to the fundamental physicochemical basis of the conformational behavior of single molecules and ultimately to cellular function is a key challenge facing the life sciences. Here we outline the emerging principles of allosteric interactions in cell signaling, with emphasis on the following points. (1) Allosteric efficacy is not a function of the chemical composition of the allosteric pocket but reflects the extent of the population shift between the inactive and active states. That is, the allosteric effect is determined by the extent of preferred binding, not by the overall binding affinity. (2) Coupling between the allosteric and active sites does not decide the allosteric effect; however, it does define the propagation pathways, the allosteric binding sites, and key on-path residues. (3) Atoms of allosteric effectors can act as “driver” or “anchor” and create attractive “pulling” or repulsive “pushing” interactions. Deciphering, quantifying, and integrating the multiple co-occurring events present daunting challenges to our scientific community.

Inhibition of F1-ATPase Rotational Catalysis by the Carboxyl-terminal Domain of the ϵ Subunit

Escherichia coli ATP synthase (F0F1) couples catalysis and proton transport through subunit rotation. The ϵ subunit, an endogenous inhibitor, lowers F1-ATPase activity by decreasing the rotation speed and extending the duration of the inhibited state (Sekiya, M., Hosokawa, H., Nakanishi-Matsui, M., Al-Shawi, M. K., Nakamoto, R. K., and Futai, M. (2010) Single molecule behavior of inhibited and active states of Escherichia coli ATP synthase F1 rotation. J. Biol. Chem. 285, 42058-42067). In this study, we constructed a series of ϵ subunits truncated successively from the carboxyl-terminal domain (helix 1/loop 2/helix 2) and examined their effects on rotational catalysis (ATPase activity, average rotation rate, and duration of inhibited state). As expected, the ϵ subunit lacking helix 2 caused about ½-fold reduced inhibition, and that without loop 2/helix 2 or helix 1/loop 2/helix 2 showed a further reduced effect. Substitution of ϵSer(108) in loop 2 and ϵTyr(114) in helix 2, which possibly interact with the β and γ subunits, respectively, decreased the inhibitory effect. These results suggest that the carboxyl-terminal domain of the ϵ subunit plays a pivotal role in the inhibition of F1 rotation through interaction with other subunits.

Abstract 4925: Targeted metabolic profiling of gliomas and glioma stem cells identifies methionine as a differentially regulated metabolite: Proof of principle supporting methionine-pet imaging

Abstract Purpose: At present, 18F-Fluorodeoxyglucose(FDG)-positron emission tomography (PET) is the most commonly used diagnostic imaging modality in clinical practice for predicting prognosis in malignant brain tumors. The diffusive nature of glioblastoma (GBM) often makes FDG-PET detection inaccurate and there is no diagnostic gold-standard for GBM. Thus, it is necessary to find new criteria independent of conventional FDG-PET diagnosis to delineate tumor versus normal tissues. Molecular imaging using 11C-Methionine-PET (Met-PET) is one of the most promising approaches to this challenge. Methods: We employed a targeted metabolomic approach to study the extracellular and intracellular levels of methionine in established (ATCC) and patient derived primary glioblastoma (GBM) cell lines, glioma stem cells (GSC) and normal human astrocytes (NHA) to gain a better understanding of methionine uptake. Methylthioadenosine phosphorylase (MTAP) is an enzyme that plays a vital role in polyamine metabolism and methionine salvage. Therefore, we determined the expression profile of MTAP in these cells using RT-qPCR. We then extracted the intracellular and extracellular metabolites of GBM, GSC and NHA to estimate their methionine levels using LC-MS Quadropole Time of Flight (Q-TOF) and LC-MS Triple Quad (QQQ) instruments. Thus, by using metabolic fingerprints of methionine, we quantified methionine accumulation / uptake in GBM, GSC and NHA. Results and Conclusion: PET provides real-time information of metabolism through the behavior of single molecules. Despite clinical trials advocating Met-PET imaging as a superior imaging modality compared to FDG-PET for brain tumors, to date Met-PET has not been widely used in clinical diagnostics. Through this metabolic profiling of gliomas, we have shown for the first time that GBM and GSC accumulate methionine when compared to NHA. Uptake studies through exogenous addition of methionine in GBM, GSC and NHA show a significant uptake of methionine by GBM and GSC when compared to NHA. The results of our study establish the scientific basis for Met-PET imaging in GBM and provide a rationale for incorporating Met-PET imaging in future brain tumor clinical trials to evaluate its prognostic value. Citation Format: Kamalakannan Palanichamy, Thirumoorthy Krishnan, Nikhil Sebastian, Rajbir Singh, Suman Kanji, Arnab Chakravarti. Targeted metabolic profiling of gliomas and glioma stem cells identifies methionine as a differentially regulated metabolite: Proof of principle supporting methionine-pet imaging. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 4925. doi:10.1158/1538-7445.AM2014-4925

Real time ligand-induced motion mappings of AChBP and nAChR using X-ray single molecule tracking.

We observed the dynamic three-dimensional (3D) single molecule behaviour of acetylcholine-binding protein (AChBP) and nicotinic acetylcholine receptor (nAChR) using a single molecule tracking technique, diffracted X-ray tracking (DXT) with atomic scale and 100 μs time resolution. We found that the combined tilting and twisting motions of the proteins were enhanced upon acetylcholine (ACh) binding. We present the internal motion maps of AChBP and nAChR in the presence of either ACh or α-bungarotoxin (αBtx), with views from two rotational axes. Our findings indicate that specific motion patterns represented as biaxial angular motion maps are associated with channel function in real time and on an atomic scale.

The structural basis for cancer treatment decisions.

Cancer treatment decisions rely on genetics, large data screens and clinical pharmacology. Here we point out that genetic analysis and treatment decisions may overlook critical elements in cancer development, progression and drug resistance. Two critical structural elements are missing in genetics-based decision-making: the mechanisms of oncogenic mutations and the cellular network which is rewired in cancer. These lay the foundation for the structural basis for cancer treatment decisions, which is rooted in the physical principles of the molecular conformational behavior of single molecules and their interactions. Improved tumor mutational analysis platforms and knowledge of the redundant pathways which can take over in cancer, may not only supplement known actionable findings, but forecast possible cancer progression and resistance. Such forward-looking can be powerful, endowing the oncologist with mechanistic insight and cancer prognosis, and consequently more informed treatment options. Examples include redundant pathways taking over after inhibition of EGFR constitutive activation, mutations in PIK3CA p110α and p85, and the non-hotspot AKT1 mutants conferring constitutive membrane localization.

Measurement and understanding of single-molecule break junction rectification caused by asymmetric contacts.

The contact effects of single-molecule break junctions on rectification behaviors were experimentally explored by a systematic control of anchoring groups of 1,4-disubstituted benzene molecular junctions. Single-molecule conductance and I-V characteristic measurements reveal a strong correlation between rectifying effects and the asymmetry in contacts. Analysis using energy band models and I-V calculations suggested that the rectification behavior is mainly caused by asymmetric coupling strengths at the two contact interfaces. Fitting of the rectification ratio by a modified Simmons model we developed suggests asymmetry in potential drop across the asymmetric anchoring groups as the mechanism of rectifying I-V behavior. This study provides direct experimental evidence and sheds light on the mechanisms of rectification behavior induced simply by contact asymmetry, which serves as an aid to interpret future single-molecule electronic behavior involved with asymmetric contact conformation.

Instrumental requirements for the detection of electron beam-induced object excitations at the single atom level in high-resolution transmission electron microscopy

This contribution touches on essential requirements for instrument stability and resolution that allows operating advanced electron microscopes at the edge to technological capabilities. They enable the detection of single atoms and their dynamic behavior on a length scale of picometers in real time. It is understood that the observed atom dynamic is intimately linked to the relaxation and thermalization of electron beam-induced sample excitation. Resulting contrast fluctuations are beam current dependent and largely contribute to a contrast mismatch between experiments and theory if not considered. If explored, they open the possibility to study functional behavior of nanocrystals and single molecules at the atomic level in real time.

Chemically cross-linked microtubule assembly shows enhanced dynamic motions on kinesins

Biomolecular motors have cooperative functions that facilitate various important cellular functions such as signaling and deformation. However, the multiple-molecular behavior is poorly understood when compared with the well-studied behavior of single molecules. Kinesin and microtubules (MTs) are a combination of motor and rail proteins that have been reported to show cooperativity in moving patterns. Here, we report that cross-linking of isotropic MTs enhances the movement of the local network temporally, which can elicit the effective conveyance of 5 μm diameter silica microbeads. The average velocity of the beads reached 143 nm s−1 for over 15 min, which is 2.7 times higher than the beads on kinesin-driven MTs without cross-links. Although the velocity of the beads fluctuated over the time course, the maximum velocity calculated from 5 s displacement reached 1.2 μm s−1, which was ca. 3-fold higher than the intrinsic driving velocity of kinesins, i.e., the sliding velocity of the MTs without cross-links. This technique provides such an enhanced movement by simply cross-linking the isotropic MTs, but without specific control or alignment over network polarity. The enhancement in the local motion of the MTs is discussed in terms of the “mechanochemical” effect coupled between dynamically stored elastic energies in the MT network and the collective motion of MTs driven by an ATP-fueled kinesin motor.

Coupling of fluorescence correlation spectroscopy with capillary and microchannel analytical systems and its applications

In the past decade, fluorescence correlation spectroscopy (FCS) has become an ultrasensitive and noninvasive single-molecule detection technique, which is widely applied in the physical, chemical, and life-science research. The coupling of FCS with narrow channel flow systems including the ones based on capillary provide the important, convenient, and sensitive assay platforms for probing and understanding the behavior of single molecules or nanoparticles with improved temporal and spatial resolution and need for less sample volume among other advantages. This review focus on different approaches for FCS with capillary and microchannel analytical systems and its applications in confined diffusion study, flow profiles, and imaging of narrow channel, multicomponent analyses such as protein, DNA analysis, and characterization on nanoparticles.

Fluorescence Behavior of Single Guest Molecules in Nonpolar Oil Droplets Covered with Stabilizing Surfactants

By using confocal and wide-field microscopic techniques we have investigated fluorescence behaviors of a single perylenediimide derivative, N,N′-dipropyl-1,6,7,12-tetrakis(4-tert-butylphenoxy)-3,4,9,10-perylenetetracarboxydiimide (BP-PDI), in ca. 200–300 nm sized droplets of n-octane covered with surfactant molecules. The guest dye did not show distinguishable blinking in the droplets prepared with a nonionic surfactant, Triton X-100, while frequent blinking was observed in the droplets with a cationic surfactant, trimethylstearylammonium chloride (STAC). The survival time of the guest dye before the permanent photodegradation was much longer in the droplets with Triton X-100 than those with STAC. On the basis of the analysis with a model assuming the transition from the S1 state to the dark state (DS) and the permanent photodegradation during the dark state, it was deduced that the probability of the transition from the S1 state to the DS in the droplet with STAC, 7.2 × 10–6, was significantly larger than that with Triton X-100, 1.1 × 10–6. On the other hand, the probability of the photodegradation during the dark state was comparable between these two systems. These results indicate that the probability of transition from the S1 state to the DS essentially dominates the survival time of the guest dyes. Fluorescence polarization measurements also revealed that guest dyes frequently stay in the vicinity of polar interface in the droplets prepared with STAC.

TrackArt: the user friendly interface for single molecule tracking data analysis and simulation applied to complex diffusion in mica supported lipid bilayers.

BackgroundSingle molecule tracking (SMT) analysis of fluorescently tagged lipid and protein probes is an attractive alternative to ensemble averaged methods such as fluorescence correlation spectroscopy (FCS) or fluorescence recovery after photobleaching (FRAP) for measuring diffusion in artificial and plasma membranes. The meaningful estimation of diffusion coefficients and their errors is however not straightforward, and is heavily dependent on sample type, acquisition method, and equipment used. Many approaches require advanced computing and programming skills for their implementation.FindingsHere we present TrackArt software, an accessible graphic interface for simulation and complex analysis of multiple particle paths. Imported trajectories can be filtered to eliminate spurious or corrupted tracks, and are then analyzed using several previously described methodologies, to yield single or multiple diffusion coefficients, their population fractions, and estimated errors. We use TrackArt to analyze the single-molecule diffusion behavior of a sphingolipid analog SM-Atto647N, in mica supported DOPC (1,2-dioleoyl-sn-glycero-3-phosphocholine) bilayers. Fitting with a two-component diffusion model confirms the existence of two separate populations of diffusing particles in these bilayers on mica. As a demonstration of the TrackArt workflow, we characterize and discuss the effective activation energies required to increase the diffusion rates of these populations, obtained from Arrhenius plots of temperature-dependent diffusion. Finally, TrackArt provides a simulation module, allowing the user to generate models with multiple particle trajectories, diffusing with different characteristics. Maps of domains, acting as impermeable or permeable obstacles for particles diffusing with given rate constants and diffusion coefficients, can be simulated or imported from an image. Importantly, this allows one to use simulated data with a known diffusion behavior as a comparison for results acquired using particular algorithms on actual, “natural” samples whose diffusion behavior is to be extracted. It can also serve as a tool for demonstrating diffusion principles.ConclusionsTrackArt is an open source, platform-independent, Matlab-based graphical user interface, and is easy to use even for those unfamiliar with the Matlab programming environment. TrackArt can be used for accurate simulation and analysis of complex diffusion data, such as diffusion in lipid bilayers, providing publication-quality formatted results.

Single-Particle Fluorescence Intensity Fluctuations of Carbon Nanodots

Fluorescent carbon nanodots (CNDs) were synthesized in oxidized and reduced forms and were analyzed at the single-particle level. Images of single CNDs at different excitation energies revealed significant heterogeneity in the lower energy trap sites between particles. We observed that a high percentage of reduced CND particles transitioned between multiple fluorescence intensity levels indicative of multichromophoric systems. Despite this behavior, individual CNDs exhibit single-step photobleaching and transient blinking to the background level suggesting single-molecule behavior.

Accurate High Throughput Bright Field Bead Tracking using Gpu Hardware

With the recent advances in camera technology, the potential of microscopy to study the behaviour of single molecules can make great strides. Here, we present a highly-optimized method to track magnetic beads in a bright-field microscopy setup that takes full advantage of both new camera technology and fast graphics hardware to do parallel computations. Using large field-of-view cameras, we can carry out real-time tracking of hundreds of magnetic beads in parallel while maintaining nanometer-scale accuracy. Additionally, we can use fast CMOS camera's to perform real-time bead tracking at an acquisition rate of several kHz. These advances allow us to use the magnetic tweezer as a tool to acquire detailed statistical knowledge at high temporal resolution of the behaviour of single molecules or molecular complexes.

Simulations of DNA stretching by flow field in microchannels with complex geometry.

Recently, we have reported the experimental results of DNA stretching by flow field in three microchannels (C. H. Lee and C. C. Hsieh, Biomicrofluidics 7(1), 014109 (2013)) designed specifically for the purpose of preconditioning DNA conformation for easier stretching. The experimental results do not only demonstrate the superiority of the new devices but also provides detailed observation of DNA behavior in complex flow field that was not available before. In this study, we use Brownian dynamics-finite element method (BD-FEM) to simulate DNA behavior in these microchannels, and compare the results against the experiments. Although the hydrodynamic interaction (HI) between DNA segments and between DNA and the device boundaries was not included in the simulations, the simulation results are in fairly good agreement with the experimental data from either the aspect of the single molecule behavior or from the aspect of ensemble averaged properties. The discrepancy between the simulation and the experimental results can be explained by the neglect of HI effect in the simulations. Considering the huge savings on the computational cost from neglecting HI, we conclude that BD-FEM can be used as an efficient and economic designing tool for developing new microfluidic device for DNA manipulation.