RESUMEN
Intro: Force measurements of the nucleus, the strongest organelle, have propelled the field of mechanobiology to understand the basic mechanical components of the nucleus and how these components properly support nuclear morphology and function. Micromanipulation force measurement provides separation of the relative roles of nuclear mechanical components chromatin and lamin A. Methods: To provide access to this technique, we have developed a universal micromanipulation apparatus for inverted microscopes. We outline how to engineer and utilize this apparatus through dual micromanipulators, fashion and calibrate micropipettes, and flow systems to isolate a nucleus and provide force vs. extensions measurements. This force measurement approach provides the unique ability to measure the separate contributions of chromatin at short extensions and lamin A strain stiffening at long extensions. We then investigated the apparatus' controllable and programmable micromanipulators through compression, isolation, and extension in conjunction with fluorescence to develop new assays for nuclear mechanobiology. Results: Using this methodology, we provide the first rebuilding of the micromanipulation setup outside of its lab of origin and recapitulate many key findings including spring constant of the nucleus and strain stiffening across many cell types. Furthermore, we have developed new micromanipulation-based techniques to compress nuclei inducing nuclear deformation and/or rupture, track nuclear shape post-isolation, and fluorescence imaging during micromanipulation force measurements. Conclusion: We provide the workflow to build and use a micromanipulation apparatus with any inverted microscope to perform nucleus isolation, force measurements, and various other biophysical techniques. Supplementary Information: The online version contains supplementary material available at 10.1007/s12195-022-00734-y.
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Structural Maintenance of Chromosomes (SMC) complexes play essential roles in genome organization across all domains of life. To determine how the activities of these large (≈50 nm) complexes are controlled by ATP binding and hydrolysis, we developed a molecular dynamics model that accounts for conformational motions of the SMC and DNA. The model combines DNA loop capture with an ATP-induced 'power stroke' to translocate the SMC complex along DNA. This process is sensitive to DNA tension: at low tension (0.1 pN), the model makes loop-capture steps of average 60 nm and up to 200 nm along DNA (larger than the complex itself), while at higher tension, a distinct inchworm-like translocation mode appears. By tethering DNA to an experimentally-observed additional binding site ('safety belt'), the model SMC complex can perform loop extrusion (LE). The dependence of LE on DNA tension is distinct for fixed DNA tension vs. fixed DNA end points: LE reversal occurs above 0.5 pN for fixed tension, while LE stalling without reversal occurs at about 2 pN for fixed end points. Our model matches recent experimental results for condensin and cohesin, and makes testable predictions for how specific structural variations affect SMC function.
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Cromosomas , Simulación de Dinámica Molecular , Adenosina Trifosfato/metabolismo , Proteínas de Ciclo Celular/metabolismo , Cromosomas/metabolismo , ADN/química , Humanos , Conformación Molecular , Translocación GenéticaRESUMEN
Supercoiling impacts DNA replication, transcription, protein binding to DNA, and the three-dimensional organization of chromosomes. However, there are currently no methods to directly interrogate or map positive supercoils, so their distribution in genomes remains unknown. Here, we describe a method, GapR-seq, based on the chromatin immunoprecipitation of GapR, a bacterial protein that preferentially recognizes overtwisted DNA, for generating high-resolution maps of positive supercoiling. Applying this method to Escherichia coli and Saccharomyces cerevisiae, we find that positive supercoiling is widespread, associated with transcription, and particularly enriched between convergently oriented genes, consistent with the 'twin-domain' model of supercoiling. In yeast, we also find positive supercoils associated with centromeres, cohesin-binding sites, autonomously replicating sites, and the borders of R-loops (DNA-RNA hybrids). Our results suggest that GapR-seq is a powerful approach, likely applicable in any organism, to investigate aspects of chromosome structure and organization not accessible by Hi-C or other existing methods.
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Proteínas Bacterianas/genética , Inmunoprecipitación de Cromatina , Estructuras Cromosómicas , Cromosomas/metabolismo , Proteínas Bacterianas/metabolismo , Sitios de Unión , Proteínas de Ciclo Celular , Proteínas Cromosómicas no Histona , Cromosomas Bacterianos , ADN/metabolismo , Replicación del ADN , ADN Bacteriano , Escherichia coli/genética , Unión Proteica , Saccharomyces cerevisiae/genética , Transcripción Genética , CohesinasRESUMEN
Chromatin, which consists of DNA and associated proteins, contains genetic information and is a mechanical component of the nucleus. Heterochromatic histone methylation controls nucleus and chromosome stiffness, but the contribution of heterochromatin protein HP1α (CBX5) is unknown. We used a novel HP1α auxin-inducible degron human cell line to rapidly degrade HP1α. Degradation did not alter transcription, local chromatin compaction, or histone methylation, but did decrease chromatin stiffness. Single-nucleus micromanipulation reveals that HP1α is essential to chromatin-based mechanics and maintains nuclear morphology, separate from histone methylation. Further experiments with dimerization-deficient HP1αI165E indicate that chromatin crosslinking via HP1α dimerization is critical, while polymer simulations demonstrate the importance of chromatin-chromatin crosslinkers in mechanics. In mitotic chromosomes, HP1α similarly bolsters stiffness while aiding in mitotic alignment and faithful segregation. HP1α is therefore a critical chromatin-crosslinking protein that provides mechanical strength to chromosomes and the nucleus throughout the cell cycle and supports cellular functions.
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Núcleo Celular/metabolismo , Cromatina , Proteínas Cromosómicas no Histona , Cromosomas , Mitosis/fisiología , Línea Celular , Núcleo Celular/química , Cromatina/química , Cromatina/metabolismo , Homólogo de la Proteína Chromobox 5 , Proteínas Cromosómicas no Histona/química , Proteínas Cromosómicas no Histona/metabolismo , Cromosomas/química , Cromosomas/metabolismo , Humanos , MetilaciónRESUMEN
Cytosine methylated at the five-carbon position is the most widely studied reversible DNA modification. Prior findings indicate that methylation can alter mechanical properties. However, those findings were qualitative and sometimes contradictory, leaving many aspects unclear. By applying single-molecule magnetic force spectroscopy techniques allowing for direct manipulation and dynamic observation of DNA mechanics and mechanically driven strand separation, we investigated how CpG and non-CpG cytosine methylation affects DNA micromechanical properties. We quantitatively characterized DNA stiffness using persistence length measurements from force-extension curves in the nanoscale length regime and demonstrated that cytosine methylation results in longer contour length and increased DNA flexibility (i.e., decreased persistence length). In addition, we observed the preferential formation of plectonemes over unwound single-stranded "bubbles" of DNA under physiologically relevant stretching forces and supercoiling densities. The flexibility and high structural stability of methylated DNA is likely to have significant consequences on the recruitment of proteins recognizing cytosine methylation and DNA packaging.
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Citosina , ADN , ADN/metabolismo , Metilación de ADN , Micromanipulación , NanotecnologíaRESUMEN
Nuclear compartmentalization of active and inactive chromatin is thought to occur through microphase separation mediated by interactions between loci of similar type. The nature and dynamics of these interactions are not known. We developed liquid chromatin Hi-C to map the stability of associations between loci. Before fixation and Hi-C, chromosomes are fragmented, which removes strong polymeric constraint, enabling detection of intrinsic locus-locus interaction stabilities. Compartmentalization is stable when fragments are larger than 10-25 kb. Fragmentation of chromatin into pieces smaller than 6 kb leads to gradual loss of genome organization. Lamin-associated domains are most stable, whereas interactions for speckle- and polycomb-associated loci are more dynamic. Cohesin-mediated loops dissolve after fragmentation. Liquid chromatin Hi-C provides a genome-wide view of chromosome interaction dynamics.
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Cromatina/química , Cromatina/metabolismo , Cromosomas Humanos/química , Compartimento Celular , Proteínas de Ciclo Celular/metabolismo , Núcleo Celular/química , Núcleo Celular/genética , Cromatina/genética , Ensamble y Desensamble de Cromatina , Proteínas Cromosómicas no Histona/metabolismo , Cromosomas Humanos/metabolismo , Semivida , Humanos , Células K562 , Cinética , CohesinasRESUMEN
The structural organization of chromosomes is a crucial feature that defines the functional state of genes and genomes. The extent of structural changes experienced by genomes of eukaryotic cells can be dramatic and spans several orders of magnitude. At the core of these changes lies a unique group of ATPases-the SMC proteins-that act as major effectors of chromosome behavior in cells. The Smc5/6 proteins play essential roles in the maintenance of genome stability, yet their mode of action is not fully understood. Here we show that the human Smc5/6 complex recognizes unusual DNA configurations and uses the energy of ATP hydrolysis to promote their compaction. Structural analyses reveal subunit interfaces responsible for the functionality of the Smc5/6 complex and how mutations in these regions may lead to chromosome breakage syndromes in humans. Collectively, our results suggest that the Smc5/6 complex promotes genome stability as a DNA micro-compaction machine.
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Proteínas de Ciclo Celular/genética , Proteínas Cromosómicas no Histona/genética , Inestabilidad Genómica/genética , Complejos Multiproteicos/ultraestructura , Adenosina Trifosfatasas/genética , Adenosina Trifosfato/genética , Rotura Cromosómica , Humanos , Complejos Multiproteicos/genética , Mutación/genética , Conformación de Ácido Nucleico , Proteínas de Saccharomyces cerevisiae/genéticaRESUMEN
Damaged or mismatched DNA bases result in the formation of physical defects in double-stranded DNA. In vivo, defects in DNA must be rapidly and efficiently repaired to maintain cellular function and integrity. Defects can also alter the mechanical response of DNA to bending and twisting constraints, both of which are important in defining the mechanics of DNA supercoiling. Here, we use coarse-grained molecular dynamics (MD) simulation and supporting statistical-mechanical theory to study the effect of mismatched base pairs on DNA supercoiling. Our simulations show that plectoneme pinning at the mismatch site is deterministic under conditions of relatively high force (>2 pN) and high salt concentration (>0.5 M NaCl). Under physiologically relevant conditions of lower force (0.3 pN) and lower salt concentration (0.2 M NaCl), we find that plectoneme pinning becomes probabilistic and the pinning probability increases with the mismatch size. These findings are in line with experimental observations. The simulation framework, validated with experimental results and supported by the theoretical predictions, provides a way to study the effect of defects on DNA supercoiling and the dynamics of supercoiling in molecular detail.
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Disparidad de Par Base , ADN/química , Simulación de Dinámica MolecularRESUMEN
Meiosis produces four haploid cells after two successive divisions in sexually reproducing organisms. A critical event during meiosis is construction of the synaptonemal complex (SC), a large, protein-based bridge that physically links homologous chromosomes. The SC facilitates meiotic recombination, chromosome compaction, and the eventual separation of homologous chromosomes at metaphase I. We present experiments directly measuring physical properties of captured mammalian meiotic prophase I chromosomes. Mouse meiotic chromosomes are about ten-fold stiffer than somatic mitotic chromosomes, even for genetic mutants lacking SYCP1, the central element of the SC. Meiotic chromosomes dissolve when treated with nucleases, but only weaken when treated with proteases, suggesting that the SC is not rigidly connected, and that meiotic prophase I chromosomes are a gel meshwork of chromatin, similar to mitotic chromosomes. These results are consistent with a liquid- or liquid-crystal SC, but with SC-chromatin stiff enough to mechanically drive crossover interference.
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Cromatina/metabolismo , Cromosomas de los Mamíferos , Profase Meiótica I , Espermatocitos , Animales , Cromatina/ultraestructura , Cromosomas de los Mamíferos/metabolismo , Cromosomas de los Mamíferos/ultraestructura , Técnica del Anticuerpo Fluorescente , Geles , Masculino , Ratones , Ratones Endogámicos C57BL , Espermatocitos/metabolismo , Espermatocitos/ultraestructuraRESUMEN
SMC complexes, such as condensin or cohesin, organize chromatin throughout the cell cycle by a process known as loop extrusion. SMC complexes reel in DNA, extruding and progressively growing DNA loops. Modeling assuming two-sided loop extrusion reproduces key features of chromatin organization across different organisms. In vitro single-molecule experiments confirmed that yeast condensins extrude loops, however, they remain anchored to their loading sites and extrude loops in a 'one-sided' manner. We therefore simulate one-sided loop extrusion to investigate whether 'one-sided' complexes can compact mitotic chromosomes, organize interphase domains, and juxtapose bacterial chromosomal arms, as can be done by 'two-sided' loop extruders. While one-sided loop extrusion cannot reproduce these phenomena, variants can recapitulate in vivo observations. We predict that SMC complexes in vivo constitute effectively two-sided motors or exhibit biased loading and propose relevant experiments. Our work suggests that loop extrusion is a viable general mechanism of chromatin organization.
The different molecules of DNA in a cell are called chromosomes, and they change shape dramatically when cells divide. Ordinarily, chromosomes are packaged by proteins called histones to make thick fibres called chromatin. Chromatin fibres are further folded into a sparse collection of loops. These loops are important not only to make genetic material fit inside a cell, but also to make distant regions of the chromosomes interact with each other, which is important to regulate gene activities. The fibres compact to prepare for cell division: they fold into a much denser series of loops. This is a remarkable physical feat in which tiny protein machines wrangle lengthy strands of DNA. A process called loop extrusion could explain how chromatin folding works. In this process, ring-like protein complexes known as SMC complexes would act as motors that can form loops. SMC complexes could bind a chromatin fibre and reel it in to form the loops, with the density of loops increasing before cell division to further compact the chromosomes. Looping by SMC complexes has been observed in a variety of cell types, including mammalian and bacterial cells. From these studies, loop extrusion is generally assumed to be 'two-sided'. This means that each SMC complex reels in the chromatin on both sides of it, thus growing the chromatin loop. However, imaging individual SMC complexes bound to single molecules of DNA showed that extrusion can be asymmetric, or 'one-sided'. These observations show the SMC complex remains anchored in place and the chromatin is reeled in and extruded by only one side of the complex. So Banigan, van den Berg, Brandão et al. created a computer model to test whether the mechanism of one-sided extrusion could produce chromosomes that are organised, compact, and ready for cell division, like two-sided extrusion can. To answer this question, Banigan, van den Berg, Brandão et al. analysed imaging experiments and data that had been collected using a technique that captures how chromatin fibres are arranged inside cells. This was paired with computer simulations of chromosomes bound by SMC protein complexes. The simulations and analysis found that the simplest one-sided loop extrusion complexes generally cannot reproduce the same patterns of chromatin loops as two-sided complexes. However, a few specific variations of one-sided extrusion can actually recapitulate correct chromatin folding and organisation. These results show that some aspects of chromosome organization can be attained by one-sided extrusion, but many require two-sided extrusion. Banigan, van den Berg, Brandão et al. explain how the simulated mechanisms of loop extrusion could be consistent with seemingly contradictory observations from different sets of experiments. Altogether, they demonstrate that loop extrusion is a viable general mechanism to explain chromatin organisation, and that it likely possesses physical capabilities that have yet to be observed experimentally.
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Cromosomas/química , Proteínas Bacterianas/química , Proteínas Bacterianas/fisiología , Proteínas de Ciclo Celular/química , Proteínas de Ciclo Celular/fisiología , Cromatina/química , Cromosomas Bacterianos/química , Interfase , Modelos Moleculares , Conformación Molecular , Simulación de Dinámica MolecularRESUMEN
Advances in molecular biology, optics, genetics, and bioinformatics have opened the door to mapping, in molecular detail, processes inside living cells. With the ability to observe the individual moving parts of cellular machinery, concepts formerly confined to physics are entering mainstream biology. This article discusses a few ideas of this sort related to chromosome biology, to illustrate what kinds of insights physics might yet bring to our understanding of living systems.
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Cromosomas/genética , Biología Molecular/métodos , Física/métodos , Biofisica/métodos , Cromosomas/ultraestructura , Biología Computacional/métodos , HumanosRESUMEN
Eukaryote cell division features a chromosome compaction-decompaction cycle that is synchronized with their physical and topological segregation. It has been proposed that lengthwise compaction of chromatin into mitotic chromosomes via loop extrusion underlies the compaction-segregation/resolution process. We analyze this disentanglement scheme via considering the chromosome to be a succession of DNA/chromatin loops-a polymer "brush"-where active extrusion of loops controls the brush structure. Given type-II DNA topoisomerase (Topo II)-catalyzed topology fluctuations, we find that interchromosome entanglements are minimized for a certain "optimal" loop that scales with the chromosome size. The optimal loop organization is in accord with experimental data across species, suggesting an important structural role of genomic loops in maintaining a less entangled genome. Application of the model to the interphase genome indicates that active loop extrusion can maintain a level of chromosome compaction with suppressed entanglements; the transition to the metaphase state requires higher lengthwise compaction and drives complete topological segregation. Optimized genomic loops may provide a means for evolutionary propagation of gene-expression patterns while simultaneously maintaining a disentangled genome. We also find that compact metaphase chromosomes have a densely packed core along their cylindrical axes that explains their observed mechanical stiffness. Our model connects chromosome structural reorganization to topological resolution through the cell cycle and highlights a mechanism of directing Topo II-mediated strand passage via loop extrusion-driven lengthwise compaction.
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Cromatina , Cromosomas , Animales , Cromatina/química , Cromatina/metabolismo , Cromosomas/química , Cromosomas/genética , Cromosomas/metabolismo , ADN/química , ADN/metabolismo , Genoma/genética , Humanos , Metafase/genética , Mitosis/genética , Modelos Genéticos , Schizosaccharomyces/genéticaRESUMEN
Dissociation of a protein from DNA is often assumed to be described by an off rate that is independent of other molecules in solution. Recent experiments and computational analyses have challenged this view by showing that unbinding rates (residence times) of DNA-bound proteins can depend on concentrations of nearby molecules that are competing for binding. This 'facilitated dissociation' (FD) process can occur at the single-binding site level via formation of a ternary complex, and can dominate over 'spontaneous dissociation' at low (submicromolar) concentrations. In the crowded intracellular environment FD introduces new regulatory possibilities at the level of individual biomolecule interactions.
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Proteínas de Unión al ADN/metabolismo , Unión Competitiva , ADN/metabolismo , Proteínas de Unión al ADN/química , Sales (Química)/farmacologíaRESUMEN
The nucleus houses, organizes, and protects chromatin to ensure genome integrity and proper gene expression, but how the nucleus adapts mechanically to changes in the extracellular environment is poorly understood. Recent studies have revealed that extracellular physical stresses induce chromatin compaction via mechanotransductive processes. We report that increased extracellular multivalent cations lead to increased heterochromatin levels through activation of mechanosensitive ion channels (MSCs), without large-scale cell stretching. In cells with perturbed chromatin or lamins, this increase in heterochromatin suppresses nuclear blebbing associated with nuclear rupture and DNA damage. Through micromanipulation force measurements, we show that this increase in heterochromatin increases chromatin-based nuclear rigidity, which protects nuclear morphology and function. In addition, transduction of elevated extracellular cations rescues nuclear morphology in model and patient cells of human diseases, including progeria and the breast cancer model cell line MDA-MB-231. We conclude that nuclear mechanics, morphology, and function can be modulated by cell sensing of the extracellular environment through MSCs and consequent changes to histone modification state and chromatin-based nuclear rigidity.
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Heterocromatina/metabolismo , Mecanorreceptores/fisiología , Mecanotransducción Celular/fisiología , Animales , Fenómenos Biomecánicos/fisiología , Línea Celular , Línea Celular Tumoral , Núcleo Celular/metabolismo , Forma de la Célula/fisiología , Cromatina/metabolismo , Ensamble y Desensamble de Cromatina , Heterocromatina/fisiología , Histonas/metabolismo , Humanos , Lamina Tipo A/metabolismo , Mecanorreceptores/metabolismoRESUMEN
The nucleus houses, organizes, and protects chromatin to ensure genome integrity and proper gene expression, but how the nucleus adapts mechanically to changes in the extracellular environment is poorly understood. Recent studies have revealed that extracellular physical stresses induce chromatin compaction via mechanotransductive processes. We report that increased extracellular multivalent cations lead to increased heterochromatin levels through activation of mechanosensitive ion channels, without large-scale cell stretching. In cells with perturbed chromatin or lamins, this increase in heterochromatin suppresses nuclear blebbing associated with nuclear rupture and DNA damage. Through micromanipulation force measurements, we show that this increase in heterochromatin increases chromatin-based nuclear rigidity, which protects nuclear morphology and function. In addition, transduction of elevated extracellular cations rescues nuclear morphology in model and patient cells of human diseases, including progeria and the breast cancer model cell line MDA-MB-231. We conclude that nuclear mechanics, morphology, and function can be modulated by cell sensing of the extracellular environment through mechanosensitive ion channels and consequent changes to histone modification state and chromatin-based nuclear rigidity.
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Cells possess remarkable control of the folding and entanglement topology of long and flexible chromosomal DNA molecules. It is thought that structural maintenance of chromosome (SMC) protein complexes play a crucial role in this, by organizing long DNAs into series of loops. Experimental data suggest that SMC complexes are able to translocate on DNA, as well as pull out lengths of DNA via a 'loop extrusion' process. We describe a Brownian loop-capture-ratchet model for translocation and loop extrusion based on known structural, catalytic, and DNA-binding properties of the Bacillus subtilis SMC complex. Our model provides an example of a new class of molecular motor where large conformational fluctuations of the motor 'track'-in this case DNA-are involved in the basic translocation process. Quantitative analysis of our model leads to a series of predictions for the motor properties of SMC complexes, most strikingly a strong dependence of SMC translocation velocity and step size on tension in the DNA track that it is moving along, with 'stalling' occuring at subpiconewton tensions. We discuss how the same mechanism might be used by structurally related SMC complexes (Escherichia coli MukBEF and eukaryote condensin, cohesin and SMC5/6) to organize genomic DNA.
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Proteínas Bacterianas/metabolismo , Proteínas de Ciclo Celular/metabolismo , Proteínas Cromosómicas no Histona/metabolismo , ADN/química , Modelos Químicos , Proteínas Motoras Moleculares/metabolismo , Complejos Multiproteicos/metabolismo , Conformación de Ácido Nucleico , Adenosina Difosfato/metabolismo , Adenosina Trifosfato/metabolismo , Animales , ADN/metabolismo , Células Eucariotas/metabolismo , Cinética , Unión Proteica , Conformación Proteica , Estrés Mecánico , Termodinámica , CohesinasRESUMEN
Understanding the relationship between intracellular motion and macromolecular structure remains a challenge in biology. Macromolecular structures are assembled from numerous molecules, some of which cannot be labeled. Most techniques to study motion require potentially cytotoxic dyes or transfection, which can alter cellular behavior and are susceptible to photobleaching. Here we present a multimodal label-free imaging platform for measuring intracellular structure and macromolecular dynamics in living cells with a sensitivity to macromolecular structure as small as 20 nm and millisecond temporal resolution. We develop and validate a theory for temporal measurements of light interference. In vitro, we study how higher-order chromatin structure and dynamics change during cell differentiation and ultraviolet (UV) light irradiation. Finally, we discover cellular paroxysms, a near-instantaneous burst of macromolecular motion that occurs during UV induced cell death. With nanoscale sensitive, millisecond resolved capabilities, this platform could address critical questions about macromolecular behavior in live cells.
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Apoptosis/efectos de la radiación , Microscopía Intravital/métodos , Microscopía de Interferencia/métodos , Imagen Multimodal/métodos , Rayos Ultravioleta/efectos adversos , Citoesqueleto de Actina/metabolismo , Diferenciación Celular , Cromatina/metabolismo , Células HeLa , Humanos , Microscopía Intravital/instrumentación , Células Madre Mesenquimatosas , Microscopía de Interferencia/instrumentación , Imagen Multimodal/instrumentación , Nanosferas , Fantasmas de Imagen , Fosfatidilserinas/metabolismo , Factores de TiempoRESUMEN
The simplest model of DNA mechanics describes the double helix as a continuous rod with twist and bend elasticity. Recent work has discussed the relevance of a little-studied coupling G between twisting and bending, known to arise from the groove asymmetry of the DNA double helix. Here the effect of G on the statistical mechanics of long DNA molecules subject to applied forces and torques is investigated. We present a perturbative calculation of the effective torsional stiffness C_{eff} for small twist-bend coupling. We find that the "bare" G is "screened" by thermal fluctuations, in the sense that the low-force, long-molecule effective free energy is that of a model with G=0 but with long-wavelength bending and twisting rigidities that are shifted by G-dependent amounts. Using results for torsional and bending rigidities for freely fluctuating DNA, we show how our perturbative results can be extended to a nonperturbative regime. These results are in excellent agreement with numerical calculations for Monte Carlo "triad" and molecular dynamics "oxDNA" models, characterized by different degrees of coarse graining, validating the perturbative and nonperturbative analyses. While our theory is in generally good quantitative agreement with experiment, the predicted torsional stiffness does systematically deviate from experimental data, suggesting that there are as-yet-uncharacterized aspects of DNA twisting-stretching mechanics relevant to low-force, long-molecule mechanical response, which are not captured by widely used coarse-grained models.
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ADN , Modelos Moleculares , Modelos Estadísticos , Algoritmos , Fenómenos Biomecánicos , Simulación por Computador , ADN/química , Elasticidad , Modelos Químicos , Modelos Genéticos , Método de Montecarlo , Conformación de Ácido Nucleico , Torsión MecánicaRESUMEN
Rebinding kinetics of molecular ligands plays a key role in the operation of biomachinery, from regulatory networks to protein transcription, and is also a key factor in design of drugs and high-precision biosensors. In this study, we investigate initial release and rebinding of ligands to their binding sites grafted on a planar surface, a situation commonly observed in single-molecule experiments and that occurs in vivo, e.g., during exocytosis. Via scaling arguments and molecular dynamic simulations, we analyze the dependence of nonequilibrium rebinding kinetics on two intrinsic length scales: the average separation distance between the binding sites and the total diffusible volume (i.e., height of the experimental reservoir in which diffusion takes place or average distance between receptor-bearing surfaces). We obtain time-dependent scaling laws for on rates and for the cumulative number of rebinding events. For diffusion-limited binding, the (rebinding) on rate decreases with time via multiple power-law regimes before the terminal steady-state (constant on-rate) regime. At intermediate times, when particle density has not yet become uniform throughout the diffusible volume, the cumulative number of rebindings exhibits a novel, to our knowledge, plateau behavior because of the three-dimensional escape process of ligands from binding sites. The duration of the plateau regime depends on the average separation distance between binding sites. After the three-dimensional diffusive escape process, a one-dimensional diffusive regime describes on rates. In the reaction-limited scenario, ligands with higher affinity to their binding sites (e.g., longer residence times) delay entry to the power-law regimes. Our results will be useful for extracting hidden timescales in experiments such as kinetic rate measurements for ligand-receptor interactions in microchannels, as well as for cell signaling via diffusing molecules.
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Simulación de Dinámica Molecular , Proteínas/química , Sitios de Unión , Difusión , Cinética , Ligandos , Unión Proteica , Conformación ProteicaRESUMEN
The cell nucleus encloses, organizes, and protects the genome. Chromatin maintains nuclear mechanical stability and shape in coordination with lamins and the cytoskeleton. Abnormal nuclear shape is a diagnostic marker for human diseases, and it can cause nuclear dysfunction. Chromatin mechanics underlies this link, as alterations to chromatin and its physical properties can disrupt or rescue nuclear shape. The cell can regulate nuclear shape through mechanotransduction pathways that sense and respond to extracellular cues, thus modulating chromatin compaction and rigidity. These findings reveal how chromatin's physical properties can regulate cellular function and drive abnormal nuclear morphology and dysfunction in disease.