Single-Molecule Fluorescence Imaging Reveals Coassembly of CTPS and P5CS.

The cellular compartmentation induced by self-assembly of natural proteins has recently attracted widespread attention due to its structural-functional significance. Among them, as a highly conserved metabolic enzyme and one of the potential targets for cancers and parasitic diseases in drug development, CTP synthase (CTPS) has also been reported to self-assemble into filamentous structures termed cytoophidia. To elucidate the dynamical mechanism of cytoophidium filamentation, we utilize single-molecule fluorescence imaging to observe the real-time self-assembly dynamics of CTPS and the coordinated assembly between CTPS and its interaction partner, Δ1-pyrroline-5-carboxylate synthase (P5CS). Significant differences exist in the direction of growth and extension when the two proteins self-assemble. The oligomer state distribution analysis of the CTPS minimum structural subunit under different conditions and the stoichiometry statistics of binding CTPS and P5CS by single-molecule fluorescence photobleach counting further confirm that the CTPS cytoophidia are mainly stacked with tetramers. CTPS can act as the nucleation core to induce the subsequent growth of the P5CS filaments. Our work not only provide evidence from the molecular level for the self-assembly and coordinated assembly (coassembly) of CTPS with its interaction partner P5CS in vitro but also offer new experimental perspectives for the dynamics research of coordinated regulation between other protein polymers.

Monocular and Binocular Visual Function Assessments and Activities of Daily Living Performance in Age-Related Macular Degeneration.

PURPOSE: To evaluate the relationship between specific monocular and binocular visual function (VF) assessments with binocularly performed activities of daily living task tests (ADLTTs) in patients with age-related macular degeneration (AMD) and healthy controls. DESIGN: Prospective case-control cohort study. SUBJECTS: Thirty-six AMD patients and 36 controls. METHOD: Visual field assessments included monocular and binocular best-corrected visual acuity (BCVA), contrast sensitivity (CS), and monocular microperimetry testing for mean macula sensitivity, mean retina sensitivity (MRS), fixation area, and fixation distance from fovea (FDF). Age-related macular degeneration lesion area and sensitivity were measured on OCT and microperimetry, respectively. Participants performed 4 validated ADLTTs with binocular BCVA: (1) reading; (2) item-search; (3) money-counting; and (4) multi-step drink-making tasks. MAIN OUTCOME MEASURES: Spearman correlations and multivariate regression analysis, adjusted for age, sex, and potential correlation between the 2 eyes, were used to assess the relationship between monocular and binocular VF assessments, and ADLTT performance in both groups. RESULTS: Age-related macular degeneration patients had poorer VF (BCVA, CS, mean macula sensitivity, and MRS) compared with healthy controls. Monocular BCVA in both better- and worse-vision eyes was moderately correlated with the binocular reading speed and money-counting tasks in participants with AMD. In AMD, monocular worse eye CS, MRS, AMD lesion area on OCT, and lesion sensitivity on microperimetry showed moderate correlations to various ADLTTs, such as reading, money-counting, and drink-making. Similar findings were found in our AMD cohort on multivariate regression analysis. Fewer significant correlations were observed for the better-vision eye, whereas no correlations were observed for healthy controls between VF parameters and ADLTTs. In contrast, significant associations were observed between binocular BCVA and CS with binocular ADLTTs (reading and item-search tasks) but not in AMD patients. CONCLUSION: Although monocular BCVA remains the most common measure of VF, CS and microperimetry testing also show significant correlations with ADLTTs performance in AMD patients, and should be considered as complimentary VF-outcome measures in both clinical and research settings. Unlike healthy subjects, AMD patients do not rely on binocular VF for ADLTT function, with the worse-vision eye impacting binocular ADLTT function more than the better-vision eye. Therefore, the worse-vision eye should not be neglected during the management of AMD. FINANCIAL DISCLOSURE(S): Proprietary or commercial disclosure may be found in the Footnotes and Disclosures at the end of this article.

Transition Time Determination of Single-Molecule FRET Trajectories via Wasserstein Distance Analysis in Steady-State Variations in smFRET (WAVE).

Many biological molecules respond to external stimuli that can cause their conformational states to shift from one steady state to another. Single-molecule FRET (Fluorescence Resonance Energy Transfer) is of particular interest to not only define the steady-state conformational ensemble usually averaged out in the ensemble of molecules but also characterize the dynamics of biomolecules. To study steady-state transitions, i.e., non-equilibrium transitions, a data analysis methodology is necessary to analyze single-molecule FRET photon trajectories, which contain mixtures of contributions from two steady-state statuses and include non-equilibrium transitions. In this study, we introduce a novel methodology called WAVE (Wasserstein distance Analysis in steady-state Variations in smFRET) to detect and locate non-equilibrium transition positions in FRET trajectories. Our method first utilizes a combined STaSI-HMM (Stepwise Transitions with State Inference Hidden Markov Model) algorithm to convert the original FRET trajectories into discretized trajectories. We then apply Maximum Wasserstein Distance analysis to differentiate the FRET state compositions of the fitting trajectories before and after the non-equilibrium transition. Forward and backward algorithms, based on the Minimum Description Length (MDL) principle, are used to find the refined positions of the non-equilibrium transitions. This methodology allows us to observe changes in experimental conditions in chromophore-tagged biomolecules or vice versa.

Direct experimental observation of blue-light-induced conformational change and intermolecular interactions of cryptochrome.

Cryptochromes are blue light receptors that mediate circadian rhythm and magnetic sensing in various organisms. A typical cryptochrome consists of a conserved photolyase homology region domain and a varying carboxyl-terminal extension across species. The structure of the flexible carboxyl-terminal extension and how carboxyl-terminal extension participates in cryptochrome's signaling function remain mostly unknown. In this study, we uncover the potential missing link between carboxyl-terminal extension conformational changes and downstream signaling functions. Specifically, we discover that the blue-light induced opening of carboxyl-terminal extension in C. reinhardtii animal-like cryptochrome can structurally facilitate its interaction with Rhythm Of Chloroplast 15, a circadian-clock-related protein. Our finding is made possible by two technical advances. Using single-molecule Förster resonance energy transfer technique, we directly observe the displacement of carboxyl-terminal extension by about 15 Å upon blue light excitation. Combining structure prediction and solution X-ray scattering methods, we propose plausible structures of full-length cryptochrome under dark and lit conditions. The structures provide molecular basis for light active conformational changes of cryptochrome and downstream regulatory functions.

Rational Design of Adenylate Kinase Thermostability through Coevolution and Sequence Divergence Analysis.

Protein engineering is actively pursued in industrial and laboratory settings for high thermostability. Among the many protein engineering methods, rational design by bioinformatics provides theoretical guidance without time-consuming experimental screenings. However, most rational design methods either rely on protein tertiary structure information or have limited accuracies. We proposed a primary-sequence-based algorithm for increasing the heat resistance of a protein while maintaining its functions. Using adenylate kinase (ADK) family as a model system, this method identified a series of amino acid sites closely related to thermostability. Single- and double-point mutants constructed based on this method increase the thermal denaturation temperature of the mesophilic Escherichia coli (E. coli) ADK by 5.5 and 8.3 °C, respectively, while preserving most of the catalytic function at ambient temperatures. Additionally, the constructed mutants have improved enzymatic activity at higher temperature.

Engineering single-molecule fluorescence with asymmetric nano-antennas.

As a powerful tool for studying molecular dynamics in bioscience, single-molecule fluorescence detection provides dynamical information buried in ensemble experiments. Fluorescence in the near-infrared (NIR) is particularly useful because it offers higher signal-to-noise ratio and increased penetration depth in tissue compared with visible fluorescence. The low quantum yield of most NIR fluorophores, however, makes the detection of single-molecule fluorescence difficult. Here, we use asymmetric plasmonic nano-antenna to enhance the fluorescence intensity of AIEE1000, a typical NIR dye, by a factor up to 405. The asymmetric nano-antenna achieve such an enhancement mainly by increasing the quantum yield (to ~80%) rather than the local field, which degrades the molecules' photostability. Our coupled-mode-theory analysis reveals that the enhancements stem from resonance-matching between antenna and molecule and, more importantly, from optimizing the coupling between the near- and far-field modes with designer asymmetric structures. Our work provides a universal scheme for engineering single-molecule fluorescence in the near-infrared regime.

State-of-the-Art Technologies for Understanding Brassinosteroid Signaling Networks.

Brassinosteroids, the steroid hormones of plants, control physiological and developmental processes through its signaling pathway. The major brassinosteroid signaling network components, from the receptor to transcription factors, have been identified in the past two decades. The development of biotechnologies has driven the identification of novel brassinosteroid signaling components, even revealing several crosstalks between brassinosteroid and other plant signaling pathways. Herein, we would like to summarize the identification and improvement of several representative brassinosteroid signaling components through the development of new technologies, including brassinosteroid-insensitive 1 (BRI1), BRI1-associated kinase 1 (BAK1), BR-insensitive 2 (BIN2), BRI1 kinase inhibitor 1 (BKI1), BRI1-suppressor 1 (BSU1), BR signaling kinases (BSKs), BRI1 ethyl methanesulfonate suppressor 1 (BES1), and brassinazole resistant 1 (BZR1). Furthermore, improvement of BR signaling knowledge, such as the function of BKI1, BES1 and its homologous through clustered regularly interspaced short palindromic repeats (CRISPR), the regulation of BIN2 through single-molecule methods, and the new in vivo interactors of BIN2 identified by proximity labeling are described. Among these technologies, recent advanced methods proximity labeling and single-molecule methods will be reviewed in detail to provide insights to brassinosteroid and other phytohormone signaling pathway studies.

Reactive oxygen species-mediated BIN2 activity revealed by single-molecule analysis.

Much evidence has shown that reactive oxygen species (ROS) regulate several plant hormone signaling cascades, but little is known about the real-time kinetics and the underlying molecular mechanisms of the target proteins in the brassinosteroid (BR) signaling pathway. In this study, we used single-molecule techniques to investigate the true signaling timescales of the major BR signaling components BRI1-EMS-SUPPRESSOR 1 (BES1) and BRASSINOSTEROID INSENSITIVE 2 (BIN2) of Arabidopsis thaliana. The rate constants of BIN2 associating with ATP and phosphorylating BES1 were determined to be 0.7 ± 0.4 mM-1  s-1 and 2.3 ± 1.4 s-1 , respectively. Interestingly, we found that the interaction of BIN2 and BES1 was oxygen-dependent, and oxygen can directly modify BIN2. The activity of BIN2 was switched on via modification of specific cysteine (Cys) residues, including C59, C95, C99 and C162. The mutation of these Cys residues inhibited the BR signaling outputs. These findings demonstrate the power of using single-molecule techniques to study the dynamic interactions of signaling components, which is difficult to be discovered by conventional physiological and biochemical methods.

Quantifying Intramolecular Protein Conformational Dynamics Under Lipid Interaction Using smFRET and FCCS.

FÓ§rster-type resonance energy transfer (FRET) with fluorescence cross-correlation spectroscopy (FCCS) is a powerful combination for observing intramolecular conformational dynamics on the micro- to millisecond timescale. Owing to its sensitivity to various physical parameters, FRET-FCCS has also been used to detect the reagent effects on proteins dynamics. However, FRET-FCCS alone cannot acquire the exact measurements of rate constants. Moreover, this technique is highly model dependent and can be unreliable when determining too many parameters at once. On the contrary, single-molecular FRET (smFRET) can measure the conformational states and their populations directly, although it is extremely challenging for probing fast dynamics under 1 ms. In this chapter, we describe how to realize sub-millisecond conformational dynamics measurements of a SNARE protein Ykt6 under lipid environments by smFRET and FRET-FCCS. This protocol includes sample preparation, microscope designs, data acquisition, and analysis methodology.

Single-Molecule Fluorescence Methods to Study Plant Hormone Signal Transduction Pathways.

Plant-hormone-initiated signaling pathways are extremely vital for plant growth, differentiation, development, and adaptation to environmental stresses. Hormonal perception by receptors induces downstream signal transduction mechanisms that lead to plant responses. However, conventional techniques-such as genetics, biochemistry, and physiology methods-that are applied to elucidate these signaling pathways can only provide qualitative or ensemble-averaged quantitative results, and the intrinsic molecular mechanisms remain unclear. The present study developed novel methodologies based on in vitro single-molecule fluorescence assays to elucidate the complete and detailed mechanisms of plant hormone signal transduction pathways. The proposed methods are based on multicolor total internal reflection fluorescence microscopy and a flow cell model for gas environment control. The methods validate the effectiveness of single-molecule approaches for the extraction of abundant information, including oligomerization, specific gas dependence, and the interaction kinetics of different components.

Lipid Regulated Intramolecular Conformational Dynamics of SNARE-Protein Ykt6.

Cellular informational and metabolic processes are propagated with specific membrane fusions governed by soluble N-ethylmaleimide sensitive factor attachment protein receptors (SNARE). SNARE protein Ykt6 is highly expressed in brain neurons and plays a critical role in the membrane-trafficking process. Studies suggested that Ykt6 undergoes a conformational change at the interface between its longin domain and the SNARE core. In this work, we study the conformational state distributions and dynamics of rat Ykt6 by means of single-molecule Förster Resonance Energy Transfer (smFRET) and Fluorescence Cross-Correlation Spectroscopy (FCCS). We observed that intramolecular conformational dynamics between longin domain and SNARE core occurred at the timescale ~200 µs. Furthermore, this dynamics can be regulated and even eliminated by the presence of lipid dodecylphoshpocholine (DPC). Our molecular dynamic (MD) simulations have shown that, the SNARE core exhibits a flexible structure while the longin domain retains relatively stable in apo state. Combining single molecule experiments and theoretical MD simulations, we are the first to provide a quantitative dynamics of Ykt6 and explain the functional conformational change from a qualitative point of view.

Temperature-feedback upconversion nanocomposite for accurate photothermal therapy at facile temperature.

Photothermal therapy (PTT) at present, following the temperature definition for conventional thermal therapy, usually keeps the temperature of lesions at 42-45 °C or even higher. Such high temperature kills cancer cells but also increases the damage of normal tissues near lesions through heat conduction and thus brings about more side effects and inhibits therapeutic accuracy. Here we use temperature-feedback upconversion nanoparticle combined with photothermal material for real-time monitoring of microscopic temperature in PTT. We observe that microscopic temperature of photothermal material upon illumination is high enough to kill cancer cells when the temperature of lesions is still low enough to prevent damage to normal tissue. On the basis of the above phenomenon, we further realize high spatial resolution photothermal ablation of labelled tumour with minimal damage to normal tissues in vivo. Our work points to a method for investigating photothermal properties at nanoscale, and for the development of new generation of PTT strategy.

Septin ring assembly is regulated by Spt20, a structural subunit of the SAGA complex.

Accurate cell division requires the proper assembly of high-order septin structures. In fission yeast (Schizosaccharomyces pombe), Spn1-Spn4 are assembled into a primary septin ring at the division site, and the subsequent recruitment of Mid2 to the structure results in a stable septin ring. However, not much is known about the regulation of this key process. Here, we found that deletion of Spt20, a structural subunit of the Spt-Ada-Gcn5-acetyltransferase (SAGA) transcriptional activation complex, caused a severe cell separation defect. The defect was mainly due to impaired septin ring assembly, as 80% of spt20Δ cells lost septin rings at the division sites. Spt20 regulates septin ring assembly partially through the transcriptional activation of mid2(+). Spt20 also interacted with Spn2 and Mid2 in vitro and was associated with other components of the ring in vivo. Spt20 colocalized with the septin ring, but did not separate when the septin ring split. Importantly, Spt20 regulated the stability of the septin ring and was required for the recruitment of Mid2. The transcription-dependent and -independent roles of Spt20 in septin ring assembly highlight a multifaceted regulation of one process by a SAGA subunit.

Confocal single-molecule FRET for protein conformational dynamics.

Single-molecule FÓ§rster-type resonance energy transfer (smFRET) is a unique technique capable of following conformational motions of individual protein molecules. The direct observation of individual proteins provides rich information that would be washed away in ensemble measurements, hence opening up new avenues for establishing the protein structure-function relationships through dynamics. Retrieving dynamics information of biomolecular motions via smFRET, though, requires careful experiment design and rigorous treatment of single-molecule statistics. Here, we describe the rudimentary steps for an smFRET experiment, including sample preparation for the microscope, building of critical parts for single-molecule FRET detection, and a robust methodology for photon-by-photon data analysis.

Surface-modified silicon nanoparticles with ultrabright photoluminescence and single-exponential decay for nanoscale fluorescence lifetime imaging of temperature.

In this Communication, we report fabrication of ultrabright water-dispersible silicon nanoparticles (SiNPs) with quantum yields (QYs) up to 75% through a novelly designed chemical surface modification. A simple one-pot surface modification was developed that improves the photoluminescent QYs of SiNPs from 8% to 75% and meanwhile makes SiNPs water-dispersible. Time-correlated single photon counting and femtosecond time-resolved photoluminescence techniques demonstrate the emergence of a single and uncommonly highly emissive recombination channel across the entire NP ensemble induced by surface modification. The extended relatively long fluorescence lifetime (FLT), with a monoexponential decay, makes such surface-modified SiNPs suitable for applications involving lifetime measurements. Experimental results demonstrate that the surface-modified SiNPs can be utilized as an extraordinary nanothermometer through FLT imaging.

Revisiting the Stöber method: inhomogeneity in silica shells.

We demonstrate that the silica shell on nanoparticles formed by a typical Stöber method is inhomogeneous in nature. The outer layer of the shell is chemically more robust than the inner layer, which can be selectively etched by hot water. Methods are developed to "harden" the soft silica shells. These new understandings are exploited to develop versatile and template-free approaches for fabricating sophisticated yolk-shell nanostructures.

Seeing the forest for the trees: fluorescence studies of single enzymes in the context of ensemble experiments.

Enzymes are remarkable molecular machines that make many difficult biochemical reactions possible under mild biological conditions with incredible precision and efficiency. Our understanding of the working principles of enzymes, however, has not reached the level where one can readily deduce the mechanism and the catalytic rates from an enzyme's structure. Resolving the dynamics that relate the three-dimensional structure of an enzyme to its function has been identified as a key issue. While still challenging to implement, single-molecule techniques have emerged as one of the most useful methods for studying enzymes. We review enzymes studied using single-molecule fluorescent methods but placing them in the context of results from other complementary experimental work done on bulk samples. This review primarily covers three enzyme systems--flavoenzymes, dehydrofolate reductase, and adenylate kinase--with additional enzymes mentioned where appropriate. When the single-molecule experiments are discussed together with other methods aiming at the same scientific question, the weakness, strength, and unique contributions become clear.

Direct Mg(2+) binding activates adenylate kinase from Escherichia coli.

We report evidence that adenylate kinase (AK) from Escherichia coli can be activated by the direct binding of a magnesium ion to the enzyme, in addition to ATP-complexed Mg(2+). By systematically varying the concentrations of AMP, ATP, and magnesium in kinetic experiments, we found that the apparent substrate inhibition of AK, formerly attributed to AMP, was suppressed at low magnesium concentrations and enhanced at high magnesium concentrations. This previously unreported magnesium dependence can be accounted for by a modified random bi-bi model in which Mg(2+) can bind to AK directly prior to AMP binding. A new kinetic model is proposed to replace the conventional random bi-bi mechanism with substrate inhibition and is able to describe the kinetic data over a physiologically relevant range of magnesium concentrations. According to this model, the magnesium-activated AK exhibits a 23- +/- 3-fold increase in its forward reaction rate compared with the unactivated form. The findings imply that Mg(2+) could be an important affecter in the energy signaling network in cells.

Spin texture and magnetoroton excitations at nu=1/3.

Neutral spin texture (ST) excitations at nu=1/3 are directly observed for the first time by resonant inelastic light scattering. They are determined to involve two simultaneous spin flips. At low magnetic fields, the ST energy is below that of the magnetoroton minimum. With increasing in-plane magnetic field these mode energies cross at a critical ratio of the Zeeman and Coulomb energies of eta(c)=0.020+/-0.001. Surprisingly, the intensity of the ST mode grows with temperature in the range in which the magnetoroton modes collapse. The temperature dependence is interpreted in terms of a competition between coexisting phases supporting different excitations. We consider the role of the ST excitations in activated transport at nu=1/3.