RESUMEN
In cell membranes, proteins and lipids are organized into submicrometric nanodomains of varying sizes, shapes, and compositions, performing specific functions. Despite their biological importance, the detailed morphology of these nanodomains remains unknown. Not only can they hardly be observed by conventional microscopy due to their small size, but there is no full consensus on the theoretical models to describe their structuring and their shapes. Here, we use a combination of analytical calculations and Monte Carlo simulations based upon a model coupling membrane composition and shape to show that increasing protein concentration leads to an elongation of membrane nanodomains. The results are corroborated by single-particle tracking measurements on HIV receptors, whose level of expression in the membrane of specifically designed living cells can be tuned. These findings highlight that protein abundance can modulate nanodomain shape and potentially their biological function. Beyond biomembranes, this mesopatterning mechanism is of relevance in several soft-matter systems because it relies on generic physical arguments.
Asunto(s)
Microscopía , Imagen Individual de Molécula , Membrana Celular/metabolismo , Microdominios de Membrana/metabolismoRESUMEN
G protein-coupled receptors (GPCRs) are signaling hubs in cell membranes that regulate a wide range of physiological processes and are popular drug targets. Serotonin1A receptors are important members of the GPCR family and are implicated in neuropsychiatric disorders. Cholesterol is a key constituent of higher eukaryotic membranes and is believed to contribute to the segregated distribution of membrane constituents into domains. To explore the role of cholesterol in lateral dynamics of GPCRs, we utilized single particle tracking (SPT) to monitor diffusion of serotonin1A receptors under acute and chronic cholesterol-depleted conditions. Our results show that the short-term diffusion coefficient of the receptor decreases upon cholesterol depletion, irrespective of the method of cholesterol depletion. Analysis of SPT trajectories revealed that relative populations of receptors undergoing various modes of diffusion change upon cholesterol depletion. Notably, in cholesterol-depleted cells, we observed an increase in the confined population of the receptor accompanied by a reduction in diffusion coefficient for chronic cholesterol depletion. These results are supported by our recent work and present observations that show polymerization of G-actin in response to chronic cholesterol depletion. Taken together, our results bring out the interdependence of cholesterol and actin cytoskeleton in regulating diffusion of GPCRs in membranes.
Asunto(s)
Receptor de Serotonina 5-HT1A , Serotonina , Membrana Celular/metabolismo , Colesterol/metabolismo , Difusión , Receptor de Serotonina 5-HT1A/metabolismo , Receptores Acoplados a Proteínas G/metabolismo , Serotonina/metabolismo , Imagen Individual de MoléculaRESUMEN
High-throughput single-molecule techniques are expected to challenge the demand for rapid, simple, and sensitive detection methods in health and environmental fields. Based on a single-DNA-molecule biochip for the parallelization of tethered particle motion analyses by videomicroscopy coupled to image analysis and its smart combination with aptamers, we successfully developed an aptasensor enabling the detection of single target molecules by a sandwich assay. One aptamer is grafted to the nanoparticles tethered to the surface by a long DNA molecule bearing the second aptamer in its middle. The detection and quantification of the target are direct. The recognition of the target by a pair of aptamers leads to a looped configuration of the DNA-particle complex associated with a restricted motion of the particles, which is monitored in real time. An analytical range extending over 3 orders of magnitude of target concentration with a limit of detection in the picomolar range was obtained for thrombin.
Asunto(s)
Aptámeros de Nucleótidos , Técnicas Biosensibles , Técnicas Biosensibles/métodos , ADN , Límite de Detección , Análisis por Micromatrices , Trombina/análisisRESUMEN
The occurrence of pharmaceutical residues in surface water is raising environmental concern. To accompany the evolution of measures for natural resources protection, sensing methods enabling sensitive and rapid water quality monitoring are needed. We recently managed the parallelization of the Tethered Particle Motion (TPM), a single molecule technique, sensitive to the conformational changes of DNA. Here, we investigate the capacity of high throughput TPM (htTPM) to detect drugs that intercalate into DNA. As a proof-of-concept we analyze the htTPM signal for two DNA intercalating dyes, namely, YOYO-1 and SYTOX orange. The efficient detection of intercalating drugs is then demonstrated with doxorubicin. We further evaluate the possibility to detect carbamazepine, an antiepileptic massively prescribed and persistent in water, which had been described to interact with DNA through intercalation. Our results corroborated by other techniques show that, in fact, carbamazepine is not a DNA intercalator. The comparison of the results obtained with different aqueous buffers and solutions allows us to identify optimal conditions for the monitoring of intercalation compounds by htTPM.
Asunto(s)
Antibióticos Antineoplásicos/análisis , Benzoxazoles/química , ADN/química , Doxorrubicina/análisis , Colorantes Fluorescentes/química , Compuestos de Quinolinio/química , Compuestos Orgánicos/química , Agua/químicaRESUMEN
Water pollution is a global concern for human and environmental health. As technology and industries have developed over the past decades, increasingly more complex and diverse pollutants are found even in treated waters. For better management of water resources, continuous and efficient monitoring is needed to detect the broad range of contaminants. Biosensors have the potential to meet this challenge and to overcome the limitations of the conventional methods used for water analysis. They combine a biological recognition element to a transducer in a sensitive and robust device, capable of specific detection of molecules of interest. DNA-based sensing technologies meet this set of specifications and benefit from the progress made in nanoscience and nanotechnology. This mini-review proposes an overview of this upcoming new generation of DNA-based biosensors, focusing on promising innovations having for portable, stable, rapid and sensitive devices for water quality monitoring.
Asunto(s)
Técnicas Biosensibles , ADN , Monitoreo del Ambiente/métodos , Calidad del AguaRESUMEN
G protein-coupled receptors (GPCRs) are important membrane proteins in higher eukaryotes that carry out a vast array of cellular signaling and act as major drug targets. The serotonin1A receptor is a prototypical member of the GPCR family and is implicated in neuropsychiatric disorders such as anxiety and depression, besides serving as an important drug target. With an overall goal of exploring the functional consequence of altered receptor dynamics, in this work, we probed the role of the actin cytoskeleton in the dynamics, ligand binding, and signaling of the serotonin1A receptor. We monitored receptor dynamics utilizing single particle tracking, which provides information on relative distribution of receptors in various diffusion modes in addition to diffusion coefficient. We show here that the short-term diffusion coefficient of the receptor increases upon actin destabilization by cytochalasin D. In addition, analysis of individual trajectories shows that there are changes in relative populations of receptors undergoing various types of diffusion upon actin destabilization. The release of dynamic constraint was evident by an increase in the radius of confinement of the receptor upon actin destabilization. The functional implication of such actin destabilization was manifested as an increase in specific agonist binding and downstream signaling, monitored by measuring reduction in cellular cAMP levels. These results bring out the interdependence of GPCR dynamics with cellular signaling.
Asunto(s)
Receptor de Serotonina 5-HT1A , Serotonina , Citoesqueleto de Actina , Actinas , Receptores Acoplados a Proteínas GRESUMEN
Tethering beads to DNA offers a panel of single molecule techniques for the refined analysis of the conformational dynamics of DNA and the elucidation of the mechanisms of enzyme activity. Recent developments include the massive parallelization of these techniques achieved by the fabrication of dedicated nanoarrays by soft nanolithography. We focus here on two of these techniques: the Tethered Particle motion and Magnetic Tweezers allowing analysis of the behavior of individual DNA molecules in the absence of force and under the application of a force and/or a torque, respectively. We introduce the experimental protocols for the parallelization and discuss the benefits already gained, and to come, for these single molecule investigations.
Asunto(s)
ADN/química , Pinzas Ópticas , Imagen Individual de Molécula/métodos , ADN Polimerasa Dirigida por ADN/química , ADN Polimerasa Dirigida por ADN/metabolismo , Magnetismo/métodos , Movimiento (Física) , Nanotecnología/métodos , Conformación de Ácido NucleicoRESUMEN
Even though the persistence length L_{P} of double-stranded DNA plays a pivotal role in cell biology and nanotechnologies, its dependence on ionic strength I lacks a consensual description. Using a high-throughput single-molecule technique and statistical physics modeling, we measure L_{P} in the presence of monovalent (Li^{+}, Na^{+}, K^{+}) and divalent (Mg^{2+}, Ca^{2+}) metallic and alkyl ammonium ions, over a large range 0.5 mM≤I≤5 M. We show that linear Debye-Hückel-type theories do not describe even part of these data. By contrast, the Netz-Orland and Trizac-Shen formulas, two approximate theories including nonlinear electrostatic effects and the finite DNA radius, fit our data with divalent and monovalent ions, respectively, over the whole I range. Furthermore, the metallic ion type does not influence L_{P}(I), in contrast to alkyl ammonium monovalent ions at high I.
RESUMEN
The double stranded DNA molecule undergoes drastic structural changes during biological processes such as transcription during which it opens locally under the action of RNA polymerases. Local spontaneous denaturation could contribute to this mechanism by promoting it. Supporting this idea, different biophysical studies have found an unexpected increase in the flexibility of DNA molecules with various sequences as a function of the temperature, which would be consistent with the formation of a growing number of locally denatured sequences. Here, we take advantage of our capacity to detect subtle changes occurring on DNA by using high throughput tethered particle motion to question the existence of bubbles in double stranded DNA under physiological salt conditions through their conformational impact on DNA molecules ranging from several hundreds to thousands of base pairs. Our results strikingly differ from previously published ones, as we do not detect any unexpected change in DNA flexibility below melting temperature. Instead, we measure a bending modulus that remains stable with temperature as expected for intact double stranded DNA.
Asunto(s)
ADN/química , Temperatura , Tampones (Química) , Movimiento (Física) , Conformación de Ácido Nucleico , Temperatura de Transición , ViscosidadRESUMEN
In bacteria, the FtsK/Xer/dif (chromosome dimer resolution site) system is essential for faithful vertical genetic transmission, ensuring the resolution of chromosome dimers during their segregation to daughter cells. This system is also targeted by mobile genetic elements that integrate into chromosomal dif sites. A central question is thus how Xer/dif recombination is tuned to both act in chromosome segregation and stably maintain mobile elements. To explore this question, we focused on pathogenic Neisseria species harboring a genomic island in their dif sites. We show that the FtsK DNA translocase acts differentially at the recombination sites flanking the genomic island. It stops at one Xer/dif complex, activating recombination, but it does not stop on the other site, thus dismantling it. FtsK translocation thus permits cis discrimination between an endogenous and an imported Xer/dif recombination complex.
Asunto(s)
Proteínas Bacterianas/fisiología , Neisseria gonorrhoeae/fisiología , Recombinasas/metabolismo , Recombinación GenéticaRESUMEN
Epidermal Growth Factor Receptor (EGFR) is an important target of anticancer therapy. Nowadays, the search for new molecules inhibiting this receptor is turning toward natural substances. One of the most promising natural compounds that have shown an anti-EGFR activity is curcumin, a polyphenol found in turmeric. Its effect on the receptor kinase activity and on the receptor autophosphorylation has been already described, but the mechanism of how curcumin interacts with EGFR is not fully elucidated. We demonstrate that the mode of action of curcumin is dual. This polyphenol is able to inhibit directly but partially the enzymatic activity of the EGFR intracellular domain. The present work shows that curcumin also influences the cell membrane environment of EGFR. Using biomimetic membrane models, we show that curcumin insertion into the lipid bilayer leads to its rigidification. Single particle tracking analyses performed in the membrane of A431 cancer cells confirmed that this effect of curcumin on the membrane slows down the receptor diffusion. This is likely to affect the receptor dimerization and, in turn, its activation.
Asunto(s)
Curcumina/uso terapéutico , Receptores ErbB/genética , Neoplasias/tratamiento farmacológico , Línea Celular Tumoral , Membrana Celular/química , Membrana Celular/efectos de los fármacos , Curcumina/química , Receptores ErbB/antagonistas & inhibidores , Humanos , Membrana Dobles de Lípidos/química , Neoplasias/genéticaRESUMEN
Being capable of characterizing DNA local bending is essential to understand thoroughly many biological processes because they involve a local bending of the double helix axis, either intrinsic to the sequence or induced by the binding of proteins. Developing a method to measure DNA bend angles that does not perturb the conformation of the DNA itself or the DNA-protein complex is a challenging task. Here, we propose a joint theory-experiment high-throughput approach to rigorously measure such bend angles using the Tethered Particle Motion (TPM) technique. By carefully modeling the TPM geometry, we propose a simple formula based on a kinked Worm-Like Chain model to extract the bend angle from TPM measurements. Using constructs made of 575 base-pair DNAs with in-phase assemblies of one to seven 6A-tracts, we find that the sequence CA6CGG induces a bend angle of 19° ± 4°. Our method is successfully compared to more theoretically complex or experimentally invasive ones such as cyclization, NMR, FRET or AFM. We further apply our procedure to TPM measurements from the literature and demonstrate that the angles of bends induced by proteins, such as Integration Host Factor (IHF) can be reliably evaluated as well.
Asunto(s)
ADN/química , Secuencia de Bases , ADN/metabolismo , Factores de Integración del Huésped/metabolismo , Modelos Químicos , Movimiento (Física) , Conformación de Ácido Nucleico , Física/métodosRESUMEN
The initial steps of the Human Immunodeficiency Virus (HIV) replication cycle play a crucial role that arbitrates viral tropism and infection efficiency. Before the release of its genome into the host cell cytoplasm, viruses operate a complex sequence of events that take place at the plasma membrane of the target cell. The first step is the binding of the HIV protein envelope (Env) to the cellular receptor CD4. This triggers conformational changes of the gp120 viral protein that allow its interaction with a co-receptor that can be either CCR5 or CXCR4, defining the tropism of the virus entering the cell. This sequential interaction finally drives the fusion of the viral and host cell membrane or to the endocytosis of the viruses. Here, we discuss how the membrane composition and organization of both the virus and the target cell can affect these steps and thus influence the capability of the viruses to infect cells.
Asunto(s)
Membrana Celular/virología , VIH-1/fisiología , Interacciones Huésped-Patógeno , Internalización del Virus , Antígenos CD4/metabolismo , Membrana Celular/química , Membrana Celular/metabolismo , Proteína gp120 de Envoltorio del VIH/metabolismo , VIH-1/metabolismo , Humanos , Modelos Biológicos , Unión Proteica , Receptores CCR5/metabolismoRESUMEN
The dynamic organization of G protein-coupled receptors in the plasma membrane is suspected of playing a role in their function. The regulation of the diffusion mode of the mu-opioid (MOP) receptor was previously shown to be agonist-specific. Here we investigate the regulation of MOP receptor diffusion by heterologous activation of other G protein-coupled receptors and characterize the dynamic properties of the MOP receptor within the heterodimer MOP/neuropeptide FF (NPFF2) receptor. The data show that the dynamics and signaling of the MOP receptor in SH-SY5Y cells are modified by the activation of α2-adrenergic and NPFF2 receptors, but not by the activation of receptors not described to interact with the opioid receptor. By combining, for the first time, fluorescence recovery after photobleaching at variable radius experiments with bimolecular fluorescence complementation, we show that the MOP/NPFF2 heterodimer adopts a specific diffusion behavior that corresponds to a mix of the dynamic properties of both MOP and NPFF2 receptors. Altogether, the data suggest that heterologous regulation is accompanied by a specific organization of receptors in the membrane.
Asunto(s)
Analgésicos Opioides/farmacología , Transporte de Proteínas/efectos de los fármacos , Receptor Cross-Talk/efectos de los fármacos , Receptores Adrenérgicos alfa 2/metabolismo , Receptores de Neuropéptido/metabolismo , Receptores Opioides mu/metabolismo , Línea Celular Tumoral , Membrana Celular/efectos de los fármacos , Membrana Celular/metabolismo , Clonidina/farmacología , Difusión , Encefalina Ala(2)-MeFe(4)-Gli(5)/farmacología , Recuperación de Fluorescencia tras Fotoblanqueo , Colorantes Fluorescentes , Regulación de la Expresión Génica , Humanos , Neuronas/citología , Neuronas/efectos de los fármacos , Neuronas/metabolismo , Neuropéptido Y/farmacología , Oligopéptidos/farmacología , Multimerización de Proteína , Receptores Adrenérgicos alfa 2/genética , Receptores de Neuropéptido/genética , Receptores Opioides mu/genética , Transducción de SeñalRESUMEN
Circular chromosomes can form dimers during replication and failure to resolve those into monomers prevents chromosome segregation, which leads to cell death. Dimer resolution is catalysed by a highly conserved site-specific recombination system, called XerCD-dif in Escherichia coli. Recombination is activated by the DNA translocase FtsK, which is associated with the division septum, and is thought to contribute to the assembly of the XerCD-dif synapse. In our study, direct observation of the assembly of the XerCD-dif synapse, which had previously eluded other methods, was made possible by the use of Tethered Particle Motion, a single molecule approach. We show that XerC, XerD and two dif sites suffice for the assembly of XerCD-dif synapses in absence of FtsK, but lead to inactive XerCD-dif synapses. We also show that the presence of the γ domain of FtsK increases the rate of synapse formation and convert them into active synapses where recombination occurs. Our results represent the first direct observation of the formation of the XerCD-dif recombination synapse and its activation by FtsK.
Asunto(s)
Proteínas de Escherichia coli/metabolismo , Integrasas/metabolismo , Proteínas de la Membrana/metabolismo , Recombinación Genética , Proteínas de Escherichia coli/química , Cinética , Proteínas de la Membrana/química , Movimiento (Física) , Estructura Terciaria de ProteínaRESUMEN
Tethered particle motion (TPM) monitors the variations in the effective length of a single DNA molecule by tracking the Brownian motion of a bead tethered to a support by the DNA molecule. Providing information about DNA conformations in real time, this technique enables a refined characterization of DNA-protein interactions. To increase the output of this powerful but time-consuming single-molecule assay, we have developed a biochip for the simultaneous acquisition of data from more than 500 single DNA molecules. The controlled positioning of individual DNA molecules is achieved by self-assembly on nanoscale arrays fabricated through a standard microcontact printing method. We demonstrate the capacity of our biochip to study biological processes by applying our method to explore the enzymatic activity of the T7 bacteriophage exonuclease. Our single molecule observations shed new light on its behaviour that had only been examined in bulk assays previously and, more specifically, on its processivity.
Asunto(s)
Proteínas de Unión al ADN/metabolismo , ADN/química , Análisis por Micromatrices , Bacteriófago T7/enzimología , ADN/metabolismo , Exodesoxirribonucleasas/metabolismo , Ensayos Analíticos de Alto Rendimiento , Movimiento (Física) , Conformación de Ácido Nucleico , Análisis de Secuencia por Matrices de OligonucleótidosRESUMEN
We investigated the lateral diffusion of the HIV receptor CD4 at the surface of T lymphocytes at 20°C and 37°C by Single Particle Tracking using Quantum Dots. We found that the receptors presented two major distinct behaviors that were not equally affected by temperature changes. About half of the receptors showed a random diffusion with a diffusion coefficient increasing upon raising the temperature. The other half of the receptors was permanently or transiently confined with unchanged dynamics on raising the temperature. These observations suggest that two distinct subpopulations of CD4 receptors with different environments are present at the surface of living T lymphocytes.
Asunto(s)
Antígenos CD4/análisis , Membrana Celular/química , VIH/inmunología , Linfocitos T/química , Membrana Celular/inmunología , Humanos , Células Jurkat , Linfocitos T/inmunologíaRESUMEN
During the orchestrated process leading to mature erythrocytes, reticulocytes must synthesize large amounts of hemoglobin, while eliminating numerous cellular components. Exosomes are small secreted vesicles that play an important role in this process of specific elimination. To understand the mechanisms of proteolipidic sorting leading to their biogenesis, we have explored changes in the composition of exosomes released by reticulocytes during their differentiation, in parallel to their physical properties. By combining proteomic and lipidomic approaches, we found dramatic alterations in the composition of the exosomes retrieved over the course of a 7-day in vitro differentiation protocol. Our data support a previously proposed model, whereby in reticulocytes the biogenesis of exosomes involves several distinct mechanisms for the preferential recruitment of particular proteins and lipids and suggest that the respective prominence of those pathways changes over the course of the differentiation process.
Asunto(s)
Diferenciación Celular/fisiología , Endosomas/metabolismo , Lípidos de la Membrana/biosíntesis , Proteínas de la Membrana/biosíntesis , Reticulocitos/metabolismo , Animales , Hemoglobinas/biosíntesis , Masculino , Proteómica/métodos , Ratas , Ratas Sprague-Dawley , Reticulocitos/citologíaRESUMEN
The tethered particle motion (TPM) technique informs about conformational changes of DNA molecules, e.g. upon looping or interaction with proteins, by tracking the Brownian motion of a particle probe tethered to a surface by a single DNA molecule and detecting changes of its amplitude of movement. We discuss in this context the time resolution of TPM, which strongly depends on the particle-DNA complex relaxation time, i.e. the characteristic time it takes to explore its configuration space by diffusion. By comparing theory, simulations and experiments, we propose a calibration of TPM at the dynamical level: we analyze how the relaxation time grows with both DNA contour length (from 401 to 2080 base pairs) and particle radius (from 20 to 150 nm). Notably we demonstrate that, for a particle of radius 20 nm or less, the hydrodynamic friction induced by the particle and the surface does not significantly slow down the DNA. This enables us to determine the optimal time resolution of TPM in distinct experimental contexts which can be as short as 20 ms.
Asunto(s)
ADN/química , Conformación de Ácido Nucleico , Calibración , Difusión , Sondas Moleculares , Método de MontecarloRESUMEN
Tight regulation of transposition activity is essential to limit damage transposons may cause by generating potentially lethal DNA rearrangements. Assembly of a bona fide protein-DNA complex, the transpososome, within which transposition is catalysed, is a crucial checkpoint in this regulation. In the case of IS911, a member of the large IS3 bacterial insertion sequence family, the transpososome (synaptic complex A; SCA) is composed of the right and left inverted repeated DNA sequences (IRR and IRL) bridged by the transposase, OrfAB (the IS911-encoded enzyme that catalyses transposition). To characterise further this important protein-DNA complex in vitro, we used different tagged and/or truncated transposase forms and analysed their interaction with IS911 ends using gel electrophoresis. Our results allow us to propose a model in which SCA is assembled with a dimeric form of the transposase. Furthermore, we present atomic force microscopy results showing that the terminal inverted repeat sequences are probably assembled in a parallel configuration within the SCA. These results represent the first step in the structural description of the IS911 transpososome, and are discussed in comparison with the very few other transpososome examples described in the literature.