RESUMEN
Here, we use single-molecule techniques to study the aggregation of α-synuclein, the protein whose misfolding and deposition is associated with Parkinson's disease. We identify a conformational change from the initially formed oligomers to stable, more compact proteinase-K-resistant oligomers as the key step that leads ultimately to fibril formation. The oligomers formed as a result of the structural conversion generate much higher levels of oxidative stress in rat primary neurons than do the oligomers formed initially, showing that they are more damaging to cells. The structural conversion is remarkably slow, indicating a high kinetic barrier for the conversion and suggesting that there is a significant period of time for the cellular protective machinery to operate and potentially for therapeutic intervention, prior to the onset of cellular damage. In the absence of added soluble protein, the assembly process is reversed and fibrils disaggregate to form stable oligomers, hence acting as a source of cytotoxic species.
Asunto(s)
alfa-Sinucleína/química , alfa-Sinucleína/metabolismo , Animales , Células Cultivadas , Endopeptidasa K/metabolismo , Transferencia Resonante de Energía de Fluorescencia , Humanos , Cinética , Modelos Moleculares , Neuronas/metabolismo , Estrés Oxidativo , RatasRESUMEN
Optogenetic actuators have revolutionized the resolution at which biological processes can be controlled. In plants, deployment of optogenetics is challenging due to the need for these light-responsive systems to function in the context of horticultural light environments. Furthermore, many available optogenetic actuators are based on plant photoreceptors that might crosstalk with endogenous signaling processes, while others depend on exogenously supplied cofactors. To overcome such challenges, we have developed Highlighter, a synthetic, light-gated gene expression system tailored for in planta function. Highlighter is based on the photoswitchable CcaS-CcaR system from cyanobacteria and is repurposed for plants as a fully genetically encoded system. Analysis of a re-engineered CcaS in Escherichia coli demonstrated green/red photoswitching with phytochromobilin, a chromophore endogenous to plants, but also revealed a blue light response likely derived from a flavin-binding LOV-like domain. We deployed Highlighter in transiently transformed Nicotiana benthamiana for optogenetic control of fluorescent protein expression. Using light to guide differential fluorescent protein expression in nuclei of neighboring cells, we demonstrate unprecedented spatiotemporal control of target gene expression. We implemented the system to demonstrate optogenetic control over plant immunity and pigment production through modulation of the spectral composition of broadband visible (white) light. Highlighter is a step forward for optogenetics in plants and a technology for high-resolution gene induction that will advance fundamental plant biology and provide new opportunities for crop improvement.
Asunto(s)
Aracnodactilia , Optogenética , Nicotiana/genética , Escherichia coli/genética , Expresión GénicaRESUMEN
Directly examining subcellular mechanics whilst avoiding excessive strain of a live cell requires the precise control of light stress on very small areas, which is fundamentally difficult. Here we use a glass nanopipet out of contact with the plasma membrane to both exert the stress on the cell and also accurately monitor cellular compression. This allows the mapping of cell stiffness at a lateral resolution finer than 100 nm. We calculate the stress a nanopipet exerts on a cell as the sum of the intrinsic pressure between the tip face and the plasma membrane plus its direct pressure on any glycocalyx, both evaluated from the gap size in terms of the ion current decrease. A survey of cell types confirms that an intracellular pressure of approximately 120 Pa begins to detach the plasma membrane from the cytoskeleton and reveals that the first 0.66 ± 0.09 µm of compression of a neuron cell body is much softer than previous methods have been able to detect.
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Membrana Celular/fisiología , Microscopía/métodos , Animales , Línea Celular , Células Cultivadas , Citoplasma , Citoesqueleto , Fibroblastos/citología , Humanos , Iones , Neuronas/citología , RatasRESUMEN
In this work we show how hydrodynamic forces can be used to locally trap molecules in a supported lipid bilayer (SLB). The method uses the hydrodynamic drag forces arising from a flow through a conical pipette with a tip radius of 1-1.5 µm, placed approximately 1 µm above the investigated SLB. This results in a localized forcefield that acts on molecules protruding from the SLB, yielding a hydrodynamic trap with a size approximately given by the size of the pipette tip. We demonstrate this concept by trapping the protein streptavidin, bound to biotin receptors in the SLB. It is also shown how static and kinetic information about the intermolecular interactions in the lipid bilayer can be obtained by relating how the magnitude of the hydrodynamic forces affects the accumulation of protein molecules in the trap.
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Membrana Dobles de Lípidos , Colorantes Fluorescentes/química , CinéticaRESUMEN
Herpes simplex viruses display hundreds of gD glycoproteins, and yet their neutralization requires tens of thousands of antibodies per virion, leading us to ask whether a wild-type virion with just a single free gD is still infective. By quantitative analysis of fluorescently labeled virus particles and virus neutralization assays, we show that entry of a wild-type HSV virion to a cell does indeed require just one or two of the approximately 300 gD glycoproteins to be left unbound by monoclonal antibody. This indicates that HSV entry is an extraordinarily efficient process, functioning at the level of single molecular complexes.
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Glicoproteínas/metabolismo , Simplexvirus/metabolismo , Proteínas Virales/metabolismo , Animales , Anticuerpos Neutralizantes/inmunología , Células CHO , Chlorocebus aethiops , Cricetinae , Cricetulus , Glicoproteínas/inmunología , Simplexvirus/fisiología , Células Vero , Proteínas Virales/inmunología , Virión/metabolismo , Internalización del VirusRESUMEN
There is an intrinsic repulsion between glass and cell surfaces that allows noninvasive scanning ion conductance microscopy (SICM) of cells and which must be overcome in order to form the gigaseals used for patch clamping investigations of ion channels. However, the interactions of surfaces in physiological solutions of electrolytes, including the presence of this repulsion, for example, do not obviously agree with the standard Derjaguin-Landau-Verwey-Overbeek (DLVO) colloid theory accurate at much lower salt concentrations. In this paper we investigate the interactions of glass nanopipettes in this high-salt regime with a variety of surfaces and propose a way to resolve DLVO theory with the results. We demonstrate the utility of this understanding to SICM by topographically mapping a live cell's cytoskeleton. We also report an interesting effect whereby the ion current though a nanopipette can increase under certain conditions upon approaching an insulating surface, rather than decreasing as would be expected. We propose that this is due to electroosmotic flow separation, a high-salt electrokinetic effect. Overall these experiments yield key insights into the fundamental interactions that take place between surfaces in strong solutions of electrolytes.
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Nanoporos , Animales , Células COS , Chlorocebus aethiops , Electrólitos/química , Microscopía de Sonda de Barrido , Propiedades de SuperficieRESUMEN
We have experimentally determined the optimal flow velocities to characterize or count single molecules by using a simple microfluidic device to perform two-color coincidence detection (TCCD) and single pair Förster resonance energy transfer (spFRET) using confocal fluorescence spectroscopy on molecules traveling at speeds of up to 10 cm s(-1). We show that flowing single fluorophores at ≥0.5 cm s(-1) reduces the photophysical processes competing with fluorescence, enabling the use of high excitation irradiances to partially compensate for the short residence time within the confocal volume (10-200 µs). Under these conditions, the data acquisition rate can be increased by a maximum of 38-fold using TCCD at 5 cm s(-1) or 18-fold using spFRET at 2 cm s(-1), when compared with diffusion. While structural characterization requires more photons to be collected per event and so necessitates the use of slower speeds (2 cm s(-1) for TCCD and 1 cm s(-1) for spFRET), a considerable enhancement in the event rate could still be obtained (33-fold for TCCD and 16-fold for spFRET). Using flow under optimized conditions, analytes could be rapidly quantified over a dynamic range of up to 4 orders of magnitude by direct molecule counting; a 50 fM dual-labeled model sample can be detected with 99.5% statistical confidence in around 8 s using TCCD and a flow velocity of 5 cm s(-1).
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Fluorescencia , Transferencia Resonante de Energía de Fluorescencia , MicrofluídicaRESUMEN
The use of Förster resonance energy transfer (FRET) as a tool to study biomolecules has been greatly enhanced by new advances in single-molecule fluorescence (SMF) techniques. This has allowed new insights into the structure and dynamics of complex biomolecular machinery. However, there are still technical drawbacks in the application of conventional SMF-FRET. Herein, we review the use of single-molecule coincidence spectroscopy to study FRET systems, an analytical variation of the conventional scheme, using one or two confocal lasers of different colours. We highlight the advantages of the coincidence spectroscopy and illustrate this with examples of its application to some biological systems of interest.
Asunto(s)
Transferencia Resonante de Energía de Fluorescencia/métodos , Espectrometría de Fluorescencia , Conformación de Ácido Nucleico , Pliegue de Proteína , Proteínas/química , ARN/químicaRESUMEN
A key issue in understanding the pathogenic conditions associated with the aberrant aggregation of misfolded proteins is the identification and characterization of species formed during the aggregation process. Probing the nature of such species has, however, proved to be extremely challenging to conventional techniques because of their transient and heterogeneous character. We describe here the application of a two-color single-molecule fluorescence technique to examine the assembly of oligomeric species formed during the aggregation of the SH3 domain of PI3 kinase. The single-molecule experiments show that the species formed at the stage of the reaction where aggregates have previously been found to be maximally cytotoxic are a heterogeneous ensemble of oligomers with a median size of 38 +/- 10 molecules. This number is remarkably similar to estimates from bulk measurements of the critical size of species observed to seed ordered fibril formation and of the most infective form of prion particles. Moreover, although the size distribution of the SH3 oligomers remains virtually constant as the time of aggregation increases, their stability increases substantially. These findings together provide direct evidence for a general mechanism of amyloid aggregation in which the stable cross-beta structure emerges via internal reorganization of disordered oligomers formed during the lag phase of the self-assembly reaction.
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Amiloide/química , Amiloide/metabolismo , Fluorescencia , Pliegue de Proteína , Animales , Bovinos , Cinética , Fosfatidilinositol 3-Quinasas/química , Fosfatidilinositol 3-Quinasas/metabolismo , Espectrometría de Fluorescencia , Dominios Homologos srcRESUMEN
There are three regimes of cell membrane interaction with glass: Tight and loose adhesion, separated by repulsion. Explicitly including hydration, this paper evaluates the pressure between the surfaces as functions of distance for ion correlation and ion-screened electrostatics and electromagnetic fluctuations. The results agree with data for tight adhesion energy (0.5-3 vs 0.4-4 mJ/m^{2}), detachment pressure (7.9 vs. 9 MPa), and peak repulsion (3.4-7.5 vs. 5-10 kPa), also matching the repulsion's distance dependence on renormalization by steric pressure mainly from undulations.
RESUMEN
Single-molecule fluorescence resonance energy transfer (FRET) is commonly used to probe different conformations and conformational dynamics of single biomolecules. However, the analysis of raw burst traces is not always straightforward. The presence of a "zero peak" and the skewness of peaks at high and low FRET efficiencies in proximity ratio histograms make the accurate evaluation of the histogram a challenging task. This is further compounded by the difficulty associated with siting two fluorophores in optimal range of each other. Here we present an alternative method of analysis, based on handling coincident FRET photon bursts, that addresses these problems. In addition, we demonstrate methods to enhance coincidence levels and thus the accuracy of FRET determination: the use of dual-color excitation, including direct excitation of the acceptor fluorophore; the addition of a remote dye to the biomolecule, not involved in the FRET process; or a combination of the two. We show the advantages of dual excitation by studying several labeled double-stranded DNA samples as FRET models. This method extends the application of single-molecule FRET to more complicated biological systems where only a small fraction of complexes are fully assembled.
Asunto(s)
Transferencia Resonante de Energía de Fluorescencia/métodos , Color , Colorantes/química , Sensibilidad y EspecificidadRESUMEN
This paper discusses physical and structural aspects of the mechanisms herpes simplex virus (HSV) uses for membrane fusion. Calculations show that herpes simplex virus glycoprotein D has such avidity for its receptors that it can hold the virion against the plasma membrane of a neuron strongly enough for glycoprotein B (gB) to disrupt both leaflets of the bilayer. The strong electric field generated by the cell potential across perforations at this disruption would break the hydrogen bonds securing the gB fusion loops, leading to fusion of the plasma and viral membranes. This mechanism agrees with the high stability of the tall trimeric spike structure of gB and is consistent with the probable existence of a more compact initial conformation that would allow it to closely approach the plasma membrane. The release of the fusion domains by disruption of hydrogen bonds is shared with the endocytotic entry pathway where, for some cell types not punctured by gB, the virus is able to induce inward forces that cause endocytosis and the fusion loops are released by acidification. The puncture-fusion mechanism requires low critical strain or high tissue strain, matching primary tropism of neural processes at the vermillion border. In support of this mechanism, this paper proposes a functional superstructure of the antigens essential to entry and reviews its consistency with experimental evidence.
RESUMEN
We discuss how the ideal formalism of computational mechanics can be adapted to apply to a noninfinite series of corrupted and correlated data, that is typical of most observed natural time series. Specifically, a simple filter that removes the corruption that creates rare unphysical causal states is demonstrated, and the concept of effective soficity is introduced. We believe that computational mechanics cannot be applied to a noisy and finite data series without invoking an argument based upon effective soficity. A related distinction between noise and unresolved structure is also defined: Noise can only be eliminated by increasing the length of the time series, whereas the resolution of previously unresolved structure only requires the finite memory of the analysis to be increased. The benefits of these concepts are demonstrated in a simulated times series by (a) the effective elimination of white noise corruption from a periodic signal using the expletive filter and (b) the appearance of an effectively sofic region in the statistical complexity of a biased Poisson switch time series that is insensitive to changes in the word length (memory) used in the analysis. The new algorithm is then applied to an analysis of a real geomagnetic time series measured at Halley, Antarctica. Two principal components in the structure are detected that are interpreted as the diurnal variation due to the rotation of the Earth-based station under an electrical current pattern that is fixed with respect to the Sun-Earth axis and the random occurrence of a signature likely to be that of the magnetic substorm. In conclusion, some useful terminology for the discussion of model construction in general is introduced.
RESUMEN
In recent genome-wide association studies, the extracellular chaperone protein, clusterin, has been identified as a newly-discovered risk factor in Alzheimer's disease. We have examined the interactions between human clusterin and the Alzheimer's disease-associated amyloid-ß(1-40) peptide (Aß(1-40)), which is prone to aggregate into an ensemble of oligomeric intermediates implicated in both the proliferation of amyloid fibrils and in neuronal toxicity. Using highly sensitive single-molecule fluorescence methods, we have found that Aß(1-40) forms a heterogeneous distribution of small oligomers (from dimers to 50-mers), all of which interact with clusterin to form long-lived, stable complexes. Consequently, clusterin is able to influence both the aggregation and disaggregation of Aß(1-40) by sequestration of the Aß oligomers. These results not only elucidate the protective role of clusterin but also provide a molecular basis for the genetic link between clusterin and Alzheimer's disease.
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Péptidos beta-Amiloides/metabolismo , Clusterina/metabolismo , Chaperonas Moleculares/metabolismo , Fragmentos de Péptidos/metabolismo , Algoritmos , Enfermedad de Alzheimer/metabolismo , Amiloide/química , Amiloide/metabolismo , Amiloide/ultraestructura , Péptidos beta-Amiloides/química , Clusterina/química , Espacio Extracelular/metabolismo , Fluorescencia , Fluorometría/instrumentación , Fluorometría/métodos , Humanos , Cinética , Microscopía Confocal , Microscopía Electrónica de Transmisión , Chaperonas Moleculares/química , Fragmentos de Péptidos/química , Unión Proteica , Multimerización de Proteína , TermodinámicaAsunto(s)
Inmunoglobulina G/química , Nanotubos/química , Proteínas del Tejido Nervioso/química , ADN/química , Electrodos , Electroforesis , Colorantes Fluorescentes/química , Inmunoglobulina G/fisiología , Microscopía Fluorescente , Nanotecnología/instrumentación , Nanotecnología/métodos , Proteínas del Tejido Nervioso/fisiología , Tamaño de la Partícula , Sensibilidad y EspecificidadRESUMEN
Two-color coincidence detection is a single-molecule fluorescence technique that is capable of resolving subpopulations of biomolecular complexes at very low concentrations. In this paper, we have developed a method that automatically determines appropriate thresholds for the analysis of sets of two-color coincidence data. This has the distinct advantage of allowing the rapid determination of optimized thresholds in a reproducible fashion. The trade-offs involved in such selections are that the thresholds should be high enough both to exceed the background photon count rates and to ensure a low rate of chance coincident events and that they should be low enough to give reasonably high rates of fluorescence events. Previously, thresholds were selected by judgment to balance these various separate considerations. The method reported in this paper incorporates the three factors into the maximization of a single value derived from the data as a function of the thresholds used in the two channels. The value that is maximized is a ratio of event rates, specifically the rate of coincident events above that expected by chance, divided by the total event rate; this is called the association quotient. In this paper, we demonstrate that maximization of the association quotient selects appropriate thresholds for data derived from dual-labeled duplex DNA samples over a range of concentrations and laser powers. This method should allow the application of two-color coincidence detection to more complex biological systems and cells where the sample concentration and background levels are more variable and where it is not possible to run separate control experiments to determine the latter statistics.
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ADN/análisis , Espectrometría de Fluorescencia/métodos , Sensibilidad y EspecificidadRESUMEN
We report the measurement of the surface conductivity of biological macromolecules by dielectrophoretic trapping at the tip of a glass nanopipet. We find that the threshold voltage for trapping is a function of salt concentration and can be directly linked to the effective conductivity of the biomolecule and its solvation shell. The surface conductivities obtained for 20-mer single-stranded DNA, 40-mer double-stranded DNA, and yellow fluorescent protein are 7.9+/-1.9 nS, 5.3+/-0.7 nS, and 21.5+/-1.6 nS, respectively.
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Proteínas Bacterianas/química , ADN de Cadena Simple/química , ADN/química , Conductividad Eléctrica , Proteínas Luminiscentes/química , Sales (Química)/química , Electroforesis/métodos , Microelectrodos , Nanoestructuras/químicaRESUMEN
We present a single virion method to determine absolute distributions of copy number in the protein composition of viruses and apply it to herpes simplex virus type 1. Using two-color coincidence fluorescence spectroscopy, we determine the virion-to-virion variability in copy numbers of fluorescently labeled tegument and envelope proteins relative to a capsid protein by analyzing fluorescence intensity ratios for ensembles of individual dual-labeled virions and fitting the resulting histogram of ratios. Using EYFP-tagged capsid protein VP26 as a reference for fluorescence intensity, we are able to calculate the mean and also, for the first time to our knowledge, the variation in numbers of gD, VP16, and VP22 tegument. The measurement of the number of glycoprotein D molecules was in good agreement with independent measurements of average numbers of these glycoproteins in bulk virus preparations, validating the method. The accuracy, straightforward data processing, and high throughput of this technique make it widely applicable to the analysis of the molecular composition of large complexes in general, and it is particularly suited to providing insights into virus structure, assembly, and infectivity.
Asunto(s)
Proteínas de la Cápside/metabolismo , Proteína Vmw65 de Virus del Herpes Simple/metabolismo , Herpesvirus Humano 1/metabolismo , Proteínas del Envoltorio Viral/metabolismo , Proteínas Estructurales Virales/metabolismo , Anticuerpos/metabolismo , Proteínas de la Cápside/genética , AMP Cíclico/análogos & derivados , Fluorescencia , Colorantes Fluorescentes , Proteínas Fluorescentes Verdes/genética , Proteína Vmw65 de Virus del Herpes Simple/genética , Proteínas Luminiscentes/genética , Proteínas Recombinantes de Fusión/genética , Proteínas del Envoltorio Viral/inmunología , Proteínas Estructurales Virales/genética , Virión/metabolismoRESUMEN
The T cell receptor (TCR) expressed on most T cells is a protein complex consisting of TCRalphabeta heterodimers that bind antigen and cluster of differentiation (CD) 3epsilondelta, epsilongamma, and zetazeta dimers that initiate signaling. A long-standing controversy concerns whether there is one, or more than one, alphabeta heterodimer per complex. We used a form of single-molecule spectroscopy to investigate this question on live T cell hybridomas. The method relies on detecting coincident fluorescence from single molecules labeled with two different fluorophores, as the molecules diffuse through a confocal volume. The fraction of events that are coincident above the statistical background is defined as the "association quotient," Q. In control experiments, Q was significantly higher for cells incubated with wheat germ agglutinin dual-labeled with Alexa488 and Alexa647 than for cells incubated with singly labeled wheat germ agglutinin. Similarly, cells expressing the homodimer, CD28, gave larger values of Q than cells expressing the monomer, CD86, when incubated with mixtures of Alexa488- and Alexa647-labeled antibody Fab fragments. T cell hybridomas incubated with mixtures of anti-TCRbeta Fab fragments labeled with each fluorophore gave a Q value indistinguishable from the Q value for CD86, indicating that the dominant form of the TCR comprises single alphabeta heterodimers. The values of Q obtained for CD86 and the TCR were low but nonzero, suggesting that there is transient or nonrandom confinement, or diffuse clustering of molecules at the T cell surface. This general method for analyzing the subunit composition of protein complexes could be extended to other cell surface or intracellular complexes, and other living cells.
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Receptores de Antígenos de Linfocitos T/química , Receptores de Antígenos de Linfocitos T/inmunología , Linfocitos T/inmunología , Animales , Antígeno B7-2/química , Antígenos CD28/química , Dimerización , Hibridomas/inmunología , Ratones , Modelos Moleculares , Fragmentos de Péptidos/química , Subunidades de Proteína/química , Complejo Receptor-CD3 del Antígeno de Linfocito T/química , Receptores de Antígenos de Linfocitos T alfa-beta/químicaRESUMEN
Telomerase is a nonclassical DNA polymerase that uses its integral RNA as a template to synthesize telomeric repeats onto chromosome ends. The molecular mechanism of telomerase is unique and involves a translocation step after the synthesis of each telomeric repeat. To directly measure the enzymatic turnover of substrate and the efficiency of the translocation step we have extended our two-color single molecule fluorescence coincidence method (Anal.Chem. 2003, 75, 1664-1670). The method employs Cy5-dATP incorporation into a DNA primer that has been prelabeled with a reference fluorophore. Measurements are performed in the single molecule regime and products, which necessarily have both fluorophores, are excited by two independent lasers, and give rise to coincident events. By counting the number of coincident events and using the coincidence detection efficiency, it is possible to determine the number of the extended products generated by attomole quantities of telomerase, without separation or the use of PCR or radioactivity. Histograms of the logarithms of the ratios of the Cy5 to the reference fluorophore fluorescence can be used to determine the length distribution of the products and hence the enzyme processivity. The mean processivity obtained from the single molecule fluorescence coincidence assay is 0.32 +/- 0.04, in good agreement with the value of 0.37 +/- 0.05 derived from the direct radioactive assay approach. The function of the alignment domain of human telomerase RNA in sustaining catalytic activity in vitro has been reevaluated using this method. Together with our previous results (Nucleic Acids Res. 2002, 30, 4470-4480) these experiments identify the essential residues in the alignment domain of human telomerase RNA that contribute to the activity and processivity of telomerase.