RESUMO
Efficient tools for controlling molecular functions with exquisite spatiotemporal resolution are much in demand to investigate biological processes in living systems. Here we report an easily synthesized caged dexamethasone for photo-activating cytoplasmic proteins fused to the glucocorticoid receptor. In the dark, it is stable inâ vitro as well as inâ vivo in both zebrafish (Danio rerio) and Xenopus sp, two significant models of vertebrates. In contrast, it liberates dexamethasone upon UV illumination, which has been harnessed to interfere with developmental steps in embryos of these animals. Interestingly, this new system is biologically orthogonal to the one for photo-activating proteins fused to the estrogen ERT receptor, which brings great prospect for activating two distinct proteins down to the single cell level.
RESUMO
The hybridization kinetic of an oligonucleotide to its template is a fundamental step in many biological processes such as replication arrest, CRISPR recognition, DNA sequencing, DNA origami, etc. Although single kinetic descriptions exist for special cases of this problem, there are no simple general prediction schemes. In this work, we have measured experimentally, with no fluorescent labelling, the displacement of an oligonucleotide from its substrate in two situations: one corresponding to oligonucleotide binding/unbinding on ssDNA and one in which the oligonucleotide is displaced by the refolding of a dsDNA fork. In this second situation, the fork is expelling the oligonucleotide thus significantly reducing its residence time. To account for our data in these two situations, we have constructed a mathematical model, based on the known nearest neighbour dinucleotide free energies, and provided a good estimate of the residence times of different oligonucleotides (DNA, RNA, LNA) of various lengths in different experimental conditions (force, temperature, buffer conditions, presence of mismatches, etc.). This study provides a foundation for the dynamics of oligonucleotide displacement, a process of importance in numerous biological and bioengineering contexts.
Assuntos
DNA , Oligonucleotídeos , DNA/genética , Hibridização de Ácido Nucleico , DNA de Cadeia Simples , Sondas de OligonucleotídeosRESUMO
The zebrafish is one of the most widely adopted animal models in both basic and translational research. This popularity of the zebrafish results from several advantages such as a high degree of similarity to the human genome, the ease of genetic and chemical perturbations, external fertilization with high fecundity, transparent and fast-developing embryos, and relatively low cost-effective maintenance. In particular, body translucency is a unique feature of zebrafish that is not adequately obtained with other vertebrate organisms. The animal's distinctive optical clarity and small size therefore make it a successful model for optical modulation and observation. Furthermore, the convenience of microinjection and high embryonic permeability readily allow for efficient delivery of large and small molecules into live animals. Finally, the numerous number of siblings obtained from a single pair of animals offers large replicates and improved statistical analysis of the results. In this review, we describe the development of opto-chemical tools based on various strategies that control biological activities with unprecedented spatiotemporal resolution. We also discuss the reported applications of these tools in zebrafish and highlight the current challenges and future possibilities of opto-chemical approaches, particularly at the single cell level.
Assuntos
Peixe-Zebra , Animais , Humanos , MicroinjeçõesRESUMO
Homeoproteins of the Engrailed family are involved in the patterning of mesencephalic boundaries through a mechanism classically ascribed to their transcriptional functions. In light of recent reports on the paracrine activity of homeoproteins, including Engrailed, we asked whether Engrailed intercellular transfer was also involved in brain patterning and boundary formation. Using time-controlled activation of Engrailed combined with tools that block its transfer, we show that the positioning of the diencephalic-mesencephalic boundary (DMB) requires Engrailed paracrine activity. Both zebrafish Eng2a and Eng2b are competent for intercellular transfer in vivo, but only extracellular endogenous Eng2b, and not Eng2a, participates in DMB positioning. In addition, disruption of the Pbx-interacting motif in Engrailed, known to strongly reduce the gain-of-function phenotype, also downregulates Engrailed transfer, thus revealing an unsuspected participation of the Pbx interaction domain in this pathway.
Assuntos
Encéfalo/embriologia , Encéfalo/metabolismo , Proteínas de Peixe-Zebra/metabolismo , Peixe-Zebra/embriologia , Peixe-Zebra/metabolismo , Animais , Proteínas de Homeodomínio/genética , Proteínas de Homeodomínio/metabolismo , Proteínas do Tecido Nervoso/genética , Proteínas do Tecido Nervoso/metabolismo , Proteínas de Peixe-Zebra/genéticaRESUMO
The use of light to control the expression of genes and the activity of proteins is a rapidly expanding field. Whereas many of these approaches use fusion between a light-activable protein and the protein of interest to control the activity of the latter, it is also possible to control the activity of a protein by uncaging a specific ligand. In that context, controlling the activation of a protein fused to the modified estrogen receptor (ERT) by uncaging its ligand cyclofen-OH has emerged as a generic and versatile method to control the activation of proteins quantitatively, quickly, and locally in a live organism. We present that approach and its uses in a variety of physiological contexts.
Assuntos
Optogenética/métodos , Compostos Policíclicos/metabolismo , Receptores de Estrogênio/genética , Animais , Regulação da Expressão Gênica/efeitos da radiação , Proteínas de Fluorescência Verde/genética , Proteínas de Fluorescência Verde/metabolismo , Humanos , Ligantes , Compostos Policíclicos/química , Receptores de Estrogênio/metabolismo , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/metabolismoRESUMO
Helicases are a broad family of enzymes that perform crucial functions in DNA replication and in the maintenance of DNA and RNA integrity. A detailed mechanical study of helicases on DNA and RNA is possible using single molecule manipulation methods. Among those, magnetic tweezers (or traps) present a convenient, moderate throughput assay (tens of enzymes can be monitored simultaneously) that allow for high resolution (single base-pair) studies of these enzymes in various conditions and on various substrates (double and single stranded DNA and RNA). Here we discuss various implementation of the basic assay relevant for these studies.
Assuntos
DNA Helicases/química , DNA Cruciforme/química , Magnetismo/métodos , Pinças Ópticas , DNA/química , DNA/genética , DNA Helicases/genética , Replicação do DNA/genética , DNA Cruciforme/genética , RNA/química , RNA/genética , Imagem Individual de Molécula/métodosRESUMO
The possibility offered by photocontrolling the activity of biomolecules in vivo while recording physiological parameters is opening up new opportunities for the study of physiological processes at the single-cell level in a living organism. For the last decade, such tools have been mainly used in neuroscience, and their application in freely moving animals has revolutionized this field. New photochemical approaches enable the control of various cellular processes by manipulating a wide range of protein functions in a noninvasive way and with unprecedented spatiotemporal resolution. We are at a pivotal moment where biologists can adapt these cutting-edge technologies to their system of study. This user-oriented review presents the state of the art and highlights technical issues to be resolved in the near future for wide and easy use of these powerful approaches.
Assuntos
Células Eucarióticas/metabolismo , Sondas Moleculares/química , Optogenética/métodos , Análise de Célula Única/métodos , Animais , Células Eucarióticas/citologia , Expressão Gênica , Humanos , Luz , Opsinas/genética , Opsinas/metabolismo , Processos Fotoquímicos , Rodopsinas Microbianas/genética , Rodopsinas Microbianas/metabolismo , Análise Espaço-TemporalRESUMO
The maintenance of cooperation in populations where public goods are equally accessible to all but inflict a fitness cost on individual producers is a long-standing puzzle of evolutionary biology. An example of such a scenario is the secretion of siderophores by bacteria into their environment to fetch soluble iron. In a planktonic culture, these molecules diffuse rapidly, such that the same concentration is experienced by all bacteria. However, on solid substrates, bacteria form dense and packed colonies that may alter the diffusion dynamics through cell-cell contact interactions. In Pseudomonas aeruginosa microcolonies growing on solid substrate, we found that the concentration of pyoverdine, a secreted iron chelator, is heterogeneous, with a maximum at the center of the colony. We quantitatively explain the formation of this gradient by local exchange between contacting cells rather than by global diffusion of pyoverdine. In addition, we show that this local trafficking modulates the growth rate of individual cells. Taken together, these data provide a physical basis that explains the stability of public goods production in packed colonies.
Assuntos
Ferro/metabolismo , Oligopeptídeos/metabolismo , Pseudomonas aeruginosa/fisiologia , Transporte Biológico/fisiologiaRESUMO
All-trans retinoic acid (RA) is a key player in many developmental pathways. Most methods used to study its effects in development involve continuous all-trans RA activation by incubation in a solution of all-trans RA or by implanting all-trans RA-soaked beads at desired locations in the embryo. Here we show that the UV-driven photo-isomerization of 13-cis RA to the trans-isomer (and vice versa) can be used to non-invasively and quantitatively control the concentration of all-trans RA in a developing embryo in time and space. This facilitates the global or local perturbation of developmental pathways with a pulse of all-trans RA of known concentration or its inactivation by UV illumination. In zebrafish embryos in which endogenous synthesis of all-trans RA is impaired, incubation for as little as 5 minutes in 1 nM all-trans RA (a pulse) or 5 nM 13-cis RA followed by 1-minute UV illumination is sufficient to rescue the development of the hindbrain if performed no later than bud stage. However, if subsequent to this all-trans RA pulse the embryo is illuminated (no later than bud stage) for 1 minute with UV light (to isomerize, i.e. deactivate, all-trans RA), the rescue of hindbrain development is impaired. This suggests that all-trans RA is sequestered in embryos that have been transiently exposed to it. Using 13-cis RA isomerization with UV light, we further show that local illumination at bud stage of the head region (but not the tail) is sufficient to rescue hindbrain formation in embryos whose all-trans RA synthetic pathway has been impaired.
Assuntos
Rombencéfalo/embriologia , Rombencéfalo/metabolismo , Tretinoína/metabolismo , Peixe-Zebra/embriologia , Peixe-Zebra/metabolismo , Animais , Isotretinoína/química , Isotretinoína/metabolismo , Rombencéfalo/efeitos da radiação , Tretinoína/química , Raios UltravioletaRESUMO
High-throughput, low-cost DNA sequencing has emerged as one of the challenges of the postgenomic era. Here we present the proof of concept for a single-molecule platform that allows DNA identification and sequencing. In contrast to most present methods, our scheme is not based on the detection of the fluorescent nucleotides but on DNA hairpin length. By pulling on magnetic beads tethered by a DNA hairpin to the surface, the molecule can be unzipped. In this open state it can hybridize with complementary oligonucleotides, which transiently block the hairpin rezipping when the pulling force is reduced. By measuring from the surface to the bead of a blocked hairpin, one can determine the position of the hybrid along the molecule with nearly single-base precision. Our approach can be used to identify a DNA fragment of known sequence in a mix of various fragments and to sequence an unknown DNA fragment by hybridization or ligation.
Assuntos
DNA/genética , Sequenciamento de Nucleotídeos em Larga Escala/métodos , Sequência de Bases , DNA/química , DNA/metabolismo , DNA Ligases/metabolismo , Sequência Rica em GC , Sequenciamento de Nucleotídeos em Larga Escala/instrumentação , Magnetismo , Conformação de Ácido Nucleico , Hibridização de Ácido Nucleico , Moldes GenéticosRESUMO
Enzymatic inhibition by product molecules is an important and widespread phenomenon. We describe an approach to study product inhibition at the single-molecule level. Individual HRP molecules are trapped within surface-tethered lipid vesicles, and their reaction with a fluorogenic substrate is probed. While the substrate readily penetrates into the vesicles, the charged product (resorufin) gets trapped and accumulates inside the vesicles. Surprisingly, individual enzyme molecules are found to stall when a few tens of product molecules accumulate. Bulk enzymology experiments verify that the enzyme is noncompetitively inhibited by resorufin. The initial reaction velocity of individual enzyme molecules and the number of product molecules required for their complete inhibition are broadly distributed and dynamically disordered. The two seemingly unrelated parameters, however, are found to be substantially correlated with each other in each enzyme molecule and over long times. These results suggest that, as a way to counter disorder, enzymes have evolved the means to correlate fluctuations at structurally distinct functional sites.
Assuntos
Regulação Alostérica , Enzimas/química , Lipídeos/química , Algoritmos , Sítio Alostérico , Enzimas/metabolismo , Corantes Fluorescentes , Peroxidase do Rábano Silvestre/antagonistas & inibidores , Peroxidase do Rábano Silvestre/química , Peroxidase do Rábano Silvestre/metabolismo , Cinética , Modelos Químicos , Modelos Moleculares , Estrutura Molecular , Oxazinas/química , Oxazinas/metabolismo , Oxazinas/farmacologia , Ligação Proteica , Estrutura Terciária de ProteínaRESUMO
Replicative holoenzymes exhibit rapid and processive primer extension DNA synthesis, but inefficient strand displacement DNA synthesis. We investigated the bacteriophage T4 and T7 holoenzymes primer extension activity and strand displacement activity on a DNA hairpin substrate manipulated by a magnetic trap. Holoenzyme primer extension activity is moderately hindered by the applied force. In contrast, the strand displacement activity is strongly stimulated by the applied force; DNA polymerization is favoured at high force, while a processive exonuclease activity is triggered at low force. We propose that the DNA fork upstream of the holoenzyme generates a regression pressure which inhibits the polymerization-driven forward motion of the holoenzyme. The inhibition is generated by the distortion of the template strand within the polymerization active site thereby shifting the equilibrium to a DNA-protein exonuclease conformation. We conclude that stalling of the holoenzyme induced by the fork regression pressure is the basis for the inefficient strand displacement synthesis characteristic of replicative polymerases. The resulting processive exonuclease activity may be relevant in replisome disassembly to reset a stalled replication fork to a symmetrical situation. Our findings offer interesting applications for single-molecule DNA sequencing.
Assuntos
DNA Polimerase Dirigida por DNA/metabolismo , DNA/biossíntese , Proteínas Virais/metabolismo , DNA/metabolismo , Replicação do DNA , Holoenzimas/metabolismo , Cinética , Análise de Sequência de DNARESUMO
Living organisms are made of cells that are capable of responding to external signals by modifying their internal state and subsequently their external environment. Revealing and understanding the spatio-temporal dynamics of these complex interaction networks is the subject of a field known as systems biology. To investigate these interactions (a necessary step before understanding or modelling them) one needs to develop means to control or interfere spatially and temporally with these processes and to monitor their response on a fast timescale (< minute) and with single-cell resolution. In 2012, an EMBO workshop on 'single-cell physiology' (organized by some of us) was held in Paris to discuss those issues in the light of recent developments that allow for precise spatio-temporal perturbations and observations. This review will be largely based on the investigations reported there. We will first present a non-exhaustive list of examples of cellular interactions and developmental pathways that could benefit from these new approaches. We will review some of the novel tools that have been developed for the observation of cellular activity and then discuss the recent breakthroughs in optical super-resolution microscopy that allow for optical observations beyond the diffraction limit. We will review the various means to photo-control the activity of biomolecules, which allow for local perturbations of physiological processes. We will end up this review with a report on the current status of optogenetics: the use of photo-sensitive DNA-encoded proteins as sensitive reporters and efficient actuators to perturb and monitor physiological processes.
Assuntos
Fenômenos Ópticos , Análise de Célula Única/métodos , Animais , Corantes Fluorescentes/metabolismo , Humanos , Luz , OptogenéticaRESUMO
The detection of covalent and noncovalent binding events between molecules and biomembranes is a fundamental goal of contemporary biochemistry and analytical chemistry. Currently, such studies are performed routinely using fluorescence methods, surface-plasmon resonance spectroscopy, and electrochemical methods. However, there is still a need for novel sensitive miniaturizable detection methods where the sample does not have to be transferred to the sensor, but the sensor can be brought into contact with the sample studied. We present a novel approach for detection and quantification of processes occurring on the surface of a lipid bilayer membrane, by monitoring the current change through the n-type GaAs-based molecularly controlled semiconductor resistor (MOCSER), on which the membrane is adsorbed. Since GaAs is susceptible to etching in an aqueous environment, a protective thin film of methoxysilane was deposited on the device. The system was found to be sensitive enough to allow monitoring changes in pH and in the concentration of amino acids in aqueous solution on top of the membrane. When biotinylated lipids were incorporated into the membrane, it was possible to monitor the binding of streptavidin or avidin. The device modified with biotin-streptavidin complex was capable of detecting the binding of streptavidin antibodies to immobilized streptavidin with high sensitivity and selectivity. The response depends on the charge on the analyte. These results open the way to facile electrical detection of protein-membrane interactions.
Assuntos
Membranas Artificiais , SemicondutoresRESUMO
In this work, we discuss the active or passive character of helicases. In the past years, several studies have used the theoretical framework proposed by Betterton and Julicher [Betterton, M.D. and Julicher, F. (2005) Opening of nucleic-acid double strands by helicases: active versus passive opening. Phys. Rev. E, 71, 11904-11911.] to analyse the unwinding data and assess the mechanism of the helicase under study (active versus passive). However, this procedure has given rise to apparently contradictory interpretations: helicases exhibiting similar behaviour have been classified as both active and passive enzymes [Johnson, D.S., Bai, L. Smith, B.Y., Patel, S.S. and Wang, M.D. (2007) Single-molecule studies reveal dynamics of DNA unwinding by the ring-shaped T7 helicase. Cell, 129, 1299-1309; Lionnet, T., Spiering, M.M., Benkovic, S.J., Bensimon, D. and Croquette, V. (2007) Real-time observation of bacteriophage T4 gp41 helicase reveals an unwinding mechanism Proc. Natl Acid. Sci., 104, 19790-19795]. In this work, we show that when the helicase under study has not been previously well characterized (namely, if its step size and rate of slippage are unknown) a multi-parameter fit to the afore-mentioned model can indeed lead to contradictory interpretations. We thus propose to differentiate between active and passive helicases on the basis of the comparison between their observed translocation velocity on single-stranded nucleic acid and their unwinding rate of double-stranded nucleic acid (with various GC content and under different tensions). A threshold separating active from passive behaviour is proposed following an analysis of the reported activities of different helicases. We study and contrast the mechanism of two helicases that exemplify these two behaviours: active for the RecQ helicase and passive for the gp41 helicase.
Assuntos
DNA Helicases/metabolismo , RecQ Helicases/metabolismo , Proteínas Virais/metabolismo , Biocatálise , DNA/química , DNA/metabolismo , Modelos BiológicosRESUMO
During vertebrate development, embryonic cells pass through a continuum of transitory pluripotent states that precede multi-lineage commitment and morphogenesis. Such states are referred to as "refractory/naïve" and "competent/formative" pluripotency. The molecular mechanisms maintaining refractory pluripotency or driving the transition to competent pluripotency, as well as the cues regulating multi-lineage commitment, are evolutionarily conserved. Vertebrate-specific "Developmental Potential Guardians" (vsDPGs; i.e., VENTX/NANOG, POU5/OCT4), together with MEK1 (MAP2K1), coordinate the pluripotency continuum, competence for multi-lineage commitment and morphogenesis in vivo. During neurulation, vsDPGs empower ectodermal cells of the neuro-epithelial border (NEB) with multipotency and ectomesenchyme potential through an "endogenous reprogramming" process, giving rise to the neural crest cells (NCCs). Furthermore, vsDPGs are expressed in undifferentiated-bipotent neuro-mesodermal progenitor cells (NMPs), which participate in posterior axis elongation and growth. Finally, vsDPGs are involved in carcinogenesis, whereby they confer selective advantage to cancer stem cells (CSCs) and therapeutic resistance. Intriguingly, the heterogenous distribution of vsDPGs in these cell types impact on cellular potential and features. Here, we summarize the findings about the role of vsDPGs during vertebrate development and their selective advantage in evolution. Our aim to present a holistic view regarding vsDPGs as facilitators of both cell plasticity/adaptability and morphological innovation/variation. Moreover, vsDPGs may also be at the heart of carcinogenesis by allowing malignant cells to escape from physiological constraints and surveillance mechanisms.
Assuntos
Células-Tronco Pluripotentes , Animais , Carcinogênese/metabolismo , Diferenciação Celular/fisiologia , Crista Neural , Células-Tronco Pluripotentes/metabolismo , VertebradosRESUMO
Somitogenesis, the segmentation of the antero-posterior axis in vertebrates, is thought to result from the interactions between a genetic oscillator and a posterior-moving determination wavefront. The segment (somite) size is set by the product of the oscillator period and the velocity of the determination wavefront. Surprisingly, while the segmentation period can vary by a factor three between 20 °C and 32 °C, the somite size is constant. How this temperature independence is achieved is a mystery that we address in this study. Using RT-qPCR we show that the endogenous fgf8 mRNA concentration decreases during somitogenesis and correlates with the exponent of the shrinking pre-somitic mesoderm (PSM) size. As the temperature decreases, the dynamics of fgf8 and many other gene transcripts, as well as the segmentation frequency and the PSM shortening and tail growth rates slows down as T-Tc (with Tc = 14.4 °C). This behavior characteristic of a system near a critical point may account for the temperature independence of somitogenesis in zebrafish.
Assuntos
Embrião não Mamífero/metabolismo , Desenvolvimento Embrionário/fisiologia , Fator 8 de Crescimento de Fibroblasto/metabolismo , Regulação da Expressão Gênica no Desenvolvimento/fisiologia , Animais , Desenvolvimento Embrionário/genética , Fator 8 de Crescimento de Fibroblasto/genética , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Peixe-Zebra , p-Aminoazobenzeno/análogos & derivados , p-Aminoazobenzeno/farmacologiaRESUMO
Accurate decoding of nucleic acid variation is critical to understand the complexity and regulation of genome function. Here we use a single-molecule magnetic tweezer (MT) platform to identify sequence variation and map a range of important epigenetic base modifications with high sensitivity, specificity, and precision in the same single molecules of DNA or RNA. We have also developed a highly specific amplification-free CRISPR-Cas enrichment strategy to isolate genomic regions from native DNA. We demonstrate enrichment of DNA from both E. coli and the FMR1 5'UTR coming from cells derived from a Fragile X carrier. From these kilobase-length enriched molecules we could characterize the differential levels of adenine and cytosine base modifications on E. coli, and the repeat expansion length and methylation status of FMR1. Together these results demonstrate that our platform can detect a variety of genetic, epigenetic, and base modification changes concomitantly within the same single molecules.
Assuntos
Pareamento de Bases , DNA/genética , Epigênese Genética , Proteína do X Frágil da Deficiência Intelectual/genética , Síndrome do Cromossomo X Frágil/genética , Variação Genética , RNA/genética , Imagem Individual de Molécula , Regiões 5' não Traduzidas , Sistemas CRISPR-Cas , DNA/metabolismo , Metilação de DNA , DNA Bacteriano/genética , DNA Bacteriano/metabolismo , Escherichia coli/genética , Escherichia coli/metabolismo , Proteína do X Frágil da Deficiência Intelectual/metabolismo , Síndrome do Cromossomo X Frágil/metabolismo , Humanos , Imãs , RNA/metabolismo , Imagem Individual de Molécula/instrumentação , Repetições de TrinucleotídeosRESUMO
We have implemented a noninvasive optical method for the fast control of protein activity in a live zebrafish embryo. It relies on releasing a protein fused to a modified estrogen receptor ligand binding domain from its complex with cytoplasmic chaperones, upon the local photoactivation of a nonendogenous caged inducer. Molecular dynamics simulations were used to design cyclofen-OH, a photochemically stable inducer of the receptor specific for 4-hydroxy-tamoxifen (ER(T2)). Cyclofen-OH was easily synthesized in two steps with good yields. At submicromolar concentrations, it activates proteins fused to the ER(T2) receptor. This was shown in cultured cells and in zebrafish embryos through emission properties and subcellular localization of properly engineered fluorescent proteins. Cyclofen-OH was successfully caged with various photolabile protecting groups. One particular caged compound was efficient in photoinducing the nuclear translocation of fluorescent proteins either globally (with 365 nm UV illumination) or locally (with a focused UV laser or with two-photon illumination at 750 nm). The present method for photocontrol of protein activity could be used more generally to investigate important physiological processes (e.g., in embryogenesis, organ regeneration and carcinogenesis) with high spatiotemporal resolution.