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The formation of silenced and condensed heterochromatin foci involves enrichment of heterochromatin protein 1 (HP1). HP1 can bridge chromatin segments and form liquid droplets, but the biophysical principles underlying heterochromatin compartmentalization in the cell nucleus are elusive. Here, we assess mechanistically relevant features of pericentric heterochromatin compaction in mouse fibroblasts. We find that (1) HP1 has only a weak capacity to form liquid droplets in living cells; (2) the size, global accessibility, and compaction of heterochromatin foci are independent of HP1; (3) heterochromatin foci lack a separated liquid HP1 pool; and (4) heterochromatin compaction can toggle between two "digital" states depending on the presence of a strong transcriptional activator. These findings indicate that heterochromatin foci resemble collapsed polymer globules that are percolated with the same nucleoplasmic liquid as the surrounding euchromatin, which has implications for our understanding of chromatin compartmentalization and its functional consequences.
Assuntos
Cromatina/genética , Proteínas Cromossômicas não Histona/genética , Eucromatina/genética , Heterocromatina/genética , Animais , Homólogo 5 da Proteína Cromobox , Fibroblastos , CamundongosRESUMO
Determining the label to target ratio, also known as the degree of labeling (DOL), is crucial for quantitative fluorescence microscopy and a high DOL with minimal unspecific labeling is beneficial for fluorescence microscopy in general. Yet robust, versatile and easy-to-use tools for measuring cell-specific labeling efficiencies are not available. Here we present a DOL determination technique named protein-tag DOL (ProDOL), which enables fast quantification and optimization of protein-tag labeling. With ProDOL various factors affecting labeling efficiency, including substrate type, incubation time and concentration, as well as sample fixation and cell type can be easily assessed. We applied ProDOL to investigate how human immunodeficiency virus-1 pathogenesis factor Nef modulates CD4 T cell activation measuring total and activated copy numbers of the adapter protein SLP-76 in signaling microclusters. ProDOL proved to be a versatile and robust tool for labeling calibration, enabling determination of labeling efficiencies, optimization of strategies and quantification of protein stoichiometry.
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Direct control of protein interactions by chemically induced protein proximity holds great potential for both cell and synthetic biology as well as therapeutic applications. Low toxicity, orthogonality and excellent cell permeability are important criteria for chemical inducers of proximity (CIPs), in particular for in vivo applications. Here, we present the use of the agrochemical mandipropamid (Mandi) as a highly efficient CIP in cell culture systems and living organisms. Mandi specifically induces complex formation between a sixfold mutant of the plant hormone receptor pyrabactin resistance 1 (PYR1) and abscisic acid insensitive (ABI). It is orthogonal to other plant hormone-based CIPs and rapamycin-based CIP systems. We demonstrate the applicability of the Mandi system for rapid and efficient protein translocation in mammalian cells and zebrafish embryos, protein network shuttling and manipulation of endogenous proteins.
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Amidas/farmacologia , Ácidos Carboxílicos/farmacologia , Fungicidas Industriais/farmacologia , Ácido Abscísico/metabolismo , Animais , Dimerização , Peixe-Zebra/embriologiaRESUMO
Recent developments in fluorescence microscopy call for novel small-molecule-based labels with multiple functionalities to satisfy different experimental requirements. A current limitation in the advancement of live-cell single-molecule localization microscopy is the high excitation power required to induce blinking. This is in marked contrast to the minimal phototoxicity required in live-cell experiments. At the same time, quality of super-resolution imaging depends on high label specificity, making removal of excess dye essential. Approaching both hurdles, we present the design and synthesis of a small-molecule label comprising both fluorogenic and self-blinking features. Bioorthogonal click chemistry ensures fast and highly selective attachment onto a variety of biomolecular targets. Along with spectroscopic characterization, we demonstrate that the probe improves quality and conditions for regular and single-molecule localization microscopy on live-cell samples.
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Química Click/métodos , Microscopia de Fluorescência/métodos , Imagem Óptica/métodosRESUMO
Cells typically vary in their response to extracellular ligands. Receptor transport processes modulate ligand-receptor induced signal transduction and impact the variability in cellular responses. Here, we quantitatively characterized cellular variability in erythropoietin receptor (EpoR) trafficking at the single-cell level based on live-cell imaging and mathematical modeling. Using ensembles of single-cell mathematical models reduced parameter uncertainties and showed that rapid EpoR turnover, transport of internalized EpoR back to the plasma membrane, and degradation of Epo-EpoR complexes were essential for receptor trafficking. EpoR trafficking dynamics in adherent H838 lung cancer cells closely resembled the dynamics previously characterized by mathematical modeling in suspension cells, indicating that dynamic properties of the EpoR system are widely conserved. Receptor transport processes differed by one order of magnitude between individual cells. However, the concentration of activated Epo-EpoR complexes was less variable due to the correlated kinetics of opposing transport processes acting as a buffering system.
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Transporte Biológico/fisiologia , Modelos Biológicos , Receptores de Superfície Celular/metabolismo , Análise de Célula Única/métodos , Linhagem Celular Tumoral , Biologia Computacional , Corantes Fluorescentes/análise , Corantes Fluorescentes/química , Corantes Fluorescentes/metabolismo , Humanos , Processamento de Imagem Assistida por Computador/métodos , Cinética , Microscopia Confocal , Receptores de Superfície Celular/análise , Receptores de Superfície Celular/química , Receptores da EritropoetinaRESUMO
Dinuclear CuII -patellamide complexes (patellamides are naturally occurring cyclic pseudo-octapeptides) are known to be efficient catalysts for hydrolysis reactions of biological importance, for example, those of phosphatase, carbonic anhydrase, and glycosidase. However, the biological role of patellamides is still unknown. Patellamides were originally extracted from the sea squirt Lissoclinum patella, but are now known to be ribosomally expressed by the blue-green algae Prochloron that live in symbiosis with L. patella. In a further step to unravel the metabolic significance of the patellamide complexes, the question as to whether these are also formed inside Prochloron cells is addressed. In this study, a biocompatible patellamide-fluorescent dye conjugate has been introduced into living Prochloron cells and, by means of flow cytometry and confocal microscopy, it is shown that CuII ions are coordinated to patellamides in vivo.
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Chemical fixation of living cells for microscopy is commonly achieved by crosslinking of intracellular proteins with dialdehydes prior to examination. We herein report a photocleavable protecting group for glutaraldehyde that results in a light-triggered and membrane-permeable fixative, which is nontoxic prior to photocleavage. Lipophilic ester groups allow for diffusion across the cell membrane and intracellular accumulation after enzymatic hydrolysis. Irradiation with UV light releases glutaraldehyde. The inâ situ generated fixative crosslinks intracellular proteins and preserves and stabilizes the cell so that it is ready for microscopy. In contrast to conventional glutaraldehyde fixation, tissue autofluorescence does not increase after fixation. Caged glutaraldehyde may in future enable functional experiments on living cells under a light microscope in which events of interest can be stopped in spatially confined volumes at defined time points. Samples with individually stopped events could then later be analyzed in ultrastructural studies.
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A small synthetic calcium sensor that can be site-specifically coupled to proteins in living cells by utilizing the bio-orthogonal HaloTag labeling strategy is presented. We synthesized an iodo-derivatized BAPTA chelator with a tetramethyl rhodamine fluorophore that allows further modification by Sonogashira cross-coupling. The presented calcium sensitive dye shows a 200-fold increase in fluorescence upon calcium binding. The derivatization with an aliphatic linker bearing a terminal haloalkane-function by Sonogashira cross-coupling allows the localization of the calcium sensor to Halo fusion proteins which we successfully demonstrate in in vitro and in vivo experiments. The herein reported highly sensitive tetramethyl rhodamine based calcium indicator, which can be selectively localized to proteins, is a powerful tool to determine changes in calcium levels inside living cells with spatiotemporal resolution.
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Cálcio/metabolismo , Corantes Fluorescentes/metabolismo , Proteínas/metabolismo , Rodaminas/metabolismo , Animais , Sobrevivência Celular , Células HeLa , Humanos , Camundongos , Células NIH 3T3 , Coloração e RotulagemRESUMO
The number of fluorophores within a molecule complex can be revealed by single-molecule photobleaching imaging. A widely applied strategy to analyze intensity traces over time is the quantification of photobleaching step counts. However, several factors can limit and bias the detection of photobleaching steps, including noise, high numbers of fluorophores, and the possibility that several photobleaching events occur almost simultaneously. In this study, we propose a new approach, to our knowledge, to determine the fluorophore number that correlates the intensity decay of a population of molecule complexes with the decay of the number of visible complexes. We validated our approach using single and fourfold Atto-labeled DNA strands. As an example we estimated the subunit stoichiometry of soluble CD95L using GFP fusion proteins. To assess the precision of our method we performed in silico experiments showing that the estimates are not biased for experimentally observed intensity fluctuations and that the relative precision remains constant with increasing number of fluorophores. In case of fractional fluorescent labeling, our simulations predicted that the fluorophore number estimate corresponds to the product of the true fluorophore number with the labeling fraction. Our method, denoted by spot number and intensity correlation (SONIC), is fully automated, robust to noise, and does not require the counting of photobleaching events.
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Corantes Fluorescentes/química , Modelos Estatísticos , Fotodegradação , Automação , Sequência de Bases , DNA/química , DNA/genética , Processamento de Imagem Assistida por Computador , Microscopia , Modelos Moleculares , Conformação de Ácido Nucleico , Multimerização Proteica , Estrutura Quaternária de Proteína , Receptor fas/químicaRESUMO
Blinking of the photoluminescence (PL) emitted from individual conjugated polymer chains is one of the central observations made by single-molecule spectroscopy (SMS). Important information, for example regarding excitation energy transfer, can be extracted by evaluating dynamic quenching. However, the nature of trap states, which are responsible for PL quenching, often remains obscured. We present a detailed investigation of the photon statistics of single poly(3-hexylthiophene) (P3HT) chains obtained by SMS. The photon statistics provide a measure of the number and brightness of independently emitting areas on a single chain. These observables can be followed during blinking. A decrease in PL intensity is shown to be correlated with either 1)â a decrease in the average brightness of the emitting sites; or 2)â a decrease in the number of emitting regions. We attribute these phenomena to the formation of 1)â shallow charge traps, which can weakly affect all emitting areas of a single chain at once; and 2)â deep traps, which have a strong effect on small regions within the single chains.
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Recent developments in biology demand an increasing number of simultaneously imaged structures with standard fluorescence microscopy. However, the number of multiplexed channels is limited for most multiplexing modalities, such as spectral multiplexing or fluorescence-lifetime imaging. We propose extending the number of imaging channels by using chemical reactions, controlling the emissive state of fluorescent dyes. As proof of concept, we reversibly switch a fluorescent copper sensor to enable successive imaging of two different structures in the same spectral channel. We also show that this chemical multiplexing is orthogonal to existing methods. By using two different dyes, we combine chemical with spectral multiplexing for the simultaneous imaging of four different structures with only two spectrally different channels. We characterize and discuss the approach and provide perspectives for extending imaging modalities in stimulated emission depletion microscopy, for which spectral multiplexing is technically demanding.
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Corantes Fluorescentes/química , Microscopia de Fluorescência/métodos , Cor , FluorescênciaRESUMO
Over the past decade, a vast variety of different fluorescent labeling systems have emerged for use in fluorescence microscopy and fluorescence-based analytical techniques. A difficulty frequently arising when quantifying fluorescently labeled samples is that the number of labels per protein is neither well defined, for example, due to multiple functional groups that can undergo covalent coupling with activated dyes, nor well known, for example, due to limited methods mostly estimating ensemble averages. Herein, we use a recently established method that evaluates the statistics of multiple photon detection events to measure the label number distribution of different fluorescent marker molecules at the single-molecule level. We tested five different far-red dyes frequently used for fluorescence labeling and found all of them suitable for our counting method. We used two dyes, ATTO647N and Alexa647, to investigate the label number distribution of fluorescently labeled proteins. In the experiments, we found that the label number distribution of antibodies and streptavidin has a significant fraction of molecules labeled with two, three, or more fluorophores. In contrast, the distribution of label numbers for nanobodies resembles the one acquired for SNAP-tag, which can have a maximum of one label per protein. This is also reflected in the ensemble degree of labeling, which is in good agreement for the latter samples, whereas stronger deviations were observed for antibodies and streptavidin. Our single-molecule studies enable full characterization of the label number distribution for various fluorescent markers. This work puts quantitative studies on the stoichiometry of fluorescently tagged oligomers and protein aggregates into perspective.
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The effect of solute affinity on solute diffusion in binary liquids well below the lower critical solution temperature (LCST) was studied by using fluorescence correlation spectroscopy. We measured the hydrodynamic radii of a hydrophobic and an amphiphilic fluorescent dye under systematic variation of the relative molar fractions of water/2-butoxyethanol and, for comparison, of water/methanol mixtures, which do not show phase separation. We found that the apparent hydrodynamic radius of the hydrophobic dye almost doubled in water/2-butoxyethanol, whereas it remained largely unchanged for the amphiphilic dye and in water/methanol mixtures. Our results indicate that the translational diffusion of solutes is influenced by transient local solution structures, even at temperatures well below the LCST. We conclude that, even far below LCST, different solutes can experience different environments in binary liquid mixtures depending on both the solute and solvent properties, all of which impact their reactivity.
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As a model for understanding how molecular crowding influences diffusion and transport of proteins in cellular environments, we combined experimental and theoretical approaches to study the diffusion of proteins in highly concentrated protein solutions. Bovine serum albumin and γ-Globulin were chosen as molecular crowders and as tracers. These two proteins are representatives of the main types of plasma protein and have different shapes and sizes. Solutions consisting of one or both proteins were studied. The self-diffusion coefficients of the fluorescently labeled tracer proteins were measured by means of fluorescence correlation spectroscopy at a total protein concentration of up to 400 g/L. γ-Globulin is found to have a stronger influence as a crowder on the tracer self-diffusion coefficient than Bovine serum albumin. Brownian dynamics simulations show that the excluded volume and the shape of the crowding protein have a significantly stronger influence on translational and rotational diffusion coefficients, as well as transient oligomerization, than hydrodynamic or direct interactions. Anomalous subdiffusion, which is not observed at the experimental fluorescence correlation spectroscopy timescales (>100 µs), appears only at very short timescales (<1 µs) in the simulations due to steric effects of the proteins. We envision that the combined experimental and computational approach employed here can be developed to unravel the different biophysical contributions to protein motion and interaction in cellular environments by systematically varying protein properties such as molecular weight, size, shape, and electrostatic interactions.
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Soroalbumina Bovina/metabolismo , gama-Globulinas/metabolismo , Animais , Bovinos , Difusão , Hidrodinâmica , Simulação de Dinâmica Molecular , Peso Molecular , Rotação , Soroalbumina Bovina/química , gama-Globulinas/químicaRESUMO
Obtaining quantitative information about molecular assemblies with high spatial and temporal resolution is a challenging task in fluorescence microscopy. Single-molecule techniques build on the ability to count molecules one by one. Here, a method is presented that extends recent approaches to analyze the statistics of coincidently emitted photons to enable reliable counting of molecules in the range of 1-20. This method does not require photochemistry such as blinking or bleaching. DNA origami structures are labeled with up to 36 dye molecules as a new evaluation tool to characterize this counting by a photon statistics approach. Labeled DNA origami has a well-defined labeling stoichiometry and ensures equal brightness for all dyes incorporated. Bias and precision of the estimating algorithm are determined, along with the minimal acquisition time required for robust estimation. Complexes containing up to 18 molecules can be investigated non-invasively within 150 ms. The method might become a quantifying add-on for confocal microscopes and could be especially powerful in combination with STED/RESOLFT-type microscopy.
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DNA/química , Corantes Fluorescentes/química , Microscopia de Fluorescência , FótonsRESUMO
The analysis of binding interactions between small molecules and biopolymers is important for understanding biological processes. While fluorescence correlation spectroscopy (FCS) requires fluorescence labeling on the small molecule, which often interferes with binding, in microscale thermophoresis (MST) the label can be placed on the biopolymer. Ribozymes have not been analyzed by MST so far. The Diels-Alderase ribozyme (DAse) is a true catalyst, facilitating the Diels-Alder reaction between two free small substrates, anthracene dienes, and maleimide dienophiles. Despite high efforts, the determination of the dissociation constant (KD) of maleimide dienophiles to the DAse by FCS has been unsuccessful. Here, we determined the binding interactions of the DAse to its substrates and the Diels-Alder product using MST. The results supported a positive cooperativity for substrate binding to the DAse. By varying the temperature, we furthermore studied the thermodynamics of dienophile dissociation. The entropic contribution was found to be the energetic driving force for the binding of the dienophile to the DAse.
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Reação de Cicloadição , RNA Catalítico/química , RNA Catalítico/metabolismo , Antracenos/metabolismo , Sítios de Ligação , Catálise , Maleimidas/metabolismo , Modelos Moleculares , Conformação de Ácido Nucleico , Especificidade por Substrato , TermodinâmicaRESUMO
CLEC-2 is a target for a new class of antiplatelet agent. Clustering of CLEC-2 leads to phosphorylation of a cytosolic YxxL and binding of the tandem SH2 domains in Syk, crosslinking two receptors. We have raised 48 nanobodies to CLEC-2 and crosslinked the most potent of these to generate divalent and tetravalent nanobody ligands. Fluorescence correlation spectroscopy (FCS) was used to show that the multivalent nanobodies cluster CLEC-2 in the membrane and that clustering is reduced by inhibition of Syk. Strikingly, the tetravalent nanobody stimulated aggregation of human platelets, whereas the divalent nanobody was an antagonist. In contrast, in human CLEC-2 knock-in mouse platelets, the divalent nanobody stimulated aggregation. Mouse platelets express a higher level of CLEC-2 than human platelets. In line with this, the divalent nanobody was an agonist in high-expressing transfected DT40 cells and an antagonist in low-expressing cells. FCS, stepwise photobleaching and non-detergent membrane extraction show that CLEC-2 is a mixture of monomers and dimers, with the degree of dimerisation increasing with expression thereby favouring crosslinking of CLEC-2 dimers. These results identify ligand valency, receptor expression/dimerisation and Syk as variables that govern activation of CLEC-2 and suggest that divalent ligands should be considered as partial agonists.
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Lectinas Tipo C , Anticorpos de Domínio Único , Animais , Humanos , Camundongos , Lectinas Tipo C/genética , Lectinas Tipo C/metabolismo , Glicoproteínas de Membrana/metabolismo , Transdução de Sinais/fisiologia , Anticorpos de Domínio Único/farmacologia , Quinase Syk/metabolismoRESUMO
Carba nicotinamide adenine dinucleotide (cNAD) may serve as a stable cofactor for the enzyme-based detection of glucose. Many characteristics of cNAD and its reduced form cNADH resemble those of NAD and NADH, respectively. The fluorescence lifetimes of cNADH are determined to be 0.32(2) ns and 0.66(3) ns compared to 0.28(2) ns and 0.60(3) ns for NADH, and the temperature dependence of these lifetimes hints towards identical processes for quenching. The maximum emission occurs at 464 nm for both cNADH and NADH and absorbance maxima are found at 360 nm and 340 nm, respectively. In contrast to previous suggestions the respective maximum extinction coefficient of cNADH equals that of NADH and amounts to 6.2(2) mM(-1) cm(-1). When changing from NADH to cNADH we observe a ~50% increase in quantum efficiency, which--together with the larger excitation wavelength and the higher stability--should make cNAD a well suited alternative as coenzyme for robust glucose detection.
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Técnicas Biossensoriais , Glucose/análise , NAD , Glicemia/análise , Fluorescência , Humanos , NAD/análogos & derivados , NAD/síntese química , Espectrometria de Fluorescência , TemperaturaRESUMO
Fluorogenic labeling via bioorthogonal tetrazine chemistry has proven to be highly successful in fluorescence microscopy of living cells. To date, trans-cyclooctene (TCO) and bicyclonyne have been found to be the most useful substrates for live-cell labeling owing to their fast labeling kinetics, high biocompatibility, and bioorthogonality. Recent kinetic studies of fluorogenic click reactions with TCO derivatives showed a transient fluorogenic effect but could not explain the reaction sequence and the contributions of different intermediates. More recently, fluorescence quenching by potential intermediates has been investigated, suggesting their occurrence in the reaction sequence. However, in situ studies of the click reaction that directly relate these observations to the known reaction sequence are still missing. In this study, we developed a single-molecule fluorescence detection framework to investigate fluorogenic click reactions. In combination with data from ultra-performance liquid chromatography-tandem mass spectrometry, this explains the transient intensity increase by relating fluorescent intermediates to the known reaction sequence of TCO with fluorogenic tetrazine dyes. More specifically, we confirm that the reaction of TCO with tetrazine rapidly forms a fluorescent 4,5-dihydropyridazine species that slowly tautomerizes to a weakly fluorescent 1,4-dihydropyridazine, explaining the observed drop in fluorescence intensity. On a much slower timescale of hours/days, the fluorescence intensity may be recovered by oxidation of the intermediate to a pyridazine. Our findings are of importance for quantitative applications in fluorescence microscopy and spectroscopy as the achieved peak intensity with TCO depends on the specific experimental settings. They clearly indicate the requirement for more robust benchmarking of click reactions with tetrazine dyes and the need for alternative dienophiles with fast reaction kinetics and stable fluorescence emission to further applications in advanced fluorescence microscopy.
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Cells contain numerous substructures that have been proposed to form via liquid-liquid phase separation (LLPS). It is currently debated how to reliably distinguish LLPS from other mechanisms. Here, we benchmark different methods using well-controlled model systems in vitro and in living cells. We find that 1,6-hexanediol treatment and classical FRAP fail to distinguish LLPS from the alternative scenario of molecules binding to spatially clustered binding sites without phase-separating. In contrast, the preferential internal mixing seen in half-bleach experiments robustly distinguishes both mechanisms. We introduce a workflow termed model-free calibrated half-FRAP (MOCHA-FRAP) to probe the barrier at the condensate interface that is responsible for preferential internal mixing. We use it to study components of heterochromatin foci, nucleoli, stress granules and nuage granules, and show that the strength of the interfacial barrier increases in this order. We anticipate that MOCHA-FRAP will help uncover the mechanistic basis of biomolecular condensates in living cells.